22 november 2017: Bron: Oncotarget. 2017; 8:67269-67286.

Onderstaande is een vrije vertaling met ook eigen teovoegingen uit de studie zelf van een artikel in Science Daily over deze studie.

Kankerstamcellen, die de groei van nieuwe en bestaande tumoren voeden, kunnen worden uitgeschakeld door een behandelingscombinatie van eerst antibiotica gevolgd door vitamine C. Dat stellen onderzoekers  van de Universiteit van Salford, naar aanleiding van resultaten uit hun experimenteel onderzoek.

Het antibioticum, Doxycycline, gevolgd door enkele doses ascorbinezuur (vitamine C), was verrassend effectief bij het doden van de kankerstamcellen in een experimenteel onderzoek. Of zoals de onderzoekers zeggen: "in bokstermen zou dit vergelijkbaar zijn met een combinatie van twee klappen die snel achter elkaar worden afgeleverd; een stoot van de linkerhand, gevolgd door een knockout met de rechter."

De onderzoekers zeggen dat hun methode een nieuwe behandelingsoptie biedt om te voorkomen dat kankercellen resistent worden tegen bepaalde kankerbehandelingen (chemo) en hoe combinaties van behandelingen kunnen worden ontwikkeld om resistentie tegen bepaalde geneesmiddelen (chemo) te overwinnen.

Professor Michael Lisanti, die de studie ontwierp, legt uit: "We weten nu dat een deel van de kankercellen ontsnappen aan chemotherapie en resistentie tegen geneesmiddelen ontwikkelen, we hebben deze nieuwe behandelingscombinaties opgezet om uit te vinden hoe ze het doen.

In hun studierapport staat hun aanpak geïllustreerd in veel grafieken, maar is voor leken niet interessant. Hier wel de strategie die de onderzoekers hebben toegepast. Eerst de kankerstamcellen verzwakken en uithongeren met doxycycline en daarna met vitamine C de dodelijk klap uitdelen :

Doxycycline plus vitamine C protocol

Figure 12: Vitamin C and Doxycycline: A synthetic lethal combination therapy for eradicating CSCs. Note that both OXPHOS and the glycolytic pathway jointly contribute to ATP production. Doxycycline inhibits mitochondrial biogenesis and OXPHOS, by acting via mitochondrial ribosomal proteins (MRPs); Vitamin C inhibits glycolytic metabolism by targeting and inhibiting the enzyme GAPDH. Therefore, their use together, as a sequential drug combination, will more severely target cell metabolism and energy production, thereby preventing or blocking the propagation of CSCs.

De onderzoekers in Salford hebben onderstaande combinaties uitgeprobeerd maar wel met altijd doxycycline als eerste om de kankerstamcellen te verzwakken / uit te hongeren:

Metabolic inhibitors successfully employed for the eradication of DoxyR CSCs.

Figure 9: Metabolic inhibitors successfully employed for the eradication of DoxyR CSCs. Briefly, a list of small molecules that we successfully used in conjunction with Doxycycline is shown. These include 9 known inhibitors of OXPHOS, glycolysis and autophagy. Two natural products (Vitamin C and Berberine), six clinically-approved drugs (Atovaquone, Chloroquine, Irinotecan, Sorafenib, Niclosamide, and Stiripentol) and one experimental drug (2-DG), are all highlighted.


"We vermoedden dat het antwoord ligt in het feit dat bepaalde kankercellen - die we metabolisch flexibel noemen - in staat zijn om van brandstofbron te veranderen, dus wanneer de behandeling met chemo de beschikbaarheid van een bepaalde voedingsstof vermindert, kunnen de flexibele kankercellen zichzelf voeden met een alternatieve energiebron.", aldus Professor Michael Lisanti

Deze nieuwe combinatiebehandeling voorkomt dat kankercellen hun dieet veranderen (metabolisch inflexibel) en zal de kankerstamcellen effectief uithongeren door te voorkomen dat ze andere beschikbare soorten biobrandstoffen gaan gebruiken.

De onderzoekers van het Biomedical Research Center van de Universiteit van Salford voegde Doxycycline toe in steeds hogere doses gedurende een periode van drie maanden om metabole inflexibiliteit te creeëren. 

Het doel was om de kankercellen wel levend te laten, maar deze proberen te verzwakken en uit te putten  zodat ze veel kwetsbaarder zouden zijn voor uithongering, door een tweede zogeheten metabole "punch".

Als eerste remden de onderzoekers de mitochondriën in de tumorcel, door de kankercellen alleen te voeden met glucose als brandstofbron; daarna namen ze hun glucose weg en doodden ze de kankercellen effectief met vitamine C. 

Glycolysis inhibitors reduce mammosphere formation in MCF7 DoxyR cells.

Figure 10: Glycolysis inhibitors reduce mammosphere formation in MCF7 DoxyR cells. Evaluation of mammosphere formation in MCF7 and MCF7 DoxyR cells cultured in low attachment plates and treated with Vehicle or increasing concentrations of the glycoysis inhibitor 2-deoxy-glucose (2 DG) (10 mM to 20 mM) for 5 days before counting A. Mammosphere formation is inhibited in MCF7 DoxyR cells cultured in low attachment plates and treated with increasing concentrations of the glycoysis inhibitor Ascorbic Acid (100 µM to 500 µM) for 5 days before counting B. Data shown are the mean ± SEM of 3 independent experiments performed in triplicate. (***) p < 0.001.

"In dit scenario gedraagt ​​vitamine C zich als een remmer van glycolyse, wat de energieproductie in de mitochondriën, de" krachtcentrale "van de cel, voedt, verklaart co-auteur Dr Federica Sotgia. 

Het Salford-team toonde onlangs aan dat vitamine C tot tien keer effectiever is in het tegenhouden van de groei van kankercellen dan farmaceutische producten zoals 2-DG, maar ze zeggen dat wanneer vitamine C wordt gecombineerd met een antibioticum, het tot tien keer effectiever wordt, waardoor het bijna 100 keer effectiever is dan 2-DG. 

Aangezien Doxycycline en vitamine C beide niet-toxisch zijn, kan dit de mogelijke bijwerkingen van een antikankertherapie (chemo) drastisch verminderen. gezien Doxycycline en vitamine C beide niet-toxisch zijn, kan dit de mogelijke bijwerkingen van een antikankertherapie (chemo) drastisch verminderen. 

Het team van Salford onderzocht ook acht andere medicijnen die als een "tweede klap" na het antibioticumgebruik konden worden gebruikt, waaronder berberine (een natuurlijk product) - en een aantal goedkope niet-toxische door de FDA goedgekeurde geneesmiddelen.

Professor Lisanti voegde hieraan toe: "Dit is verder bewijs dat vitamine C en andere niet-toxische stoffen een rol kunnen spelen in de strijd tegen kanker.
"Onze resultaten geven aan dat het een veelbelovend middel is voor klinische studies en een aanvulling op meer conventionele therapieën, om een tumorrecidief, verdere ziekteprogressie en uitzaaiingen te voorkomen."

Het is een interessante studie en het volledige studieverslag: Vitamin C and Doxycycline: A synthetic lethal combination therapy targeting metabolic flexibility in cancer stem cells (CSCs) met gedetailleerd uitgelegd hoe zij in het werk zijn gegaan is gratis in te zien.

Hier het abstract met uitgebreide originele conclusie

Vitamin C and antibiotics: A new one-two 'punch' for knocking-out cancer stem cells.

Journal Reference:

  1. Ernestina Marianna De Francesco, Gloria Bonuccelli, Marcello Maggiolini, Federica Sotgia, Michael P. Lisanti. Vitamin C and Doxycycline: A synthetic lethal combination therapy targeting metabolic flexibility in cancer stem cells (CSCs). Oncotarget, 2015; DOI: 10.18632/oncotarget.18428
Oncotarget. 2017; 8:67269-67286. https://doi.org/10.18632/oncotarget.18428

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Ernestina Marianna De Francesco1,2, Gloria Bonuccelli3, Marcello Maggiolini1, Federica Sotgia3 and Michael P. Lisanti3

1 Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, Italy

2 The Paterson Institute, University of Manchester, Withington, United Kingdom

3 Translational Medicine, School of Environment and Life Sciences, Biomedical Research Centre (BRC), University of Salford, Greater Manchester, United Kingdom

Correspondenc to:

Michael P. Lisanti, email: michaelp.lisanti@gmail.com

Federica Sotgia, email: fsotgia@gmail.com

Keywords: cancer stem-like cells (CSCs), doxycycline, vitamin C, mitochondrial biogenesis, mitochondrial DNA (mt-DNA)

Received: May 05, 2017 Accepted: May 17, 2017 Published: June 09, 2017

Abstract

Here, we developed a new synthetic lethal strategy for further optimizing the eradication of cancer stem cells (CSCs). Briefly, we show that chronic treatment with the FDA-approved antibiotic Doxycycline effectively reduces cellular respiration, by targeting mitochondrial protein translation. The expression of four mitochondrial DNA encoded proteins (MT-ND3, MT-CO2, MT-ATP6 and MT-ATP8) is suppressed, by up to 35-fold. This high selection pressure metabolically synchronizes the surviving cancer cell sub-population towards a predominantly glycolytic phenotype, resulting in metabolic inflexibility. We directly validated this Doxycycline-induced glycolytic phenotype, by using metabolic flux analysis and label-free unbiased proteomics.

Next, we identified two natural products (Vitamin C and Berberine) and six clinically-approved drugs, for metabolically targeting the Doxycycline-resistant CSC population (Atovaquone, Irinotecan, Sorafenib, Niclosamide, Chloroquine, and Stiripentol). This new combination strategy allows for the more efficacious eradication of CSCs with Doxycycline, and provides a simple pragmatic solution to the possible development of Doxycycline-resistance in cancer cells. In summary, we propose the combined use of i) Doxycycline (Hit-1: targeting mitochondria) and ii) Vitamin C (Hit-2: targeting glycolysis), which represents a new synthetic-lethal metabolic strategy for eradicating CSCs.

This type of metabolic Achilles’ heel will allow us and others to more effectively “starve” the CSC population.

Conclusions

Numerous functional studies have now directly shown that mitochondria are an important new therapeutic target in cancer cells [3, 5, 8-21, 40-53]. Since Doxycycline, an FDA-approved antibiotic, behaves as an inhibitor of mitochondrial protein translation, it may have therapeutic value in the specific targeting of mitochondria in cancer cells. However, in this paper, we have identified a novel metabolic mechanism by which CSCs successfully escape from the anti-mitochondrial effects of Doxycycline, by assuming a purely glycolytic phenotype. Therefore, DoxyR CSCs are then more susceptible to other metabolic perturbations, because of their metabolic inflexibility, allowing for their eradication with natural products and other FDA-approved drugs. Thus, understanding the metabolic basis of Doxycycline-resistance has ultimately helped us to develop a new synthetic lethal strategy, for more effectively targeting CSCs.

Materials and Methods

Materials

Doxycycline, Ascorbic Acid, 2-Deoxy-D-glucose (2-DG), Irinotecan, Berberine Chloride, Niclosamide, Chloroquine diphosphate, Stiripentol and Atovaquone were all purchased from Sigma Aldrich. Sorafenib was obtained from Generon. All compounds were dissolved in DMSO, except Ascorbic Acid, 2-deoxy-D-glucose (2-DG) and Chloroquine diphosphate, which were dissolved in cell culture medium.

Cell cultures

MCF7 breast cancer cells were obtained from ATCC and cultured in DMEM (Sigma Aldrich). MCF-7 cells resistant to Doxycycline (MCF7 DoxyR) were selected by a stepwise exposure to increasing concentration of Doxycycline. In particular, wild type MCF7 cells were initially exposed to 12.5 µM Doxycycline and the dose gradually increased to 50 µM over a 3-month period. The population of resistant cells, named MCF7 DoxyR, was selected after 3 weeks of treatment with 12.5 µM Doxycycline, followed by 3 weeks of treatment with 25 µM Doxycycline. MCF7 DoxyR cells were routinely maintained in regular medium supplemented with 25 µM Doxycycline.

Mammosphere formation

A single cell suspension of MCF7 or MCF7 DoxyR cells was prepared using enzymatic (1x Trypsin-EDTA, Sigma Aldrich), and manual disaggregation (25 gauge needle) [54]. Cells were then plated at a density of 500 cells/cm2 in mammosphere medium (DMEM-F12/ B27 / 20-ng/ml EGF/PenStrep) in nonadherent conditions, in culture dishes coated with (2-hydroxyethylmethacrylate) (poly-HEMA, Sigma), in the presence of treatments, were required. Cells were grown for 5 days and maintained in a humidified incubator at 37°C at an atmospheric pressure in 5% (v/v) carbon dioxide/air. After 5 days for culture, spheres > 50 μm were counted using an eye piece graticule, and the percentage of cells plated which formed spheres was calculated and is referred to as percentage mammosphere formation. Mammosphere assays were performed in triplicate and repeated three times independently.

Evaluation of mitochondrial mass and function

To measure mitochondrial mass by FACS analysis, cells were stained with MitoTracker Deep Red (Life Technologies), which localizes to mitochondria regardless of mitochondrial membrane potential. Cells were incubated with pre-warmed MitoTracker staining solution (diluted in PBS/CM to a final concentration of 10 nM) for 30-60 min at 37 °C. All subsequent steps were performed in the dark. Cells were washed in PBS, harvested, re-suspended in 300 μL of PBS and then analyzed by flow cytometry (Fortessa, BD Bioscience). Data analysis was performed using FlowJo software. Extracellular acidification rates (ECAR) and real-time oxygen consumption rates (OCR) for MCF7 cells were determined using the Seahorse Extracellular Flux (XFe-96) analyzer (Seahorse Bioscience) [15]. Briefly, 15,000 MCF7 and MCF7 DoxyR cells per well were seeded into XFe-96 well cell culture plates for 24h. Then, cells were washed in pre-warmed XF assay media (or for OCR measurement, XF assay media supplemented with 10mM glucose, 1mM Pyruvate, 2mM L-glutamine and adjusted at 7.4 pH). Cells were then maintained in 175 µL/well of XF assay media at 37C, in a non-CO2 incubator for 1 hour. During the incubation time, 5 µL of 80mM glucose, 9 µM oligomycin, and 1 M 2-deoxyglucose (for ECAR measurement) or 10µM oligomycin, 9 µM FCCP, 10 µM Rotenone, 10 µM antimycin A (for OCR measurement), were loaded in XF assay media into the injection ports in the XFe-96 sensor cartridge. Data set was analyzed by XFe-96 software after the measurements were normalized by protein content (SRB). All experiments were performed three times independently.

ALDEFLUOR assay and separation of the ALDH positive population

ALDH activity was assessed by FACS analysis (Fortessa, BD Bioscence) in MCF7 cells and MCF7 DoxyR cells. The ALDEFLUOR kit (StemCell Technologies) was used to isolate the population with high ALDH enzymatic activity. Briefly, 1 × 105 MCF7 and MCF7 DoxyR cells were incubated in 1ml ALDEFLUOR assay buffer containing ALDH substrate (5 μl/ml) for 40 minutes at 37°C. In each experiment, a sample of cells was stained under identical conditions with 30 μM of diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor, as a negative control. The ALDEFLUOR-positive population was established in according to the manufacturer’s instructions and was evaluated in 3 × 104 cells. Data analysis was performed using FlowJo software.

Anoikis assay

MCF7 and MCF7 DoxyR cells were seeded on low-attachment plates to enrich for the CSC population [54]. Under these conditions, the non-CSC population undergoes anoikis (a form of apoptosis induced by a lack of cell-substrate attachment) and CSCs are believed to survive. The surviving CSC fraction was analyzed by FACS analysis. Briefly, 1 x 105 MCF7 and MCF7 DoxyR monolayer cells were seeded for 48h in 6-well plates. Then, cells were trypsinized and seeded in low-attachment plates in mammosphere media. After 10h, cells were spun down and incubated with CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen) antibodies for 15 minutes on ice. Cells were rinsed twice and incubated with LIVE/DEAD dye (Fixable Dead Violet reactive dye; Life Technologies) for 10 minutes. Samples were then analyzed by FACS (Fortessa, BD Bioscence). Only the live population, as identified by the LIVE/DEAD dye staining, was analyzed for CD24/CD44 expression. Data were analyzed using FlowJo software.

Label-free semi-quantitative proteomics analysis

Cell lysates were prepared for trypsin digestion by sequential reduction of disulphide bonds with TCEP and alkylation with MMTS. Then, the peptides were extracted and prepared for LC-MS/MS. All LC-MS/MS analyses were performed on an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, San Jose, CA) coupled to an Ultimate 3000 RSLC nano system (Thermo Scientific, formerly Dionex, The Netherlands). Xcalibur raw data files acquired on the LTQ-Orbitrap XL were directly imported into Progenesis LCMS software (Waters Corp., Milford, MA, formerly Non-linear dynamics, Newcastle upon Tyne, UK) for peak detection and alignment. Data were analyzed using the Mascot search engine. Five technical replicates were analyzed for each sample type [8, 12].

Immuno-blot analysis

MCF7 and MCF7 DoxyR cells protein lysates were electrophoresed through a reducing SDS/10% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against phosphorylated AKT (Ser 473) and ATK (Cell Signaling), Phopshorylated ERK 1/2 (E-4), ERK2 (C-14), TOMM20 (F-10) and β-actin (C2), all purchased from Santa Cruz Biotechnology. Proteins were detected by horseradish peroxidase-linked secondary antibodies and revealed using the SuperSignal west pico chemiluminescent substrate (Fisher Scientific).

Click-iT EdU proliferation assay

48h after seeding MCF7 and MCF7 DoxyR were subjected to proliferation assay using Click-iT Plus EdU Pacific Blue Flow Cytometry Assay Kit (Life Technologies), customized for flow cytometry. Briefly, cells were treated with 10 µM EdU for 2 hours and then fixed and permeabilized. EdU was detected after permeabilization by staining cells with Click-iT Plus reaction cocktail containing the Fluorescent dye picolylazide for 30 min at RT. Samples were then washed and analyzed using flow cytometer (Fortessa, BD Bioscence). Background values were estimated by measuring non-EdU labeled, but Click-iT stained cells. Data were analyzed using FlowJo software.

Migration assay

MCF7 and MCF7 DoxyR cells were allowed to grow in regular growth medium until they were 70-80 % confluent. Next, to create a scratch of the cell monolayer, a p200 pipette tip was used. Cells were washed twice with PBS and then incubated at 37° C in regular medium for 24h. The migration assay was evaluated using Incucyte Zoom (Essen Bioscience) [55]. The rate of migration was measured by quantifying the % of wound closure area, determined using the software ImageJ, according to the formula:

% of wound closure = [(At = 0 h - At = Δ h)/At = 0 h] × 100%

Statistical analysis

Data is represented as the mean ± standard error of the mean (SEM), taken over ≥ 3 independent experiments, with ≥ 3 technical replicates per experiment, unless otherwise stated. Statistical significance was measured using the t-test. P ≤ 0.05 was considered significant.

Author contributions

Professor Michael Lisanti and Dr. Federica Sotgia conceived and initiated this collaborative project. All the experiments in this paper were performed by Dr. Ernestina M. De Francesco, with minor technical assistance from other lab members; Dr. Ernestina M. De Francesco analyzed all the data and generated the final figures and tables, and she wrote significant portions of the manuscript. Drs. Michael P. Lisanti, Ernestina M. De Francesco, Gloria Bonuccelli, Marcello Maggiolini and Federica Sotgia all contributed to the writing and the editing of the manuscript. Professor Lisanti generated the schematic summary diagrams.

Acknowledgments

We are grateful to the University of Manchester, which allocated start-up funds and administered a donation, to provide the necessary resources required to start and complete this drug discovery project (to MPL and FS). Dr. Ernestina M. De Francesco was supported by a fellowship from the Associazione Italiana per la Ricerca sul Cancro (AIRC) co-funded by the European Union. The Lisanti and Sotgia Laboratories are currently supported by private donations, and by funds from the Healthy Life Foundation (HLF) and the University of Salford (to MPL and FS). We also wish to thank Dr. Duncan Smith, who performed the proteomics analysis on whole cell lysates, within the CRUK Core Facility. MM was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC, IG 16719).

Conflicts of Interest

MPL and FS hold a minority interest in Lunella, Inc.

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4 Reacties op "Vitamine C in combinatie met Doxycycline een antibiotica doden samen kankerstamcellen en lijkt nieuwe behandelingsoptie nagenoeg zonder bijwerkingen"

  • Tineke :
    Albert Kroon schreef een boek over kankertherapie, kan het eenvoudiger? Deze therapie lijkt er op voort te borduren. Hij was destijds verbonden aan het UMCG! Er staat niet vermeld voor welke kanker vormen de therapie geschikt is...
    • Pieter :
      Tineke, volgens Kroon voor alle soorten omdat deze kwetsbaarheid in kanker algemeen over het hele spectrum van de kankersoorten gelijk is. Dit komt van Otto Warburg wederom in zijn stuk The Origen of Cancer, geschreven in 1927 waarvoor hijh in 1931 de Nobelprijs ontving en wiens theorie tot op de dag van vandaag gebruikt wordt voor het opsporen van kanker dmv Petscan. Dit is de Common denometor binnen kanker.! Zelfs James Watson (dubbele Hellix en grote man geweest achter chemo aanpak) is daar nu van overtuigd.
    • Pieter LV :
      Klopt helemal en Professor Dr Albert Kroon MD heeft ook aan de wieg gestaan om te onderkeMitonnen dachondri net zbacteria vreemde weens (als het ware) in het lichaam zijn die een "eigen" DNA hebben het zgn MDNA. Al vele jaren heeftprofessor Kroon gestreden om zijnbevindingen (en die van zijn wetenschappelijke staff) aan het voetlicht te krijgen maar met (veel te) weinig succes. Deze onderzoekers (prof Lisanti) kennen Kroon goed en zijn werk. Het is des te jammer dat hij daar in het onderzoek niet genoemd wordt net zo min als Warburg, die aan de wieg van deze methode van behandelen heeft gestaan. Kroon verdient de lof voor deze methode die trouwens niet alleen de kanker stamcellen doodt (wat tot nu toe een bottleneck was) maar ook de rest van de lankercellen. Een KUUR dus. Ssst niet verder vertellen???
      • Pieter nogmaals :
        Klopt helemaal en Professor Dr Albert Kroon MD heeft ook aan de wieg gestaan om te onderkennen dat Mitochondria net zo als bacteria vreemde wezens (als het ware) in het lichaam zijn die een "eigen" DNA hebben het zgn MDNA. Al vele jaren heeft professor Kroon gestreden om zijn bevindingen (en die van zijn wetenschappelijke staff) aan het voetlicht te krijgen maar met (veel te) weinig succes. Deze onderzoekers (prof Lisanti) kennen Kroon goed en zijn werk. Het is des te jammer dat hij daar in het onderzoek niet genoemd wordt net zo min als Warburg, die aan de wieg van deze methode van behandelen heeft gestaan. Kroon verdient de lof voor deze methode die trouwens niet alleen de kanker stamcellen doodt (wat tot nu toe een bottleneck was) maar ook de rest van de kankercellen. Een KUUR dus. Ssst niet verder vertellen??? Een ieder die op enige wijze te maken heeft met kanker moet echt het boek van Kroon lezen. Zeker Oncologen!

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