Milk Thistle Extract Demonstrates Potent Activity Against Liver Cancer

By Dena Schmidt

The compound silibinin, a flavonoid in milk thistle extract, has shown extremely promising activity against human liver cancer, according to University of California, Irvine researchers.

Both silibinin and silymarin are highly bioactive milk thistle compounds that are already linked with offering protection for the liver against damage from alcohol and drug use. Now silibinin is showing promise in protecting against a number of cancer types, including liver cancer.

Liver cancer cases have tripled in the U.S. since 1980

For the study, human cells were treated with the milk thistle extract silibinin at different doses. Its substantial anticancer effects were linked with several specific processes. These included suppressing the cell cycle of cancer cells, reduction of the cellular proliferation of cancer and increasing apoptosis (cancer cell death).

Silibinin has also shown promise against breast, prostate, lung, colon, bladder and ovarian cancers. Milk thistle has previously been linked with protecting against nonalcoholic fatty liver disease, metabolic syndrome and the hepatitis C virus.

An amazing fact: The liver is the largest internal organ and critical to all of the body’s processes and functioning. It performs over 500 life-sustaining actions for the body, including those related to the neutralization of toxins.

Unfortunately, liver cancer is on the rise in the U.S. (largely due to poor lifestyle choices), with around 40,000 new cases reported per year. This is triple the amount of cases that were reported in 1980. No doubt, the silibinin extract from milk thistle could offer protection against this rising rate of liver cancer.

Get your Tachyonised Milk thistle extract at the Tachyon shop

Milk thistle extract helps offset the hazards of a typical Western diet

As the liver does its job of detoxifying the body, milk thistle seems to have the effect of shielding liver cells from free radicals, processed sugars, toxic fats and other compounds that can cause many liver health issues.

The ‘Standard American Diet’ (SAD) offers too many refined sugars, toxic additives like MSG, unhealthy fats – loaded with unwanted chemicals and synthetic hormones and other processed foods (that don’t even deserve to be labeled as ‘food’). Clearly, these low-quality foods are largely to blame for the increase in liver problems throughout the country.

Even before doctors tend to diagnose a ‘liver problem,’ unhealthy diets can easily trigger metabolic syndrome – which is characterized by excess body weight (obesity), blood sugar imbalances (prediabetes and diabetes), hypertension and elevated blood fats. All of these conditions eventually lead to a breakdown of the cardiovascular system and many forms of cancer.

Get your Tachyonised Milk thistle extract at the Tachyon shop

Of course, metabolic syndrome (if left untreated) can lead to nonalcoholic fatty liver disease and a general weakening of the liver – which directly leads to an increased risk of liver cancer.

Take milk thistle extract to avoid liver cancer and support your liver health

Milk thistle and the compounds it contains offer multi-faceted protection for the liver against a range of stresses. Taking milk thistle allows the liver to do its job more efficiently and bounce back from exposure to toxins such as unhealthy food and alcohol.

Many of the liver studies done in recent years have involved animals or petri dishes, so it’s hard to glean information about ideal dosage. However, according to many integrative healthcare providers, consumption of milk thistle – as a nutritional supplement – is generally recommended at 420 mg/day in divided doses.

This dosage is considered safe for at least 41 months based upon clinical data. However, higher doses of milk thistle have also been used in clinical trials.

Final thought: If you have liver issues or would like to be proactive about liver health, consider asking an experienced medical professional about adding milk thistle to your supplement routine.

Get your Tachyonised Milk thistle extract at the Tachyon shop

Sources: LifeExtension.com; WJNET.com; LifeExtension.com

This article (Milk Thistle Extract Demonstrates Potent Activity Against Liver Cancer) was originally published on Natural Health365 and syndicated by The Event Chronicle. Via Rise Earth.

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Lemon peel is more powerful than chemotherapy

Autoriteiten verbieden al 30 jaar succesvolle kankerbehandeling van Dr. Hamer

Dit is het schokkende relaas van een Duitse internist, Dr. Hamer, die ondanks keihard bewijs van zijn succesvolle manier om kanker te bestrijden zijn vergunning verloor omdat hij weigerde zijn ontdekkingen over kanker te herroepen. En ook nog eens twee maal een gevangenisstraf kreeg.

http://delangemars.nl/2015/08/03/autoriteiten-verbieden-al-30-jaar-succesvolle-kankerbehandeling/

De arts verloor niet alleen zijn medische vergunning, maar ook zijn vrijheid in 1997 toen Dr. Hamer werd veroordeeld tot 19 maanden gevangenis omdat hij drie mensen, zonder artsenvergunning, gratis medische informatie had gegeven. Maar dat vonden de autoriteiten niet genoeg. Op 9 september 2004 werd Hamer in zijn woning in Spanje gearresteerd. Op basis van een Europees uitleveringsbevel werd hij uitgeleverd aan Frankrijk en daar veroordeeld tot drie jaar onvoorwaardelijke gevangenisstraf. Hij werd veroordeeld en verantwoordelijk bevonden aan de dood van Franse burgers omdat zijn publicaties in het Frans te krijgen waren. Hij had nooit een van deze personen gesproken.

Hamer ging echter onverdroten met zijn levenswerk door.  Hij heeft meer dan 40.000 gevallen geanalyseerd. Ondertussen is zijn werk bekend geworden als Germaanse Nieuwe Geneeskunde (GNG).

De pers en het medisch establishment deinsden er niet voor terug om hem en zijn werk aan te vallen. Journalisten van de roddelpers en medische “deskundigen” schilderen Dr. Hamer af als charlatan, als zelf verheerlijkte wonderheler, een sekteleider, een onzinnig buitenstaander of een dwaze crimineel, die kankerpatiënten de ‘levensreddende’ conventionele behandelingen ontzegde.

Dr. Hamer ontdekte dat elke ziekte ontstaat door een shock of trauma die ons volledig verrast.

Op het moment dat het onverwachte conflict ontstaat, slaat de schok in een specifiek hersengebied. De hersencellen die de conflictinslag ontvangen, zenden een biochemisch signaal naar de bijhorende lichaamscellen en veroorzaken de groei van een tumor (weefseltoename) of een weefselafname of functieverlies, afhankelijk in welk deel van de hersenen de schok ‘inslaat’.

Elke ziekte verloopt in twee fasen:
1. een conflict-actieve fase, die wordt gekenmerkt door emotionele stress, koude extremiteiten, geen eetlust en slapeloosheid en dan, vooropgesteld dat wij het conflict oplossen,
2. een genezingsfase, dit is de periode waarin de psyche, de hersenen en het overeenkomstige orgaan de herstelfase doorstaat, een vaak moeilijk proces met vermoeidheid, koorts, ontsteking, infectie en pijn.
Dr. Hamer benoemde op deze manier “De Vijf Biologische Wetten van de Germaanse Nieuwe Geneeskunde”. In de loop der jaren was hij in staat zijn ontdekkingen te staven aan de hand van meer dan 40.000 casussen. Het resultaat van zijn wetenschappelijke programmaverloop
werk is de opstelling van een “Psyche-Hersenen-Orgaan” tabel, die de ziekte aangeeft, de inhoud van het biologische conflict die haar veroorzaakt, waar de overeenkomstige laesie kan worden gezien op een hersenscan, hoe de ziekte zich manifesteert in de conflict-actieve fase en wat men in de genezingsfase kan verwachten.

Ziekten zoals kanker verliezen hun angstaanjagende imago en worden erkend als wat Hamer noemt ‘zinvolle speciale biologische overlevingsprogramma’s’ waarmee ieder mens wordt geboren.

Het is het lichaam dat zichzelf geneest, echter wat de conventionele geneeskunde (CG) ziet als ziekte is voor Hamer in feite het genezingsproces. Hij werkt in tegenstelling tot de CG met oorzaak en gevolg, waarbij de analyse van de oorzaak ook daadwerkelijk van belang is. De CG is meer symptoom gericht.

Elke ziekte maakt deel uit van onze biologische overlevingsprogramma’s. Alles in onze biologische natuur is gericht op behoud van leven. We zijn niet uitgerust met programma’s die ons willen vernietigen. Ook kanker vormt daarop geen uitzondering.

“Alleen de patiënt kan zijn symptomen ‘behandelen’, omdat alleen hij zijn conflict kan oplossen en alleen hij genezing teweeg kan brengen!”

Dr. Hamer: “Een persoon, die gezond eet, heeft minder kans op biologische conflicten. Dat spreekt vanzelf. Dat komt veel overeen met het feit dat rijke mensen minder kankers krijgen dan arme, omdat de rijken vele conflicten kunnen oplossen door eenvoudigweg hun chequeboek te pakken en een cheque uit te schrijven.
Maar het is onmogelijk kanker (of enige andere ziekte) door een dieet te voorkomen,omdat zelfs een gezond dieet conflicten niet kan verhinderen. In de Natuur zal een sterk, gezond dier natuurlijk minder kankers krijgen dan een zwak of oud dier. Maar dat betekent niet dat ouderdom dus kanker veroorzaakt.”
Hippocratic oath
De Germaanse Nieuwe Geneeskunde (GNG) van Hamer verwerpt de ideeën over kwaadaardige kankercellen of destructieve microben en identificeert “infectieziekten” en kankertumoren als natuurlijke biologische noodmaatregelen die al miljoenen jaren in de praktijk plaatsvinden, bedoeld om het organisme te redden en niet, zoals wordt geleerd, om het te vernietigen.
De angst van de medische wetenschap en de farmacie voor de ontdekkingen van Hamer is enorm. Kijk naar de manier waarop Hamer het leven onmogelijk gemaakt wordt en de weigering om zijn werk te toetsen.
Tot op heden (zie document 12 maart 2008) weigert de Universiteit van Tübingen, ondanks uitspraken van de rechtbank in 1986 en 1994, om Dr Hamers wetenschappelijke werk te toetsen. Ook de reguliere geneeskunde weigert om de GNG te bevestigen, ondanks de vele verificaties door zowel artsen als beroepsverenigingen.
De GNG is een nieuwe zienswijze in de geneeskunde, maar ook een nieuwe filosofie van het bewustzijn. Het is het besef dat ons organisme een onuitputtelijke creativiteit bezit en opmerkelijke zelfgenezende vermogens. Het is ook de erkenning dat elke lichaamscel een biologische wijsheid in zich draagt, die wij met alle levende wezens delen. Generaties lang hebben de medische autoriteiten het menselijke denken tot slaaf gemaakt en gevangen gehouden met angst voor ziekten.
De Vijf Biologische Wetten geven ons de kans, ons opnieuw met de Natuur te verbinden en ons vertrouwen te herwinnen in haar creatieve, intelligente kracht. Dankzij Dr Hamers onfeilbare integriteit en zijn toegewijd onderzoek tijdens de afgelopen dertig jaar, kunnen wij ons nu van deze angst bevrijden. (Bron)

GNG in een notendop

De allereerste stap in de GNG therapie is de patiënt de biologische betekenis van een symptoom, bijvoorbeeld kanker, leren begrijpen in relatie met de psychische oorzaak. Een hersenscan en een grondige medische voorgeschiedenis zijn noodzakelijk om te bepalen of een patiënt nog in de conflict-fase (conflictactief) is of al in de genezingsfase zit.

Als de patiënt nog conflictactief is dan ligt de focus op het vinden van het oorspronkelijke conflict en op het vinden van een strategie om dit op te lossen. Vervolgens wordt de patiënt voorbereid op de symptomen van de genezing en eventuele complicaties. Deze symptomen zijn heel voorspelbaar! Voor het eerst in de geschiedenis van de geneeskunde geven de ontdekkingen van Hamer ons een betrouwbaar systeem dat ons in staat stelt om de ontwikkeling en de symptomen van elke ziekte te begrijpen en te voorspellen.

hamerDr. Ryke Geerd Hamer, geboren in 1935 in Friesland (Duitsland), studeerde geneeskunde en theologie  aan de Universiteit van Tübingen. Op zijn 22e behaalde hij zijn academische graad in de theologie en kreeg vier jaar later zijn diploma als arts. De daarop volgende jaren werkte hij aan verschillende universiteitsklinieken in Duitsland. In 1972 voltooide dr. Hamer zijn specialisatie in de interne geneeskunde en begon aan de Universiteitskliniek in Tübingen te werken als internist voor kankerpatiënten. Tegelijkertijd runde hij een privépraktijk, samen met zijn vrouw dr. Sigrid Hamer die hij tijdens zijn studies in Tübingen had ontmoet. Hij bleek ook een buitengewoon talent te hebben voor het uitvinden van medische hulpmiddelen. Hij woonde tot zijn dood op zondagavond 2 juli 2017 in Sandefjord, Noorwegen.

Terminal Cancer Patient Walks Out of Hospice after Treatment with Rick Simpson oil

“After nine months of taking two different forms of cannabis oil, one, a cannabis capsule infused with organic coconut oil around 10:30am and high THC oil (Rick simpson oil) about an hour before bed, dad was given the life changing report, “No evidence of recurrent disease”.” [2]
– Corinne Malanca (Stan and Barb Rutner’s daughter)

Stan Found Cannabis after Chemo and Radiation Almost Killed Him

The trillion dollar burn, cut, poison, Cancer Industrial Complex boldly proclaims that chemotherapy and radiation routinely save lives – that their toxic interventions boost long-term survival rates and the biggest lie of all: chemo and radiation exclusively target cancerous cells and leave healthy cells alone.

All these statements by the cancer industry are patently false. Poison is poison; it kills indiscriminately. Chemo and radiation are poisons posing as medicine. In fact, mainstream cancer “treatment” is the leading cause of secondary cancers.

Dave Mihalovic a Naturopathic Doctor who specializes in vaccine research, cancer prevention and a holistic approach to cancer treatment stated:

“No chemotherapy drug has ever actually cured or resolved the underlying causes of cancer. Even what mainstream medicine considers “successful” chemotherapy treatments are only managing symptoms, usually at the cost of interfering with other precious physiological functions in patients that will cause side effects down the road.” [1]

Ironically, when chemo is killing cancer cells, it’s also stimulating healthy cells to produce a protein that supports tumor growth and makes the cells resistant to further cancer treatment.

Researchers Discover One of the Ways Chemo Enhances Cancer Growth

“The scientists found that healthy cells damaged by chemotherapy secreted more of a protein called WNT16B which boosts cancer cell survival.

“The increase in WNT16B was completely unexpected,” study co-author Peter Nelson of the Fred Hutchinson Cancer Research Center in Seattle told AFP.

The protein was taken up by tumor cells neighboring the damaged cells.
“WNT16B, when secreted, would interact with nearby tumor cells and cause them to grow, invade, and importantly, resist subsequent therapy,” said Nelson.

In cancer treatment, tumors often respond well initially, followed by rapid regrowth and then resistance to further chemotherapy.” [1]

 

The Stan Rutner Story

 

Stan and Barbara Rutner are a devoted, couple who have been together 59 years. They both have hard-won experience battling cancer. Roughly 25 years ago, Stan, a retired dentist with a lucrative mini-storage business was diagnosed with non-Hodgkin lymphoma, while Barb had successfully battled breast cancer twice. Stan remembers:”I just thought I had a cold, flu, type thing. That was it . . . I wasn’t thinking cancer at all.” [3]

After six months of aggressive, standard allopathic, treatment, Stan’s cancer was in remission – that was back in 1989. Meanwhile, Stan had moved on with his life; his bout with cancer faded into the deep dark recesses of his memory. Of course, Stan didn’t expect after all those years of remission he’d be fighting cancer again in 2011 but as mentioned above that’s not uncommon for those who undergo conventional cancer treatment. His initial symptom this time was a persistent cough, otherwise he seemed fine.

Somewhat concerned he made a doctor’s appointment. The initial diagnosis was early stage pneumonia. The doctor noted that there were some unusual looking spots on his lungs that needed follow-up, after the pneumonia had cleared. Numerous tests later and revealed the “spots” were cancerous nodes in the lungs. But there’s more. Stan’s final diagnosis: Stage 4 Lung Cancer with Metastasis to the Brain. Stan recalled how his doctor broke the news, ‘You’re in deep shit.’ Stan explained. “Yeah, he didn’t sugar coat it at all.” But it gets worse much worse. Down the road doctors discovered that the cancer had metastasized to his brain. [3]

Stan’s Health and Quality of life Rapidity Declined

This time, the chemotherapy and radiation, really took a heavy toll on Stan. Shortly after treatment began [march-April of 2012] Stan was plagued with debilitating fatigue, wasting away, weight loss and chronic nausea. Not a pretty picture especially for someone pushing 80.

Barb Rutner: “After that he was wiped out. He was very thin. So we finished radiation to his brain on June 25, and on July 13 he entered the hospital for the first time with radiation pneumonitis (inflammation of the lungs due to radiation therapy), so pneumonia. There were three hospital visits during that time, and on the third they kept him for a week and started him on oxygen 24/7. And then he went into palliative care.” [3]

Stan’s doctors gave him only weeks to live and ordered him to hospice, which he entered on August 12, 2011

The Rutners also used alternative treatments to ease Stan’s suffering and perhaps stall his “inevitable” death sentence. Stan tried Reiki energy treatments and acupuncture. He even attempted creative visualization but it wasn’t his thing and it didn’t really help him, although Barb had success with it when she was fighting breast cancer. Stan’s downward spiral continued unabated and time was running out. That’s when Corrine – Stan and Barb’s daughter and Corinne’s soon to be husband, John Malanca started researching Rick simpson oil as a possible treatment option for Stan.

Barb explains: “Corinne and John were becoming interested in Rick simpson oil but we were concerned that Stan was wasting away. He had lost so much weight and so we were anxious to get his appetite improved and help him with the nausea. So Corinne suggested Rick simpson oil. He started taking that in early November of 2011 and in the beginning, he took about a third of a dose in the morning, and it was about a week or two later that he was able to give up the extra oxygen that he had had 24/7.”[3]

Stan was ready to try Rick simpson oil – he realized better than anyone else that he had nothing to lose and maybe a lot to gain.
Stan stated: “No hesitation at all. Here I’m dying and getting nothing to change the course of things and so this thing has a pretty good track record in—what do you call it—in a non-medical world, so hey let’s try it. Got nothing to lose.” [3]

Stan’s Amazing Turnaround

Bingo! Within a couple of weeks of starting Rick simpson oil, Stan’s condition was improving in all ways. He started gaining weight, his sleep improved, he was regaining his strength. Eventually he ditched his walker, dumped his oxygen and started exercise classes. Barb described his progress as remarkable.

After several months of Cannabis Stan decided to get an MRI to check his progress

Barb: “On January 27, 2013 we received the results of Stan’s brain MRI in an email from his oncologist, stating simply: “IMPRESSION: No evidence of recurrent disease.” The lung cancer that had metastasized to his brain in the summer of 2011 (and nearly taken his life) was GONE! The doctor calls him a miracle man. We are deeply grateful to his “team” of doctors, friends and family, but we’ll always believe that Rick simpson oil turned the tide.” [3]

Barb and Stan Rutner at their Daughter’s
Wedding on September 21, 2013

“Dad never thought that he would make it to that day but low and behold, he continued on the Cannabis Oil and is STILL in remission!”- Corinne Malanca [2]

 

Cannabis Cures Cancer The Stan Rutner Story

Many Alternative Cancer Cures Exist and Have Been Suppressed – Kimberly Carter Gamble

Paul Fassa is a contributing staff writer for REALfarmacy.com. His pet peeves are the Medical Mafia’s control over health and the food industry and government regulatory agencies’ corruption. Paul’s valiant contributions to the health movement and global paradigm shift are world renowned. Visit his blog by following this link and follow him on Twitter here.

Sources:
[1] http://www.bibliotecapleyades.net/salud/salud_defeatcancer164.htm
[2] http://www.unitedpatientsgroup.com/blog/2014/03/20/dads-update-stan-rutner-a-cancer-survivor/
[3] http://ireadculture.com/article-2940-convinced-of-the-cure.html
http://cannajournal.com/2014/01/31/cannabis-cures-cancer-all-the-proof-you-need-3/
http://la.indymedia.org/news/2014/07/264847.php

If you like to obtain teh same oil as Rick simpson makes, write me an email.

Kanker is geen ziekte, maar… een vitamine B17-deficiëntie

We zijn voortdurend bang voor het woord kanker. We zijn omringd met mensen die er last van hebben, en het lijkt erop dat niemand deze dodelijke ziekte kan verslaan. Echter het is niet een echte ziekte. Kanker is een business, en wij betalen de prijs.
Miljoenen mensen lijden aan kanker, ongeacht hun leeftijd en geslacht. Echter, de meeste van hen zijn nog steeds niet bewust van het feit dat ze worden gemanipuleerd. Kanker is slechts een vitamine B17-deficiëntie (Amygdalin).
April 30, 2017

Er is een boek met de titel “World Without Cancer”, wat niet vertaald mocht worden in verschillende andere talen.

Echter, ons doel vandaag is om je ervan te overtuigen dat er geen kanker is, want het is slechts een vitamine B17-deficiëntie (Amygdalin). Daarom geen chirurgie, bestraling, chemotherapie en andere conventionele behandelingen.
In het verleden stierven zeelieden aan scheurbuik, en zo veel mogelijk mensen profiteerde daarvan, en later werd ontdekt dat het niet een echte ziekte was, maar slechts een tekort aan vitamine C.

Wij geloven dat dit hetzelfde is! De kanker-industrie is een enorme business, en miljarden euro’s worden jaarlijks verdiend. Het bloeide na de Tweede Wereldoorlog.
Echter, wanneer we rekening houden met het feit dat de genezing al lang geleden is gevonden, is de conclusie meer dan duidelijk: kanker vult de zakken van de kolonisatoren.
Toch kan kanker worden voorkomen door het volgen van deze eenvoudige tips:
Raak niet in paniek, maar onderzoek je conditie, en probeer uit te vinden wat voor type kanker je hebt.

Omdat kanker slechts een gebrek is aan vitamine B17 in het lichaam, zou je 15 tot 20 stukjes abrikoos steen / kern (fruit steen) per dag moeten eten om kanker te voorkomen.
Tarwe knoppen zijn een krachtige anti-kanker geneesmiddel, rijk aan vloeibare zuurstof en de meest krachtige anti-kanker verbinding-laetrile, een geëxtraheerde vorm van deze vitamine, word in de vrucht steen van appels gevonden.
Laetrile wordt geproduceerd in Mexico, en gesmokkeld naar de Verenigde Staten, omdat de geneesmiddel industrie bij wet verbiedt de productie van laetrile.

Dr. Harold W. Manner, de auteur van het boek “Death of Cancer”, beweert dat laetrile kanker behandelt in meer dan 90% van de gevallen.

Amygdalin (vitamine B17) is te vinden in:
De vrucht steen of graan (zaad) van fruit in de hoogste concentratie. Deze vruchten zijn: peren, appels, abrikozen, pruimen en perziken.
Zaden (granen): sesam en lijnzaad (zaad van linnen / vlaszaad).
Havermout , mortels van blok tarwe, gerst, bruine rijst, rogge, lijnzaad, gierst. Snijbonen, bonen, linzen spruit (linzen knop), granen, Lima (Lima bonen) en erwten.
Moerbei- zaden: Bittere amandelen (dat is de rijkste bron van vitamine B-17 van aard) en Indische amandel.
Moerbeien: bosbessen, zwarte moerbei, framboos en aardbei.
Zoete pompoen, biergist, en ruwe rijst (padie).
Dit zijn de rijkste bronnen van absorbeerbare B17:
Abrikozen (pitten / zaden)
bosbessen
vlierbessen
zaden van vruchten zoals kersen, appels, peren, perziken, pruimen
tuinbonen
amandelen
frambozen
Sorghum
Macadamia noten
aardbeien
bramen
tarwegras
Boekweit
limabonen
Gerst
cashewnoten
Taugé
Gierst

Bron: http://wtfbro.nl/een-geheim-is-blootgelegd-kanker-is-geen-ziekte-maar/

Italian Doctor: Cancer Is a Fungus That Can Be Treated With Sodium Bicarbonate

According to Italian doctor Tulio Simonchini, cancer is nothing but a fungus which can be eliminated with Sodium Bicarbonate (baking soda). Dr. Simonchini used this method to cure thousands of patients suffering from different types of cancer, and claims that it is 100% effective.

Sodium BicarbonateThe therapy isn’t harmful at all and let’s face it – you’ve got nothing to lose. The painful reality of more and more cancer cases is somehow connected to the failures of oncology. “We have to prove that modern oncology is unable to answer all the questions cancer patients have. It’s our moral and ethical commitment to find the real cure for the hardest and deadliest diseases of our time,” says Dr. Simonchini.

Cancer is a fungus!

“About a century ago, there was a great theory that cancer is caused by malfunctioning genes, which means that the disease is intracellular. However, in my opinion, cancer is a fungal infection and a special cellular phenomenon,” says Dr. Simonchini, who has sent shockwaves around the medical community with his claim.

CandidaIn the plant world, carcinoma is caused by fungal infections, and the same happens in humans. Fungi always carry a tumor with them – this has been proven in both in vivo and in vitro studies. However, scientists believe that they develop after the disease appeared. Dr. Simonchini believes that they were already there before – fungi create cancer, weaken our immune system and then attack the whole body.

Every type of cancer is caused by the Candida fungus, which has been confirmed by several studies, and its histological structure is a result of the defensive measures against the invasion. Over time, our tissues are weakened and tired, and they start producing unidentified cells.According to Dr. Simonchini, cancer is an “ulcer” where deformed cells accumulate and form colonies.

Baking soda

The usual antifungal drugs are ineffective against cancer as they only attack the surface of the cells. The main infection is more powerful than a single bacterium, which is why fungal infections last for so long.

“I have identified the things that can attack these colonies of fungi – for cancer, it’s Sodium Bicarbonatebaking soda, and a iodine tincture is the best substance for skin cancer,” claims Dr. Simonchini.

Many studies have confirmed baking soda’s intracellular action against cancer.

The treatment

“I have used the treatment on my patients for more than 20 years. Many of these patients have completely recovered from the disease. Even when doctors gave them no chances. The best way to eliminate a tumor is for it to come in contact with Sodium Bicarbonate, baking soda. Sodium Bicarbonate can be applied as an enema for digestive cancers, intravenous injection for brain and lung tumors and inhalation for tumors in the upper respiratory system. Breast, lymph system and subcutaneous tumors can be treated with a local perfusion. Internal organ tumors should be treated with baking soda by applying it directly into the arteries. It’s also important to treat every type of cancer with the proper dose,” Dr. Simonchini explains, and continues:

“For phleboclisis, you’ll need about 500 cm. of 5% or 8.4% solution; in some cases, the mixture only needs to be salty enough. During every treatment, it’s important to know that tumor colonies come back between the 3 and 4 day, and suffer a collapse between the 4 and 5 day, so a minimum of 6 days of treatment is required. The treatment should be repeated for 4 cycles, and has no other side-effects other than thirst and weakness.”

“For skin cancer, you should rub a 0.7% iodine tincture on the affected areas 20-30 times a day. Afterwards, the tumor will not return,” Dr. Simonchini says.

Here are the main symptoms of Candida infection: Chronic fatigue; Obsessive-compulsive disorder; Anxiety and irritability; Brain fog and nausea; Chronic skin disorders; Chronic digestive disorders; Mood changes; Starch and sugar cravings.

If you have notice at least 2 of these symptoms, you may have an advanced stage of candida infections which may result in cancer, so they should never be ignored.

Treatment and prevention of candida infections

The fungal development must be kept in check. Left untreated, candida can lead to candidiasis which can cause symptoms that mimic other diseases and result in perforation in the intestines and leaky gut syndrome. This will allow protein to attack your blood cells. In order to prevent further problems, we first need to eliminate the foods that feed candida – sugar and starch. This means no bread, candy, fresh fruit, pasta and rice for a while. Focus on eating raw fruit and steamed vegetables, and some people have had great results with grapefruit seeds. Dr. Simonchini recommends using aluminum-free baking soda for the treatment of cancer. It can be found in almost all health stores and pharmacies.

How the therapy works

Sodium Bicarbonate,Baking soda, significantly increases the alkalinity of your blood which destroys the fungi. Due to this, baking soda quickly disintegrates the tumor, leaving it without defense.

For stomach, colon, rectal and oral cancer, you need to take 1 teaspoon of Sodium Bicarbonate, baking soda, in a glass of water every morning and evening for a month. In most cases, this is enough time to eliminate the tumor. The therapy should last 3-4 weeks and not a day more. Dr. Simonchini’s therapy also usually requires intravenous injections as well. For best results, you’ll need 500 ml. of 5% baking soda solution applied in the vein directly every day. Do this for 24 days, then go for a scan. Vaginal fungal infections have become pretty common nowadays and according to Dr. Simonchini, they are the main culprit for cervical cancer and vaginal tumors. In order to treat these problems, you need to wash your vagina with a mixture made of 2 l. of filtered water and 2 tablespoons of baking soda. This will defeat the fungi that are causing the problem and prevent them from coming back in the future.

 

Source: An Italian Doctor Shocked the World: Cancer Is a Fungus That Can Be Treated With Baking Soda!(Video) – Collective Healthy

You’ll Freeze Lemons For The Rest Of Your Life!

So why should we freeze lemons?

hanging lemonA new study has shown for the first time how limonoids, natural compounds present in lemons and other citrus fruit, impede both ER+ and ER- breast cancer cell growth. This sheds new light on the importance of citrus fruit for breast cancer prevention and supports past studies which showed fruit consumption may lower breast cancer risk.(1)

All kinds of people are saying that the entire lemon should be used with nothing wasted. Not only for the obvious health benefits but also for the amazing taste! How? Simple, take an ORGANIC lemon, wash it and then put it in the freezer. Once it is frozen you get whatever is necessary to grate or shred the whole lemon without even peeling it first. Then sprinkle it on your salad, ice cream, soup, cereals, noodles, spaghetti sauce, or whatever. No holds barred. What you will experience is that whatever you sprinkle it on will take on a taste you may never have experienced before.

Why would I do this? Because the lemon peel contains 5 to 10 times more vitamins than the lemon juice itself and the peel is the part that is usually wasted. Not only that, but the peel helps to get rid of toxins in the body. But wait, there’s more. Lemon is effective in killing cancer cells because it is allegedly 10,000 stronger than chemotherapy. This has not been revealed because there are people out there that want to make a synthetic, toxic version that will bring them huge profits. Shades of Monsanto. The good news is that the taste of lemon is pleasant and does not deliver the horrific effects of chemotherapy. What’s bizarre is that people are closely guarding this fact so as to not jeopardize the income to those that profit from other’s illnesses. Another interesting aspect of the lemon is that it has a remarkable effect on cysts and tumors.

Always Freeze Your Lemons

Some say the lemon is a proven remedy against all types of cancer. It doesn’t end there. It has an anti-microbial effect against bacterial infections and fungi; it is effective against internal parasites and worms; it regulates blood pressure, which is too high; it acts as an anti-depressant; it combats stress and nervous disorders. The source of this information, although not specifically named, is one of the largest drug manufacturers in the world. They further say that after more than 20 laboratory tests since 1970, the extracts revealed that it destroys the malignant cells in 12 cancers, including colon, breast, prostate, lung and pancreas and that the compounds of the lemon tree were 10,000 times more effective than the product Adriamycin, which is a drug normally used chemotherapeutically in the world to slow the growth of cancer cells.

Even more, this type of therapy with lemon extract only destroys malignant cancer cells and does not affect healthy cells. The process is simple: buy an ORGANIC lemon, wash it, freeze it, grate it, and put it on everything you eat. It’s not rocket science. Nature has put stuff on the planet to keep the body healthy. The corporations hide this information and create synthetics to treat disease.

The synthetic chemical creates other symptoms from its ingestion requiring another drug to combat these symptoms. And so the cycle continues, which equates to enormous profits coming from an overt intention to keep a body ill and suppressing natural healing foods, minerals and modalities, all withheld by the mainstream media to not jeopardize their advertising dollar income, and payoffs to the politicians to not pass laws that will greatly benefit the people. If we do not take responsibility for ourselves and go against the mainstream grain, we will inevitably remain a “trick” our whole life.

Aloha!…

 

P.S. There are doctors who published studies and experiments in the 1940’s using liquid Vitamin C for the treatment of Cancer, and found that 40-60,000 units of liquid Vitamin C administered intraveinously not only cured the big “C”, but also left behind none of the side effects that chemotherapy does. The “frozen lemon” idea works on this same principal. You’re not likely to find too many Oncologists who are practicing the Vitamin C therapy due to two things: 1- the lack of knowledge of the natural world and how it pertains to our health, and, 2- there’s not a lot of income in the use of Lemons or Vitamin C in the treatment of Cancer. Look up Linus Pauling or Gerson Therapy (Dr. Max Gerson and the Gerson Institute, San Diego, CA. His daughter Charlotte has carried on her father’s legacy), and I’m sure you will eventually find what you are looking for. They did a lot of research with vitamin C during their lives.

 

credits: Real Farmacy, Natural News, Healthy Holistic Living

Shocking connection: 97% of all terminal cancer patients previously had a root canal procedure…

 Do you have a chronic degenerative disease? If so, have you been told, “It’s all in your head?” Well, that might not be that far from the truth… the root cause of your illness may be in your mouth.

There is a common dental procedure that nearly every dentist will tell you is completely safe, despite the fact that scientists have been warning of its dangers for more than 100 years. Every day in the United States alone, 41,000 of these dental procedures are performed on patients who believe they are safely and permanently fixing their problem.

What is this dental procedure?

The root canal. More than 25 million root canals are performed every year in this country.


Root-canaled teeth are essentially “dead” teeth that can become silent incubators for highly toxic anaerobic bacteria that can, under certain conditions, make their way into your bloodstream to cause a number of serious medical conditions—many not appearing until decades later.

Most of these toxic teeth feel and look fine for many years, which make their role in systemic disease even harder to trace back.

Sadly, the vast majority of dentists are oblivious to the serious potential health risks they are exposing their patients to, risks that persist for the rest of their patients’ lives. The American Dental Association claims root canals have been proven safe, but they have NO published data or actual research to substantiate this claim.

Fortunately, I had some early mentors like Dr. Tom Stone and Dr. Douglas Cook, who educated me on this issue nearly 20 years ago. Were it not for a brilliant pioneering dentist who, more than a century ago, made the connection between root-canaled teeth and disease, this underlying cause of disease may have remained hidden to this day. The dentist’s name was Weston Price — regarded by many as the greatest dentist of all time.

Weston A. Price: World’s Greatest Dentist

Most dentists would be doing an enormous service to public health if they familiarized themselves with the work of Dr. Weston Pricei. Unfortunately, his work continues to be discounted and suppressed by medical and dental professionals alike.

Dr. Price was a dentist and researcher who traveled the world to study the teeth, bones, and diets of native populations living without the “benefit” of modern food. Around the year 1900, Price had been treating persistent root canal infections and became suspicious that root-canaled teeth always remained infected, in spite of treatments. Then one day, he recommended to a woman, wheelchair bound for six years, to have her root canal tooth extracted, even though it appeared to be fine.

She agreed, so he extracted her tooth and then implanted it under the skin of a rabbit. The rabbit amazingly developed the same crippling arthritis as the woman and died from the infection 10 days later. But the woman, now free of the toxic tooth, immediately recovered from her arthritis and could now walk without even the assistance of a cane.

Price discovered that it’s mechanically impossible to sterilize a root-canaled (e.g. root-filled) tooth.

He then went on to show that many chronic degenerative diseases originate from root-filled teeth—the most frequent being heart and circulatory diseases. He actually found 16 different causative bacterial agents for these conditions. But there were also strong correlations between root-filled teeth and diseases of the joints, brain and nervous system.

Dr. Price went on to write two groundbreaking books in 1922 detailing his research into the link between dental pathology and chronic illness. Unfortunately, his work was deliberately buried for 70 years, until finally one endodontist named George Meinig recognized the importance of Price’s work and sought to expose the truth.

Must-read book on the subject: The Secret Poison in Your Mouth: Banish the Hidden Cause of Cancer, Heart Disease and Arthritis;

Dr. Meinig advances the work of Dr. Price

Dr. Meinig, a native of Chicago, was a captain in the U.S. Army during World War II before moving to Hollywood to become a dentist for the stars. He eventually became one of the founding members of the American Association of Endodontists (root canal specialists).

In the 1990s, he spent 18 months immersed in Dr. Price’s research. In June of 1993, Dr. Meinig published the book Root Canal Cover-Up, which continues to be the most comprehensive reference on this topic today. You can order your copy directly from the Price-Pottenger Foundation. ii

What dentists don’t know about the anatomy of your teeth

Your teeth are made of the hardest substances in your body.

In the middle of each tooth is the pulp chamber, a soft living inner structure that houses blood vessels and nerves. Surrounding the pulp chamber is the dentin, which is made of living cells that secrete a hard mineral substance. The outermost and hardest layer of your tooth is the white enamel, which encases the dentin.

The roots of each tooth descend into your jawbone and are held in place by the periodontal ligament. In dental school, dentists are taught that each tooth has one to four major canals. However, there are accessory canals that are never mentioned. Literally miles of them!

Just as your body has large blood vessels that branch down into very small capillaries, each of your teeth has a maze of very tiny tubules that, if stretched out, would extend for three miles. Weston Price identified as many as 75 separate accessory canals in a single central incisor (front tooth). For a more detailed explanation, refer to an article by Hal Huggins, DDS, MS, on the Weston A. Price Foundation website.iii (These images are borrowed from the Huggins article.)

Microscopic organisms regularly move in and around these tubules, like gophers in underground tunnels.

When a dentist performs a root canal, he or she hollows out the tooth, then fills the hollow chamber with a substance (called guttapercha), which cuts off the tooth from its blood supply, so fluid can no longer circulate through the tooth. But the maze of tiny tubules remains. And bacteria, cut off from their food supply, hide out in these tunnels where they are remarkably safe from antibiotics and your own body’s immune defenses.

The root cause of much disease

Under the stresses of oxygen and nutrient deprivation, these formerly friendly organisms morph into stronger, more virulent anaerobes that produce a variety of potent toxins. What were once ordinary, friendly oral bacteria mutate into highly toxic pathogens lurking in the tubules of the dead tooth, just awaiting an opportunity to spread.

No amount of sterilization has been found effective in reaching these tubules — and just about every single root-canaled tooth has been found colonized by these bacteria, especially around the apex and in the periodontal ligament. Oftentimes, the infection extends down into the jawbone where it creates cavitations — areas of necrotic tissue in the jawbone itself.

Cavitations are areas of unhealed bone, often accompanied by pockets of infected tissue and gangrene. Sometimes they form after a tooth extraction (such as a wisdom tooth extraction), but they can also follow a root canal. According to Weston Price Foundation, in the records of 5,000 surgical cavitation cleanings, only two were found healed.

And all of this occurs with few, if any, accompanying symptoms. So you may have an abscessed dead tooth and not know it. This focal infection in the immediate area of the root-canaled tooth is bad enough, but the damage doesn’t stop there.

Root canals can lead to heart, kidney, bone, and brain disease

As long as your immune system remains strong, any bacteria that stray away from the infected tooth are captured and destroyed. But once your immune system is weakened by something like an accident or illness or other trauma, your immune system may be unable to keep the infection in check.

These bacteria can migrate out into surrounding tissues by hitching a ride into your blood stream, where they are transported to new locations to set up camp. The new location can be any organ or gland or tissue.

Dr. Price was able to transfer diseases harbored by humans to rabbits, by implanting fragments of root-canaled teeth, as mentioned above. He found that root canal fragments from a person who had suffered a heart attack, when implanted into a rabbit, would cause a heart attack in the rabbit within a few weeks.

He discovered he could transfer heart disease to the rabbit 100 percent of the time! Other diseases were more than 80 percent transferable by this method. Nearly every chronic degenerative disease has been linked with root canals, including:

– Heart disease
– Kidney disease
– Arthritis, joint, and rheumatic diseases
– Neurological diseases (including ALS and MS)
– Autoimmune diseases (Lupus and more)

There may also be a cancer connection. Dr. Robert Jones, a researcher of therelationship between root canals and breast cancer, found an extremely high correlation between root canals and breast cancer.iv He claims to have found the following correlations in a five-year study of 300 breast cancer cases:

-93 percent of women with breast cancer had root canals
-7 percent had other oral pathology
-Tumors, in the majority of cases, occurred on the same side of the body as the root canal(s) or other oral pathology

Dr. Jones claims that toxins from the bacteria in an infected tooth or jawbone are able to inhibit the proteins that suppress tumor development. A German physician reported similar findings. Dr. Josef Issels reported that, in his 40 years of treating “terminal” cancer patients, 97 percent of his cancer patients had root canals. If these physicians are correct, the cure for cancer may be as simple as having a tooth pulled, then rebuilding your immune system.

Good bugs gone bad

How are these mutant oral bacteria connected with heart disease or arthritis? The ADA and the AAE claim it’s a “myth” that the bacteria found in and around root-canaled teeth can cause diseasev. But they base that on the misguided assumption that the bacteria in these diseased teeth are the SAME as normal bacteria in your mouth — and that’s clearly not the case.

Today, bacteria can be identified using DNA analysis, whether they’re dead or alive, from their telltale DNA signatures.

In a continuation of Dr. Price’s work, the Toxic Element Research Foundation (TERF) used DNA analysis to examine root-canaled teeth, and they found bacterial contamination in 100 percent of the samples tested. They identified 42 different species of anaerobic bacteria in 43 root canal samples. In cavitations, 67 different bacteria were identified among the 85 samples tested, with individual samples housing between 19 to 53 types of bacteria each. The bacteria they found included the following types:

– Capnocytophagaochracea
– Fusobacteriumnucleatum
– Gemellamorbillorum
– Leptotrichiabuccalis
– Porphyromonasgingivalis

Are these just benign, ordinary mouth bugs? Absolutely not. Four can affect your heart, three can affect your nerves, two can affect your kidneys, two can affect your brain, and one can infect your sinus cavities… so they are anything BUT friendly! (If you want see just how unfriendly they can be, I invite you to investigate the footnotes.)

Approximately 400 percent more bacteria were found in the blood surrounding the root canal tooth than were found in the tooth itself, suggesting the tooth is the incubatorand the periodontal ligament is the food supply. The bone surrounding root-canaled teeth was found even HIGHER in bacterial count… not surprising, since bone is virtual buffet of bacterial nutrients.

Since when is leaving a dead body part IN your body a good idea?

There is no other medical procedure that involves allowing a dead body part to remain in your body. When your appendix dies, it’s removed. If you get frostbite or gangrene on a finger or toe, it is amputated. If a baby dies in utero, the body typically initiates a miscarriage.

Your immune system doesn’t care for dead substances, and just the presence of dead tissue can cause your system to launch an attack, which is another reason to avoid root canals — they leave behind a dead tooth.

Infection, plus the autoimmune rejection reaction, causes more bacteria to collect around the dead tissue. In the case of a root canal, bacteria are given the opportunity to flush into your blood stream every time you bite down.

Why dentists cling to the belief root canals are safe

The ADA rejects Dr. Price’s evidence, claiming root canals are safe, yet they offer no published data or actual research to substantiate their claim. American Heart Association recommends a dose of antibiotics before many routine dental procedures to prevent infective endocarditis (IE) if you have certain heart conditions that predispose you to this type of infection.

So, on the one hand, the ADA acknowledges oral bacteria can make their way from your mouth to your heart and cause a life-threatening infection.

But at the same time, the industry vehemently denies any possibility that these same bacteria—toxic strains KNOWN to be pathogenic to humans — can hide out in your dead root-canaled tooth to be released into your blood stream every time you chew, where they can damage your health in a multitude of ways.

Is this really that large of a leap? Could there be another reason so many dentists, as well as the ADA and the AAE, refuse to admit root canals are dangerous? Well, yes, as a matter of fact, there is. Root canals are the most profitable procedure in dentistry.

What you need to know to AVOID a root canal

I strongly recommend never getting a root canal. Risking your health to preserve a tooth simply doesn’t make sense. Unfortunately, there are many people who’ve already have one. If you have, you should seriously consider having the tooth removed, even if it looks and feels fine.

Remember, as soon as your immune system is compromised, your risk of of developing a serious medical problem increases — and assaults on your immune system are far too frequent in today’s world.

If you have a tooth removed, there are a few options available to you.

1) Partial denture: This is a removable denture, often just called a “partial.” It’s the simplest and least expensive option.
2) Bridge: This is a more permanent fixture resembling a real tooth but is a bit more involved and expensive to build.
3) Implant: This is a permanent artificial tooth, typically titanium, implanted in your gums and jaw. There are some problems with these due to reactions to the metals used. Zirconium is a newer implant material that shows promise for fewer complications.

But just pulling the tooth and inserting some sort of artificial replacement isn’t enough.

Dentists are taught to remove the tooth but leave your periodontal ligament. But as you now know, this ligament can serve as a breeding ground for deadly bacteria. Most experts who’ve studied this recommend removing the ligament, along with one millimeter of the bony socket, in order to drastically reduce your risk of developing an infection from the bacterially infected tissues left behind.

I strongly recommend consulting a biological dentist because they are uniquely trained to do these extractions properly and safely, as well as being adept at removing mercury fillings, if necessary. Their approach to dental care is far more holistic and considers the impact on your entire body — not JUST your mouth.

If you need to find a biological dentist in your area, I recommend visiting toxicteeth.org, a resource sponsored by Consumers for Dental Choice. This organization, championed by Charlie Brown, is a highly reputable organization that has fought to protect and educate consumers so that they can make better-informed decisions about their dental care. The organization also heads up the Campaign for Mercury-Free Dentistry.

Must-read book on the subject: The Secret Poison in Your Mouth: Banish the Hidden Cause of Cancer, Heart Disease and Arthritis;

By Dr. Joseph Mercola

From the author: The existing medical establishment is responsible for killing and permanently injuring millions of Americans, but the surging numbers of visitors to Mercola.com since I began the site in 1997 – we are now routinely among the top 10 health sites on the Internet – convinces me that you, too, are fed up with their deception. You want practical health solutions without the hype, and that’s what I offer.

References:
i Weston A. Price Foundation
ii Price-Pottenger Foundation
iii Weston A. Price Foundation June 25, 2010
iv Quantum Cancer Management
v American Association of Endodontists
vi Journal of Clinical Microbiology February 2007
vii Journal of Clinical Microbiology July 2003
viii Clinical Infectious Diseases June 1996
ix Science Daily January 4, 2011
x The Wealthy Dentist July 12, 2011
xi ToxicTeeth.org

Dandelion root makes cancer cells disintegrate within 48 hours

The Plant That Everyone Has, But Nobody Knows about; Dandelion root Makes Cancer Cells Disintegrate Within 48 Hours

Quick – what’s the best use for dandelions? If you’re like most people, you probably can’t think of an answer, or maybe think of dandelion wine – but we’re here to tell you it’s something much, much better!

DandelionIt turns out dandelions are actually really effective at fighting cancer. Pretty cool, right?

Researchers at the University of Windsor in Ontario have shown that dandelion root extract can cause human melanoma cells to essentially kill themselves without leading to any type of toxicity. In fact, their initial study saw cancer cells disintegrating within 48 hours, while healthy cells remained unaffected. The study was led by Professor Siyaram Pandey, PhD.

These promising results prompted the non-profit organization Mitacs to reach out to AOR Inc., a Calgary company that produces natural health products, in order to develop a dandelion tea powder that is significantly stronger than that found in health food stores. They accomplish this by milling dandelion root, creating an extract from it, and then freeze-drying it into a powder that patients dissolve into hot water and then drink.

AOR is producing 6,000 doses of this tea for a clinical trial that will take place at the Windsor Regional Cancer Centre, and will involve 30 patients with various types of cancer, including leukemia, who have not had success with conventional therapy.

Dandelion Root Project

A statement on the website of the Dandelion Root Project at the University of Windsor says:Since the commencement of this project, we have been able to successfully assess the effect of a simple water extract of dandelion root in various human cancer cell types, in the lab and we have observed its effectiveness against human T cell leukemia, chronic myelomonocytic leukemia, pancreatic and colon cancers, with no toxicity to non-cancer cells. Furthermore, these efficacy studies have been confirmed in animal models (mice) that have been transplanted with human colon cancer cells.

Researcher and medical oncologist Dr. Caroline Hamm said that some of her patients have noted improvements after drinking dandelion root tea from health food stores. The concentrated tea could prove to be even more effective, potentially saving countless lives.

A 2008 study published in the International Journal of Oncology showed that dandelion tea reduced breast and prostate cancer cells. A subsequent report in the same journal showed that a dietary supplement that contained dandelion suppressed the growth of prostate cancer cells. Dandelion extracts have also demonstrated their efficacy in treating breast cancer and leukemia in traditional Chinese medicine as well as in Native American medicine.

How Does it Work?

Get your Tachyonized Dandelion Tonic here

This extract causes cancer cells to go through apoptosis, a natural cell process where a cell activates an intracellular death program because it isn`t needed anymore. In brief, dandelion root extract causes the cancer cell to “commit suicide” without affecting the healthy ones.

Two cells perform apoptosis which is far better than chemotherapy drugs which kill one healthy cell for every 5 to 10 cancer cell, the dandelion extract.

It is important to mention that the concentration of this extract is much higher than the one which is currently available. Even though trials are still underway, this extract may be the future of cancer treatment!If you would like to learn more about this, check out the following video. Dr. Siyaram Pandey’s explains how dandelion root causes the death of cancer cells within 24 hours.

Get your Tachyonized Dandelion Tonic here

@2017 Native American’s.

Source: The Plant That Everyone Has, But Nobody Knows That it Makes Cancer Cells Disintegrate Within 48 Hours

Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models

Abstract

Intra-lesional chemotherapy for treatment of cutaneous malignancies has been used for many decades, allowing higher local drug concentrations and less toxicity than systemic agents. Here we describe a novel diterpene ester, EBC-46, and provide preclinical data supporting its use as an intra-lesional treatment. A single injection of EBC-46 caused rapid inflammation and influx of blood, followed by eschar formation and rapid tumor ablation in a range of syngeneic and xenograft models. EBC-46 induced oxidative burst from purified human polymorphonuclear cells, which was prevented by the Protein Kinase C inhibitor bisindolylmaleimide-1. EBC-46 activated a more specific subset of PKC isoforms (PKC-βI, -βII, -α and -γ) compared to the structurally related phorbol 12-myristate 13-acetate (PMA). Although EBC-46 showed threefold less potency for inhibiting cell growth than PMA in vitro, it was more effective for cure of tumors in vivo. No viable tumor cells were evident four hours after injection by ex vivo culture. Pharmacokinetic profiles from treated mice indicated that EBC-46 was retained preferentially within the tumor, and resulted in significantly greater local responses (erythema, oedema) following intra-lesional injection compared with injection into normal skin. The efficacy of EBC-46 was reduced by co-injection with bisindolylmaleimide-1. Loss of vascular integrity following treatment was demonstrated by an increased permeability of endothelial cell monolayers in vitro and by CD31 immunostaining of treated tumors in vivo. Our results demonstrate that a single intra-lesional injection of EBC-46 causes PKC-dependent hemorrhagic necrosis, rapid tumor cell death and ultimate cure of solid tumors in pre-clinical models of cancer.

Introduction

Surgical excision and ionizing radiation of affected sites have been the mainstay for treatment of cancer patients for decades. Whilst often effective, efficacy of these treatments can be limited by various factors including the condition of the patient, the proximity of adjacent vital tissues, inaccessibility of the tumor and intolerance of normal tissue for repeated courses of treatment. In some of these cases, intra-tumoral treatment may be more appropriate, particularly when surgical intervention is not possible. There have therefore been many attempts to deliver localized therapies, such as injection of anti-cancer agents [1][3] and lethal implants [4], aiming for palliation or even cure. Intra-tumoral treatment may have the advantage of allowing for much higher drug concentrations at the tumor site, and potentially less toxicity than systemic agents. However, a limiting factor for greater use of intra-tumoral treatments appears to be lack of suitable agents rather than delivery technologies.

The protein kinase C (PKC) family are ubiquitous serine-threonine kinases found in many cell types that translocate to membranes after activation and regulate diverse downstream processes, like proliferation, apoptosis, differentiation, and migration. There are ten main human isoforms that comprise three subgroups divided according to sequence homology and cofactor requirements. The classical PKC subgroup includes -α, -βI and-βII (alternatively spliced from the same gene), and -γ, which require binding of calcium and DAG to activate the enzyme. Members of the novel PKC subgroup include -δ, -ε, -η and -θ, which require DAG for activation but are calcium-independent. The atypical PKCs, -ι and -ζ, are independent of calcium and DAG, but have been shown to be activated by distinct lipids and protein-protein interactions [5], [6].

Inhibition of PKC signaling has been targeted as an anti-cancer treatment as PKC isozymes are known to play roles in cellular proliferation and vasculature formation [7][9], important for tumor growth. However, several clinical trials performed with compounds thought to inhibit PKC signaling have had disappointing results [9]. For example, enzastaurin, an orally available specific inhibitor of PKC-β, showed limited efficacy as a single agent in Phase II studies of advanced diffuse large B-cell lymphoma or non-small cell lung cancer [10], [11]. Clinical trials of enzastaurin with additional chemotherapy agents are underway.

In contrast, previous work has shown that intravenous administration of the prototypic PKC activating compound phorbol 12-myristate 13-acetate (PMA) to patients suffering from myleocytic malignancies resistant to chemotherapy resulted in remission [12], [13]. We have previously demonstrated that three topical applications of the PKC-activating ingenol ester PEP005 (ingenol mebutate or ingenol 3-angelate) are sufficient for enduring regression of skin cancer lesions (including melanoma) in pre-clinical models [14], [15]. PEP005 has recently been approved for topical treatment of actinic keratoses [16], [17], and shows efficacy in additional models of squamous cell carcinoma [18], [19]. We now describe a novel PKC-activating compound, EBC-46, and demonstrate that a single intra-lesional injection is sufficient for enduring regression and ultimate cure of diverse tumor types in pre-clinical models of cancer.

Materials and Methods

Reagents

EBC-46 was purified from the kernels of the fruit from Fontainea picrosperma, was provided by QBiotics Ltd. (Yungaburra, Queensland) at greater than 97% purity. Phorbol 12-myristate 13-acetate (PMA), dihydroethidium and bisindolylmaleimide-1 (BIS-1) were purchased from Sigma (St. Louis, MO).

Cell lines

All cell lines used in this study were purchased from ATCC (SK-MEL-28, B16-F0, HeLa, FaDu, MCF-7, HT-29) except MC-38 [20], [21] and MM649 [22] cells, which were described previously. Cell lines were grown in complete media (RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 3 mM HEPES, 100 U/ml penicillin and 100 µg/ml streptomycin (CSL Ltd, Melbourne, Australia)). Cells were routinely checked by STR profiling, and for mycoplasma infection by Hoechst staining [23] and PCR, and were always negative.

Human ethics statement

This study was performed in strict accordance with the recommendations in the Australian National Statement on Ethical Conduct in Human Research (2007 – Updated December 2013), of the National Health and Medical Research Council of Australia. All protocols were reviewed and approved by the QIMR Berghofer Medical Research Institute Human Research Ethics Committee (QIMR-HREC), approval number P764. All participants provided written informed consent to donate their blood samples for research.

Measurement of reactive oxygen species production in PMN cells

PMN cells were isolated from peripheral blood from healthy volunteers. Aliquots of 2×106 PMN cells were stained incubated for 15 min at 37°C with 10 µg/ml dihydroethidium (DHE). 2×106 unstained PMN cells were incubated at 37°C to act as an unstained control. Where appropriate, PMN cells were also incubated with 1 µM bisindolylmaleimide-1. The stained PMN cells were treated with the either PMA or EBC-46 for 15 min, and the change in fluorescence resulting from oxidation of DHE to ethidium bromide measured on a dual beam FACSCalibur (BD Biosciences, Franklin Lakes, NJ) using CellQuest Pro (BD Biosciences) software.

PKC-EGFP translocation

pPKC-α, -βII, -γ, -θ and –ζ-EGFP were purchased from Clontech (Moutain View, CA). pPKC-βI, -δ, -ε, -η and -ι-EGFP were constructed in-house. PKC isoforms were cloned from Universal Human Tumor cDNA (Life Technologies, Carlsbad, CA) and ligated into pPKC-ζ-EGFP digested with XhoI/SacII. The identity and fidelity of all PKC isoforms was verified by Sanger sequencing. 96-well plates were seeded with 3×104 cells/well of SK-MEL-28 in complete media, and incubated at 37°C, 5% CO2, and 95% humidity. After 24 h, cells were transiently transfected with a 1∶3 ratio of pPKC-EGFP vector DNA: Lipofectamine 2000 (Life Technologies) (0.16 µg DNA: 0.48 µl Lipofectamine per well) in Opti-MEM media (Life Technologies). After 24 h incubation, cells were washed with phosphate-buffered saline (PBS) and treated with 100 ng/ml of either PMA or EBC-46 for 1 h. Cells were washed twice with ice-cold PBS, fixed with 2% formaldehyde/0.2% gluteraldehyde in PBS, washed twice again with ice-cold PBS, and overlaid with 100 µl of PBS. Fluorescent cells were examined with an AMG EvosFl (Life Technologies) inverted fluorescence microscope at 40×. Micrographs were taken such that at least 50 fluorescent cells/well were captured, and transmitted light images were overlaid with fluorescence images for control wells to calculate transfection efficiency. Cells were counted in each image using ImageJ (NIH) and classified according to the localization of fluorescence as cytoplasmic, plasma membrane, perinuclear membrane, or other membrane (mitochondria, endoplasmic reticulum, Golgi apparatus, or unknown). A total of three independent experiments were performed for each PKC isoform.

Immunofluorescence

HeLa cells were treated with either 175 nM (100 ng/ml) PMA or EBC-46, or vehicle alone for 1 h. PKC-α (Cat. No. 2056; Cell Signaling Technologies) was detected by immunofluorescence as described by the manufacturer. Images were acquired using a Leica TCS Inverted Fluoresence Microscope with a Nikon DS-Fi1C camera.

PKC kinase assay

HeLa cells were treated with 175 nM (100 ng/ml) or 17.5 µM (10 µg/ml) of either PMA or EBC-46 for 1 h. Cells were lysed and stored at −80°C, before the level of PKC-specific kinase activity was measured in 30 µg HeLa lysate using the PKC Kinase Activity Assay Kit (AbCam, Cambridge, U.K.; Cat. No. ab139437) as described by the manufacturer. Assays were performed in triplicate with the mean ± SD shown.

Cell growth assay

Cells were seeded at sub-confluence (5,000 cells/well) in 96-well microtitre plates. All drugs and inhibitors were diluted in complete media. Controls were treated with vehicle alone. Cells were treated with PMA or EBC-46 the day after seeding and cultured for 4 days. To measure inhibition of cell growth, attached cell lines were assayed using sulforhodamine B (SRB) [24], [25]. The experiments were repeated at least twice and the mean ± SD was determined in Prism 6 (GraphPad Software, San Diego, CA).

Animal ethics statement

This study was performed in strict accordance with the recommendations in the Australian Code for the Care and Use of Animals for Scientific Purposes 8th Edition (2013), of the National Health and Medical Research Council of Australia. All protocols were reviewed and approved by the QIMR Berghofer Medical Research Institute Animal Ethics Committee (QIMR-AEC), approval numbers A0106-042M and A0404-606M. All mice were housed in a specific pathogen free (SPF) facility, with 12 hours light/dark cycle and continual access to food and water. All mice were monitored daily and tumor volume measured at least twice weekly, recorded using digital calipers and expressed as mm3 according to the formula A×b×b×0.5 where A the length and b the measured breadth of the tumor. Mice were also assessed for clinical signs according to a QIMR-AEC approved clinical score sheet for distress during the period of the experiment to determine whether the treatments (i.e. tumor burden and effects of drugs) were causing distress to the mice to a degree and to where they should be euthanased (Table 1). Scores for each parameter were summed to give a possible total of 8. Less than 3 was considered a mild clinical score, between 3 and 6 was considered a moderate clinical score, and over 6 was considered a severe clinical score. The experiment was ceased when an unacceptable clinical score (>6) was reached, or the cumulative tumor burden of the mouse exceeded 1,000 mm3. Mice were humanly euthanized by asphyxiation at the end of the experiment.

Table 1

Clinical scoring for Drug Treated and Tumor Bearing Mice.

EBC-46 treatment of tumors in mice

SK-MEL-28, MM649, FaDu (2×106) or B16-F0 (1×105) cells were injected (two tumors per mouse) on the hindquarter of 5 week old immunocompromised BALB/c Foxn1nu mice or C57BL/6J mice. When the tumors reached approximately 50 mm3 (SK-MEL-28 and MM649) or 100 mm3 (FaDu and B16-F0), mice in the control group were treated with vehicle (20% propylene glycol in water, 50 µl), and the treatment group received 50 nmol (30 µg) EBC-46 in vehicle, via a single intra-tumoral injection. Mice were euthanized when the cumulative tumor burden per mouse exceeded 1,000 mm3 or at the end of the experiment.

Pharmacokinetic study of EBC-46 in tumor and non-tumor-bearing mice

Nine BALB/c Foxn1nu mice were injected with 2×106 MM649 melanoma cells, one tumor per mouse. Tumors were monitored until they reached approximately 100 mm3. Mice were then treated by injecting 50 nmol (30 µg) EBC-46 either into the tumor (tumor bearing mice) or into normal skin (sub-cutaneously, 9 tumor-free mice). Blood (maximum of 150 µl) was collected from the tail vein by nicking at the base of the tail at 30 min, 1, 2, 4, 8 and 24 h post-treatment (3 animals at 30 min and 4 h, 3 animals at 1 and 8 h, 3 animals at 4 and 24 h) into a lithium heparin Microvette CB300 blood collection system (Sarstedt, Numbrecht, Germany), and processed to plasma by centrifugation at 2,000 g for 5 min at 20°C until separation occurred. Plasma was frozen at −80°C until analysed. Samples were analyzed using a specifically developed HPLC method to detect EBC-46 in mouse serum against a spiked standard curve. Erythema and oedema were rated using a five point scale (0 to 4; none to severe) 24 h after injection. Weight of animals was determined immediately prior to, and 24 h following treatment.

Ex vivo analysis of tumor cell survival

SK-MEL-28 or FaDu cells were injected (two tumors per mouse) on the hindquarter of 5 week old immunocompromised BALB/c Foxn1nu mice. When the tumors reached approximately 100 mm3, mice in the control group were treated with 20% propylene glycol in water, and the treatment group received 50 nmol (30 µg) EBC-46 via a single intra-tumoral injection. Mice were euthanized at time of injection, 1, 2, 4, 8 and 24 h post-treatment with vehicle or EBC-46, and tumors were harvested. Tumors were dissected, briefly dissociated with collagenase A, and finally resuspended in culture medium. Serial 3-fold dilutions of the cell suspension were cultured in vitro for 6 days, and the SRB assay used to compare the growth of viable EBC-46-treated tumor cells with that of vehicle treated controls.

EBC-46 treatment in neutrophil-depleted mice

SK-MEL-28 cells (2×106) were injected (two tumor sites per mouse) into the flanks of thirty 5- to 6-week old male BALB/c Foxn1nu mice (n = 5 mice and n = 10 tumors/group). When tumors had reached >50 mm3, ten mice were given i.p. injections of rat anti-neutrophil targeting antibody mLy-6G (100 µg in PBS; clone 1A8, Cat. # BE0075-1) or rat IgG2a isotype control (clone 2A3, Cat. # BE0089) from BioXCell (West Lebanon, NH). The antibodies were injected on days −2, 0, and 2, relative to initiation of vehicle or EBC-46 (25 nmol, 15 µg) treatment on day 0. An additional ten control mice received no antibody. The tumors on a total of 15 mice (5 each of no antibody, IgG2a isotype control or anti-mLy-6G antibody) were treated with 25 nmol EBC-46 per site (15 µg in 20% propylene glycol in water at 300 µg/ml, 50 µl injection), while the tumors on the remaining 15 mice were treated with 50 µl 20% propylene glycol in water only. Blood was taken from tail tips on Days −2, 0 and 2, and then twice weekly, smeared, and air dried on glass slides before being stained with Quick Dip (Fronine Laboratory Supplies, Rivertone, NSW, Australia). Tumor size was measured with calipers twice weekly. Mice were euthanized when the cumulative tumor burden per mouse exceeded 1,000 mm3 or at the end of the experiment.

In vitro permeability assay

HUVEC cells (Invitrogen/Life Technologies) were grown as described by the manufacturer and used at passage 4 to 6. Media and supplements (M200 [Cat. No. M200PRF500] and Low Serum Growth Supplement [Cat. No. S-003-10] respectively, Life Technologies) were prepared as directed. The In vitro Vascular Permeability Kit was from Millipore (Billerica, MA; Cat. No. ECM642). All assays were performed as described by the manufacturer. Assays were performed in at least triplicate wells.

Results

EBC-46 is a novel Protein Kinase C-activating compound

EBC-46. (12-Tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one; C30H42O10; 562.65 g/mol) is a novel compound purified from a commercially-sustainable natural source. It is structurally similar to the prototypic PKC-activating compound phorbol 12-myristate 13-acetate (PMA), but considerably less hydrophobic due to short ester side-chains and hydroxylation in the B ring (Figure 1A). To investigate if EBC-46 activated PKC, we initially examined the production of reactive oxygen species following treatment of PMN cells. The induction of oxidative burst in human PMN by PKC activators has been previously described [26], [27]. Treatment of PMN cells with 175 nM (100 ng/ml) PMA lead to an increase in fluorescent signal that corresponded with the oxidation of dihydroethidium bromide to fluorescent ethidium bromide. Pre-treatment with 1 µM bisindolylmaleimide-l (pan-PKC inhibitor) prevented this increase in fluorescent signal (Figure 1B, right) indicating PKC-activation dependence. Similarly, treatment of PMN cells with 175 nM (100 ng/ml) EBC-46 also led to an increase in fluorescent signal by oxidation, that was prevented by pre-treatment with bisindolylmaleimide-l (Figure 1B, left).

Figure 1

EBC-46 is a novel Protein Kinase C-activating compound.

Transient transfection of HeLa cells with PKC isoforms tagged to EGFP and subsequent treatment with PMA resulted in efficient translocation to the plasma membrane of –α, -βI, βII, -γ, -δ, -θ, and –η subtypes (Figure 1C). Modest translocation of PKC-ε was observed following 1 h treatment, consistent with previous results from others [28]. As expected, no translocation was seen for the atypical PKC-ι or -ζ isoforms lacking a C1 DAG-binding domain after treatment with PMA. Treatment with EBC-46 resulted in high percentage of cells showing translocation of PKC-βI and –βII isoforms, with less translocation seen for the –α, –γ, –δ, and -ε isoforms. No translocation of the atypical PKC-ι or -ζ isoforms was observed following EBC-46 treatment. Interestingly, EBC-46 treatment did not result in significant translocation of the –θ and –η isoforms to the plasma membrane suggesting that PMA and EBC-46 differ in their selectivity and/or potency towards specific PKC isoforms. These results were confirmed in SK-MEL-28 cells (Figure S1). However, transient transfection of PKC-ε or -ι isoforms was toxic to SK-MEL-28 cells, while transfection of PKC-δ localized to the mitochondrial membrane and treatment with PMA or EBC-46 had no effect (Figure S1). EBC-46 treatment resulted in the translocation of endogenous PKC-α to the plasma membrane in HeLa cells, similar to that with PMA (Figure 1D). Further, an in vitro kinase assay demonstrated an increase of PKC kinase activity in HeLa cells treated with EBC-46, particularly at 17.5 µM (10 µg/ml) (Figure 1E). Taken together, these results suggest that EBC-46 is a novel activator of PKC, in particular of the –β isoforms.

EBC-46 is efficacious in vivo, independent of tumor cell sensitivity in vitro

Previous studies have shown activators of PKC to induce cell death in cancer cells in vitro and in vivo [14]. We therefore tested clonogenic-type cell survival of EBC-46 in comparison to PMA. Treatment with PMA for 4 days led to almost complete cell death at 52.5 µM for both B16-F0 and SK-MEL-28 cells, with the 50% lethal dose (LD50) of approximately 17.5 µM for each cell line (17.63±0.13 and 17.81±0.15 µM respectively). In comparison, EBC-46 was less potent for cell killing than PMA in vitro (Figure 2A). Treatment with EBC-46 for the same period led to complete cell killing at 175 µM, with an LD50 of approximately 52.5 µM for each cell line (B16-F0, 52.25±0.11; SK-MEL-28, 52.60±0.18 µM). Similar results were obtained for a panel of cell lines, including HeLa, FaDu, HT-29, MCF-7, MM649 and MC-38 (Figure S2); in each case EBC-46 was less potent for cell killing than PMA in vitro. However, these results show that EBC-46 has a direct effect on cell survival in vitro.

Figure 2

EBC-46 efficacy in vivo is independent of tumor cell sensitivity in vitro.

We tested the in vivo efficacy of both PMA and EBC-46 in the B16-F0/C57BL/6J mouse melanoma model via intra-tumoral injection. Each mouse was injected with 500,000 B16-F0 cells per flank, and the tumors allowed to reach 100 mm3. Intra-tumoral injection of vehicle alone (20% propylene glycol in water) into established tumors had no effect, with all mice reaching the maximal tumor burden 4 days after treatment (Figure 2B). Intra-lesional injection of 50 nmol (30 µg) PMA in vehicle delayed tumor growth compared to vehicle alone, with all mice reaching maximal tumor burden 20 days post-treatment. Importantly, all treated sites relapsed. In contrast, intra-lesional treatment with 50 nmol (30 µg) EBC-46 led to an initial swelling of the tumor site followed by rapid ablation of the tumors. Treatment with a single bolus injection of 50 nmol (30 µg) EBC-46 showed a significant (p = 0.0004, Log-rank (Mantel-Cox) test) extension of time to euthanasia due to tumor burden compared to treatment with PMA, indicating anti-tumor efficacy. There was more than 70% total cure in the EBC-46 treatment group that was censored in the data due to single sites recurring of the two treated per mouse (15 of 20; 75% cured).

We further evaluated the anti-tumor efficacy of EBC-46 in additional mouse models of cancer. Intra-lesional treatment of BALB/c Foxn1nu mice, xenografted with SK-MEL-28 or MM649 human melanoma tumors (>50 mm3), with 50 nmol (30 µg) EBC-46 again led to an initial swelling of the tumor treatment site followed by ablation of the tumors compared to treatment with vehicle alone (20% propylene glycol in water). There were three recurrences after EBC-46 treatment of SK-MEL-28 tumors within the 40 day observation period (2>100 mm3, 1<100 mm3), and a single recurrence of a MM649 tumor within the 150 day observation period (>100 mm3) (Figure 2C and D respectively). Differences in tumor recurrence was highly significant for both SK-MEL-28 and MM649 (p<0.0001, Log-rank (Mantel-Cox) Test). In the MM649 tumor model, all other sites treated with EBC-46 in this study remained tumor free with no recurrence for 12 months (data not shown). Additional models showed efficacy against head and neck cancer (FaDu, Figure S3 and S4A) and colon cancer (HT-29, Figure S4B; MC-38, Figure S4C). These results suggest that a single bolus intra-lesional treatment with EBC-46 can lead to an enduring ablation of tumor cell growth in vivo.

Pharmacokinetic studies suggest EBC-46 remains at the tumor injection site

We wished to determine the pharmacokinetic profile of EBC-46 following a single intra-tumoral or sub-cutaneous injection into mice either bearing or not bearing tumors, respectively. Intra-tumoral treatment with EBC-46 of BALB/c Foxn1nu mice xenografted with MM649 melanoma cells led to significant erythema and oedema of the injection sites, as assessed 24 hours following injection. Sub-cutaneous treatment of normal skin to mimic intra-tumoral treatment (non-tumor bearing mice) led to significantly less erythema (p = 0.0021, t-test; Figure 3A) and oedema (p = 0.0007, t-test; Figure 3B), as assessed 24 hours after treatment. Mice without tumors that were treated sub-cutaneously with 50 nmol (30 µg) EBC-46 lost significantly more weight in 24 h (4.73±0.91% loss) than those mice treated by intra-tumoral injection with EBC-46 (0.70±0.99% loss; p = 0.0087, t-test; Figure 3C). Additionally, blood was collected following administration of EBC-46 to mice, and a quantitative HPLC assay used to determine the level of EBC-46 in the plasma. Results show that less EBC-46 was detected in serum from tumor-bearing mice treated by intra-lesional injection compared to non-tumor bearing mice treated by sub-cutaneous injection (Figure 3D), suggesting that the compound is retained at the tumor site.

Figure 3

EBC-46 treatment induces greater effects when injected into tumors compared to normal skin.

Neutrophils have a minor role in EBC-46 anti-cancer activity

Previous studies have outlined the role of neutrophils in anti-tumor efficacy of PKC agonists [15], [29]. The importance of neutrophils for the anti-cancer activity of EBC-46 was investigated by using an antibody (anti-mLy-6G, clone 1A8) to deplete these cells in mice bearing SK-MEL-28 tumors prior to treatment. SK-MEL-28 tumors were allowed to grow to approximately 100 mm3 prior to injection of the antibodies on days −2, 0 and 2 relative to treatment with EBC-46 or vehicle alone. The percentage of neutrophils in WBC counts in peripheral blood were determined for each of the groups. Following treatment with anti-Ly-6G antibody, the percentage of neutrophils in the peripheral blood fell from 62% of total leukocytes to 6% after a single treatment (Day 0), while the isotype control antibody had no effect (data not shown). Tumors in mice receiving the anti-Ly-6G neutrophil antibody grew slightly more rapidly compared to SK-MEL-28 tumors receiving either no-antibody or isotype control (IgG2a) antibody following a single treatment with bolus vehicle (Figure 4A), consistent with previous observations [15]. Ablation of the SK-MEL-28 tumors following treatment with 25 nmol (15 µg) EBC-46 (a lower dose of EBC-46 was used to reduce efficacy) was apparent in all groups (Figure 4B). There were no tumor recurrences in the group treated with EBC-46 alone in the 48 day observation period. However, we observed 4 recurrences in the mice receiving the anti-Ly-6G antibody compared to a single recurrence in the mice treated with the isotype control (IgG2a) antibody (Figure 4B). Although these data were not statistically significant (p = 0.30, Fisher’s exact test), these results may suggest that neutrophils play only a minor role in the anti-tumor efficacy of EBC-46 in this model.

Figure 4

EBC-46 anti-cancer efficacy is PKC-dependent.

EBC-46 anti-cancer efficacy is PKC-dependent

We wished to assess if PKC activation by EBC-46 was necessary for the anti-cancer efficacy of the compound. We therefore tested the in vivo efficacy of EBC-46 in BALB/c Foxn1nu mice xenografted with B16-F0 mouse melanoma cells via intra-tumoral injection in the presence or absence of bisindolylmaleimide-1, a PKC inhibitor. Each mouse was injected with 500,000 B16-F0 cells per injection site, and the tumors allowed to reach >50 mm3. Intra-tumoral injection of vehicle alone (20% propylene glycol in water) into established tumors had no effect, with all tumors continuing to increase in size over the following 8 days (Figure 4C). Intra-lesional injection of 16.7 nmol (10 µg) EBC-46 (lower dose to reduce efficacy) in vehicle led to a rapid ablation of the tumors (p<0.0001, t-test, compared to vehicle treated sites; Figure 4C), with only 25% of treated sites showing tumor presence 8 days after treatment (9 of 12 sites with no tumor, Figure 4D). In contrast, intra-lesional treatment with 16.7 nmol (10 µg) EBC-46 after pre-treatment with 5 µM bisindolylmaleimide-1 for 1 h, resulted in the majority of tumors continuing to grow (p = 0.0024, t-test compared to EBC-46 alone treated sites; Figure 4C), with 83.3% of treated sites showing the presence of tumors (2 of 12 sites with no tumor, Figure 4D). Co-injection of EBC-46 with a PKC inhibitor significantly reduced the efficacy of treatment, indicating that the anti-cancer efficacy of EBC-46 is PKC-dependent.

Treatment with EBC-46 results in rapid loss of tumor cell viability in vivo

Treatment with EBC-46 led to a dramatic ablation of tumors following intra-lesional injection. We therefore wished to examine the timing of loss of viability of tumor cells following treatment with EBC-46 in an ex vivo assay. Two tumor models were used, namely SK-MEL-28 and FaDu human tumor cells, in BALB/c Foxn1nu mice. Tumors were allowed to reach approximately 100 mm3 before treatment with 50 nmol (30 µg) EBC-46 or vehicle (50 µl 20% propylene glycol in water), and then removed, dissociated into single cell suspension and assayed for clonogenic growth in vitro. The results show that the tumor cells had greatly reduced viability 4 hours after treatment with EBC-46. The results were similar for both SK-MEL-28 (Figure 5A) and FaDu (Figure 5B) tumors. In contrast, tumors treated with vehicle alone remained fully viable. These results suggest that tumor cell viability is compromised following treatment with 50 nmol (30 µg) EBC-46 after as little as 2 to 4 h.

Figure 5

EBC-46 kills tumor cells rapidly in vivo.

As indicated above, intra-lesional treatment with EBC-46 led to a rapid swelling of the tumor site. We therefore examined FaDu human head and neck cancer tumors xenografted into BALB/c Foxn1nu mice after treatment with EBC-46 or vehicle alone by histology. Intra-lesional injection of EBC-46 into FaDu tumors also resulted in red cell extravasation within the tumor 1 to 2 h following treatment (Figure 5D). We also observed that tumor nuclei became pale and shrunken 2 to 4 h after treatment, and the tumor cells disorganized 4 to 8 h following EBC-46 treatment (Figure 5D), mirroring the loss of viability seen in the ex vivo measurement of efficacy (Figure 5A and B). Similar to other PKC agonists, treatment of normal skin with EBC-46 led to an apparent dilation of smaller blood vessels, and some red cell extravasation in the dermis and subcutis of the skin up to 8 h following treatment (Figure S5B). However, there was no evidence of remaining red cell extravasation 24 h after treatment.

EBC-46 causes permeability of endothelial cells within tumors

It has been previously reported that activation of PKC, especially of PKC-β, can induce endothelial cell activation and permeability [30], [31]. Further, it was recently shown that PKC agonists can cause endothelial cell activation and permeability in vivo by disruption of structural integrity [29]. We therefore examined blood vessel integrity in FaDu tumors following EBC-46 treatment using detection of CD31 using immunohistochemistry. While treatment of FaDu tumors with vehicle alone led to no apparent disruption of vessel morphology, intra-lesional injection of EBC-46 resulted in the loss of structural integrity from as early as 30 min following treatment (Figure 6A). CD31 immunostaining showed vessels that were dilated and incomplete when compared to untreated tumors or tumors treated with vehicle alone (Figure 6A and B). Treatment of normal skin with EBC-46 led to less apparent damage, particularly of larger vessels, with the majority remaining structurally intact, although vessel swelling was evident (Figure S6A and B). We therefore examined the effect of EBC-46 on cultured monolayers of human umbilical vascular endothelial (HUVEC) cells. Monolayers of HUVEC cells were treated with 350 µM (200 µg/ml) EBC-46 (similar concentration to that present in the tumor; assuming 100 mm3 tumor volume and injection of 50 µl volume, a three-fold dilution of the initial 1,050 µM/600 µg/ml concentration) for 30 min, before being assessed for permeability using FITC-Dextran. The results show that short term, high dose treatment with EBC-46 leads to significantly increased permeability of the HUVEC cell monolayer (p = 0.0013, t-test; Figure 6C). Propidium iodide uptake experiments on HUVEC cells treated with EBC-46 showed that cells were compromised rapidly (5 min; data not shown). This uptake of propidium iodide by damaged HUVEC cells was PKC-dependent, as evidenced by inhibition with the pan-PKC inhibitor bisindolylmaleimide-1 (Figure 6D).

Figure 6

EBC-46 causes disruption and permeability of endothelial cells within tumor.

Discussion

Activation of specific PKC isoforms in vascular endothelial cells, particularly the PKC-β isoforms, has previously been shown to induce permeability [30], [31]. Here we show that intra-lesional treatment with EBC-46, a novel PKC-activating compound with apparent specificity for PKC-β isoforms, induces permeability of endothelial cell monolayers in vitro, as well as vascular swelling and apparent disruption of vessel morphology in vivo. Further, sub-cutaneous injection of EBC-46 into normal skin led to significant levels of the drug found in the peripheral circulation. In contrast, intra-lesional injection of EBC-46 resulted in greatly reduced levels detected in the peripheral blood of mice. Additionally, the erythema and oedema observed following EBC-46 administration was significantly higher in mice with tumors compared to mice with normal skin. These results suggest a specificity of vascular damage within tumor sites compared to normal skin leading to anti-cancer efficacy. This specificity may reflect the disorganization and inherent “leakiness” of the vasculature within a solid tumor [32]. We hypothesize that the damage to the tumor vasculature prevents EBC-46 from entering the circulation. There is also a direct effect on tumor cell survival, as no viable tumor cells were evident four hours after injection by ex vivo culture. This is supported by in vitro data which showed treatment with EBC-46 was capable of inhibiting cell survival.

Previous studies elegantly showed that another PKC-activating ingenol ester, Ing3A (ingenol 3-angelate, ingenol mebutate, also known as PEP005) was able to penetrate deeper into the dermis following topical application compared to PMA, as it is a substrate for MDR1/P-glycoprotein (P-gp)/ABCB1 [29]. The authors showed that penetration of PMA after topical application was restricted to the epidermis of the skin, thereby sparing the vasculature in the sub-epidermal compartments and resulting in a lack of anti-tumor activity. Further, Li and colleagues also demonstrated that Ing3A bound to and inhibited P-gp whereas PMA did not [29]. Our results presented here additionally show that direct intra-tumoral injection of PMA only led to a transient reduction of tumor growth followed by a rapid relapse. In contrast, intra-tumoral injection of EBC-46 had an enduring anti-tumor effect.

We were unable to definitively demonstrate that neutrophils contribute significantly to the anti-tumor efficacy of EBC-46 by intra-lesional injection. Our conclusion was that neutrophils play a minor role in overall efficacy of EBC-46 from the experiments presented in this study. This is in contrast with previous work with other PKC activators (Ing3A/PEP005), where neutrophils were required for anti-tumor efficacy [15], [29]. However, the method of delivery used in this study (intra-tumoral injection) is very different compared to that used in the previous studies with the PEP005 (topical application). Further, the anti-neutrophil antibody used here was from a different, more neutrophil-specific hybridoma clone [33] than that used in previous studies. Nevertheless, in the current study approximately 6% of the cells in the peripheral WBC counts were found to be neutrophils.

It is important to note that intra-lesional injection of the prototypic PKC-activator PMA did not lead to a cure of tumors in the current study, but rather an initial shrinking of the tumor followed by a rapid relapse. In contrast, treatment with EBC-46 led to a rapid ablation of the tumor. In greater than 70% of cases, the response and cure was enduring and long term, as demonstrated by the lack of relapse of MM649 tumors over a period of 12 months. PKC activation must clearly play an important role in the action of EBC-46, since pre-treatment with the wide-spectrum PKC inhibitor bisindolylmaleimide-1 resulted in a loss of efficacy in vivo, and prevented endothelial cell dye uptake in vitro. However, we cannot rule out other known targets of diacylglycerol analogues other than PKC isoforms. Recent work has identified that Ing3A/PEP005 binds to and activates members of the RasGRP family of Ras activators, and that this activation may be in part responsible for the anti-cancer activity of the compound [34]. Future studies will investigate if EBC-46 similarly activates molecules in addition to PKC isoforms. In summary, we have identified and characterized EBC-46 as a novel PKC-activating compound. We demonstrate that a single intra-lesional bolus injection is sufficient for the short term regression and ultimate cure of multiple different cancer types in pre-clinical models. EBC-46 is currently being prepared under GMP conditions for use in an upcoming Phase I clinical trial.

Supporting Information

Figure S1

Translocation of PKC isoforms induced by EBC-46 in SK-MEL-28 cells. PKC-EGFP isoform translocation in transiently transfected SK-MEL-28 cells following 1 h treatment with either 175 nM (100 ng/ml) PMA or EBC-46. Data was obtained from assessment of at least 50 cells per well from each of triplicate transient transfection experiments. Error bars – standard deviation. a – no data available due to mitochondrial location prior to and after treatment; b – no data available as isoform was toxic to SK-MEL-28 cells.

(TIF)

Figure S2

Cell survival assays following treatment with EBC-46. Dose response for cell killing by EBC-46 compared to PMA. Cells were treated with the indicated doses of either EBC-46 (blue) or PMA (red) for 4 days, before assay for cell survival using the sulforhodamine B assay. Data shown are mean ± SD from triplicate readings from three independent experiments, n = 3.

(TIF)

Figure S3

Treatment of FaDu tumors with 30 µg EBC-46 or vehicle alone. 2×106 FaDu tumor cells were injected were injected (two tumors per mouse) on the hindquarter of 5 week old immuno-compromised BALB/c Foxn1nu mice. When the tumors had reached approximately 100 mm3, mice in the control group were treated with vehicle (20% propylene glycol in water, 50 µl) and the treatment group received 50 nmol (30 µg) EBC-46 in vehicle via a single intra-tumoral injection. Figure shows tumor appearance prior to treatment, 1 h following treatment, and 2, 5 and 11 days post treatment of tumors treated with vehicle alone (left) or 50 nmol (30 µg) EBC-46 (right). Also shown are ablated tumors 21 days following treatment with 50 nmol (30 µg) EBC-46. No vehicle only control tumors are shown due to the animals being euthanized at day 12 following treatment due to excessive tumor volume.

(TIF)

Figure S4

EBC-46 Efficacy against head and neck or colon cancer tumors. A. Tumor volume of FaDu HNSCC line in BALB/c Foxn1nu mice. B. Kaplan Meier plot of HT-29 tumor volume reaching greater than 100 mm3 in BALB/c Foxn1nu mice. C. Kaplan Meier plot of MC-38 tumor volume reaching greater than 100 mm3 in C57BL/6J mice. Grey – vehicle (20% propylene glycol in water); Black – 50 nmol (30 µg) EBC-46 (in vehicle).

(TIF)

Figure S5

Effect of EBC-46 on normal skin. Normal skin of BALB/c Foxn1nu mice was treated with either A. 50 µl vehicle (20% propylene glycol in water) or B. 50 nmol (30 µg) EBC-46 in vehicle. Arrows indicate examples of dilated blood vessels. Scale bar  = 100 µm.

(TIF)

Figure S6

Effect of EBC-46 on normal skin vasculature. Normal skin of BALB/c Foxn1nu mice was treated with either A. 50 µl vehicle (20% propylene glycol in water) or B. 50 nmol (30 µg) EBC-46 in vehicle. Arrows indicate examples of intact blood vessels. Scale bar  = 100 µm.

(TIF)

Acknowledgments

We thank TetraQ Pty. Ltd. for performing the chromatographic assay to determine the level of EBC-46 in the plasma (under a fee-for-service agreement with QBiotics Ltd.).

Funding Statement

This work was supported by the National Health and Medical Research Council of Australia Development Grant, Number APP1017676 (PGP, GMB, PWR). MMAD was supported by the University of Queensland International Research Tuition Award (UQIRTA) and a University of Queensland Research Scholarship (UQIRS). GMB was supported by a Smart Futures Researcher-in-Residence fellowship from the Queensland Government, and is currently supported by the Wilson Fellowship for Skin Cancer Research administered by the Perpetual Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

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