Tech Possible Cancer prevention

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Our European partners have a new antidotal study that supports our product helps with cancer.

Here are some excerpts from the actual clinic study. I will put them up into significant parts.

 

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UNIVERSITA’ DEGLI STUDI DI URBINO “CARLO BO”

Dipartimento di Scienze Biomolecolari Sezione di Biochimica Clinica e Biologia Cellulare

Corso di Dottorato di Ricerca in Metodologie Biochimiche e Farmacologiche

Ciclo XXVI

The antiproliferative effect of the nutritional supplement CELLFOODTM on human cancer cell lines: from metabolism modifications to apoptosis induction.

Settore Scientifico Disciplinare: BIO-12

Relatore: Dottoranda:

Char.mo Prof. Battistelli Serafina Dott. Catalani Simona

Anno Accademico: 2012-2013

1.1 Natural compounds with antiproliferative effect

Tumorigenesis is a multistep process that can be activated by any of various environmental carcinogens, inflammatory agents, and tumor promoter. In this context, diet may have a very relevant influence. Indeed, over several decades, many studies have shown numerous dietary constituents and nutraceuticals as cancer chemopreventive agents (Aggarwal et al, 2009), and it has been generally accepted that dietary agents can suppress transformation, hyperproliferation, invasion, angiogenesis, and metastasis of various tumors (Sarkar and Li, 2006).

The aim of the modern medicine is, in fact, to develop drugs that are safe, efficacious, and affordable. However, the questions that remain to be answered are which component of these dietary agents is responsible for the anti-cancer effects and what is the mechanism by which they suppress cancer. Dietary agents consist of a wide variety of biologically active compounds that are ubiquitous in plants, many of which have been used in traditional medicines for thousands of years.

It is becoming clear, as shown in Figure 1.1, that these agents exert pleiotropic effects on tumor cells affecting various molecules involved in proliferation, invasion, and metastasis of cancer (Shanmugam et al, 2011). In this regard, documented data indicate that many dietary agents, such as curcumin, resveratrol, genistein, diallyl sulfide, capsaicin, catechins, beta-carotene and dietary fibers act modulating multiple intracellular signaling molecules, such as transcription factors, anti-apoptotic and pro-apoptotic proteins, protein kinases, cell cycle 9

proteins and growth factor signaling pathways. For instance, many dietary agents can down-regulate the expression of anti-apoptotic proteins, such as Bcl-2 and Bcl-XL and up-regulate the expression of pro-apoptotic proteins, such as caspases and PARP, in several cancer cell lines (Khan et al, 2008).

Figure 1.1: Signal transduction and apoptotic pathways modulated by dietary agents

(Shanmugam et al, 2011).

Hence, because oxidative and inflammatory stress contributes to malignant transformation, dietary agents with antioxidative, anti-inflammatory, and pro-apoptotic properties would be good candidates for preventing most human malignancies. Indeed, modulation of cell signaling and apoptotic pathways by dietary agents would provide more new opportunities for chemoprevention based on specific molecular targets.

 

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1.3 Metabolism of cancer cells

Changes in energy metabolism are one of the first identified biochemical hallmarks of cancer cells. In fact, cell proliferation, that represents the essence of cancer disease, involves not only a deregulated control of cell proliferation but also adjustments of energy metabolism in order to fuel cell growth and division. Among the metabolic changes exhibited by tumor cells, enhanced glucose metabolism and glucose dependence, compared to normal tissues, are particularly correlated with tumor aggressiveness and prognosis. Normal cells generate most of their ATP through glycolysis under anaerobic conditions and mitochondrial oxidative phosphorylation under aerobic conditions. Furthermore, normal tissues can use alternative energy sources, such as glucose, fatty acids, amino acids and other metabolic intermediates, to generate ATP in mitochondria. Tumor cells, however, generate as much as 60% of their ATP through glycolysis, regardless of the presence or absence of oxygen, and depend more on glycolysis for ATP generation (Nam et al, 2013). This phenomenon was originally observed by Otto Warburg (1930) and is now known as “Warburg effect” (Warburg, 1956) or “aerobic glycolysis”. Since this first observation, the “aerobic glycolysis” has been observed in a variety of tumor cells and cumulating studies on various proliferating cells have led to the evidence of a global metabolic reorganization concomitant to cancer progression (Moreno-Sánchez et al, 2007). It is known that proliferation of cancer cells is accompanied by activation of glycolysis and this altered glucose metabolism, or aerobic glycolysis (Figure 1.2), is recognized as one of the most common hallmarks of cancer (Cairns et al, 2011).

 

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1.10 Alterations of oncogenes and tumor suppressor genes drive cancer cells to metabolic reprogramming and apoptosis resistance

Cancer cells use aerobic glycolysis, despite it appears to be an inefficient way to generate ATP. Increasing evidence shows that the alterations of oncogenes and tumor suppressors in tumorigenesis play a key role in aerobic glycolysis of cancer (Zheng, 2012). Oncogene Ras mutations are often found in many types of human cancers and drive the metabolic phenotype of cancer cells toward aerobic glycolysis (Hu et al, 2012). Not surprisingly, this oncoprotein is very often mutated leading to its hyperactivation in cancer. It, in turn, mediates the activation of several effector pathways like PI3K/AKT, that is commonly altered in human cancers. Once activated, this pathway not only provides strong growth and survival signals to tumor cells but is also shown to have intense effects on cellular metabolism. The best studied effector downstream of PI3K is AKT, a serine threonine kinase that plays a dual role in regulating both cell proliferation and metabolism. Since AKT is known to be a major mediator involved in the uptake of glucose and its utilization, it is therefore not surprising that this kinase is associated with high glycolytic activity in tumor cells (Elstrom et al, 2004). In fact, AKT activation leads to enhanced expression of GLUT-1 and phosphofructokinase (PFK) activity; moreover, it enhances and maintains the association of HKI and HKII to mitochondria (Barthel et al, 1999) somehow preventing the release of cytochrome-c and apoptosis.

mTOR, activated by Ras via the PI3K/Akt/mTOR signaling pathway, promotes, in turn, glycolysis, inducing HIF-1 (Denko, 2008). HIF-1 transcription factors facilitate cellular adaptation to hypoxic environments and play critical roles in 34

shifting from the oxidative phosphorylation to the glycolytic phenotype in cancer

 

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2.AIM OF THE STUDY

Previous studieshaveshown that CF is a nutritional supplement with antioxidantpropertiesin vitro. The purpose of this PhD thesiswas to provide evidence that CFis also a pro-apoptoticand antiproliferative natural compound.In this regard, the effect of CF on cell growthandviability, glycolytic metabolism, cell cycle regulation and apoptosiswas evaluated, onboth hematologic cancer celllines(Jurkat, U937, and K562)and several solid tumor cell lines(mesothelioma,melanoma, lung,breast andcolon carcinoma). In particular, on leukemiccell lines,cell growth and viability,cell cycle, apoptosis,HIF-1 alpha amount, glucose transporter (GLUT-1) expression, glycolyticmetabolism and finally intracellular ROS productionwere investigated. Theapoptotic pathwaywas herein evaluated studying caspase-3 activity, DNAfragmentation and cell morphology modifications.The glycolytic metabolism wasevaluated studying the activity of severalglycolytic enzymes, such as HK,G6PDH, GAPDH,PK and LDH. Moreover, lactate release and extracellular pHmeasurements completed the metabolic studies.On solid cancer cell lines,the following investigations wereperformed: cell growth and viability assay, clonogenictestand cell cycle analysis.Theexpressionof several proteins involved in the apoptoticand survival pathways and in cellcycle regulation(caspase 3, PARP, p53, c-myc, p27 and p21, pAKT,AKT and Bcl-2) wasfinally performed.

 

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3. MATERIALS AND METHODS

EVALUATION OFMETABOLISM MODIFICATIONS AND APOPTOSISINDUCTION AFTER CF TREATMENT ON LEUKEMIC CELLS(1stPART)

Experiments on leukemic cells growing in suspensionwere principally performedat the Section of Clinical Biochemistry and Cellular Biology,Department ofBiomolecular Sciences of theUniversity of Urbino. To carry out DNAelectrophoresis and western blot analysis, I personally collaborated with Prof.Francesco Palma and his PhD student Dr. Marselina Arshakyan,while for the cell cycle analysis I relied on Dr. Barbara Canonico andDr. Francesca Luchetti, bothfrom the University ofUrbino.

3.1 Antibodies and reagents



All chemicals and cell culture reagents were purchased fromSigma-Aldrich(Milan, Italy) and from VWR International (Milan, Italy). The antibody anti GLUT-1 was from Thermo Scientific, Lymphoprep™from Axis-Shield PoC AS (Oslo,Norway)and WST-1 from Roche DiagnosticsGmbH (Mannheim, Germany).

3.2 CellfoodTM



The supplement (liquid) was kindly provided by Eurodream srl (La Spezia, Italy)and stored at room temperature. Before use, CF wasdiluted in phosphate bufferedsaline 150 mM pH 7.4(PBS) and sterilized using a 0,45 μm syringe-filter.CF was

tested at the final concentrationof 5 μl/ml.42

3.3 Cell lines and lymphocytes

Three leukemic cell lines, growing in suspension, were employed for the first part of the present study: Jurkat (acute lymphoblastic leukemia), U937 (acute myeloid leukemia) and K562 (chronic myeloid leukemia in blast crisis). Cells were grown in sterile plastic flasks and maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin/streptomycin 100 U/ml, and incubated in a CO2 incubator (37°C, 5% CO2 and humidified atmosphere). The medium was replaced three times a week.

Human lymphocytes were also used as non-tumor cells to test the effects of CF in a control cell system. Lymphocytes were isolated from blood samples provided by healthy volunteers by centrifugation in the presence of LymphoprepTM, and were cultured as described above with the addition of 10 μg/ml of phytohemagglutinin (PHA).

 

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4.9 Cell cycle analysis

To identify the distribution of cells in the different phases of the cell cycle and the sub-G1 proportion (apoptosis), the DNA content of two cancer cell lines (MSTO and HCT116) was detected by propidium iodide staining and flow cytometry. Unsynchronized MSTO and HCT116 cells were seeded on 6-well plates and incubated with CF 5 μl/ml for 24 and 48 hours. At each experimental time 1x106 cells were collected, washed with PBS, and cell pellets were fixed gently resuspending (drop by drop) them with 70% ice-cold ethanol and stored at -20°C. Cells were washed twice in PBS and cell pellets were resuspended in 400 μl PBS. 50 μl of 0,1 mg/ml RNase A and 50 μl of 200 μg/ml propidium iodide were added and then incubated at 37°C for 1 hour in the dark. Finally, the cell cycle distribution of cells was determined by flow cytometry (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, CA) equipped with an argon laser at 488 nm. Results were expressed as percentage of DNA content and compared to untreated cells. Pre-G1 picks were considered indicative of sub-G1 apoptotic population.

4.10 Western blot analysis

To evaluate the expression of several proteins characteristic of the apoptotic pathway, MSTO and HCT116 cells were seeded in full culture media, as described above, in absence (CTR) or presence of CF 5 μl/ml up to 48 hours. At each experimental time, cells were counted, 2 x 107cells/ml were collected by centrifugation, washed in PBS e resuspended in 150 μl of 1X SDS loading buffer to 20x106 cells/ml. Cell lysis was achieved by vortex, and the viscosity of lysates was 66

reduced by three passages through a syringe needle. 15 μl of each samples were run on 0.8% SDS-polyacrylamide gel and the resolved proteins were electrophoretically transferred to supported nitrocellulose membranes (Bio-Rad Laboratories S.r.l., Milan, Italy) using a Bio-Rad Semidry Transfer system. Membranes were stained with Poinceau red dye for a rapid reversible detection of protein bands and then washed with running water. Non-specific binding to membranes was blocked by incubation in blocking solution (50 mM Tris-HCl, 150 mM NaCl and 5% (w/v) non-fat dried milk, pH 7.5). After blocking solution removal, membranes were incubated in a new blocking solution at 4°C overnight with primary antibodies against: caspases-3, PARP, p53, c-myc, p21, p27, p-AKT, AKT, Bcl-2 and γ-tubulin. Membranes were then washed three times with TTBS (50 mM Tris-HCl, 150 mM NaCl and 0.05% (v/v) Tween 20, pH 7.5) and incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody diluted 1:4500 in TTBS for 1 hour at room temperature. After TTBS washes, the blots were exposed to reagents for ECL immunodetection (ECL Advance Western Blotting Detection Kit) and labelled proteins were detected by autoradiographic film (Pierce).

 

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5.RESULTS

RESULTS ON LEUKEMIC CELL LINES (1stPART)

Part ofthe experiments performed onthe leukemic cell lines and described in thepresent thesis has been recently published in the Journal of Experimental and ClinicalCancer Research2013, 32:63(Catalani S, Carbonaro V, Palma F, Arshakyan M,Galati R, Nuvoli B, Battistelli S, Canestrari F and Benedetti S:“Metabolismmodifications and apoptosis induction after Cellfood™ administration to leukemia celllines”).

5.1 Preliminary investigations



Preliminary studies were performed on Jurkat cells to evaluate the most efficientconcentration of CF as antiproliferative agent. As shown in Figure5.1, all concentrations tested (from 0,125μl/ml to 2μl/ml) induced cell growth inhibition;however,only the highest concentration (CF 2μl/ml)induced asignificantcellgrowthreduction, reaching 15% after24 hours of incubation, compared to untreated cells (CTR).

 

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A further experiment was conducted incubating cells with higher concentration of CF (1,7 μl/ml, 2,5 μl/ml and 5 μl/ml), in comparison to the previous assay, for 24, 48 and 72 hours. As shown in Figure 5.2, cell growth inhibition significantly increased reaching more than 30% after 72 hours of incubation with CF 5 μl/ml.

 

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5.2 Effect of CF on leukemic cell proliferation and viability

Since the results obtained were promising and the concentration 5 μl/ml of CF seemed to be the most efficient in reducing leukemic cell growth, further experiments were extended and conducted on three leukemic cell lines (Jurkat, U937, and K562) treated only with CF 5 μl/ml up to 72 hours.

Cell count using trypan blue dye revealed a significant inhibition of leukemic cell proliferation after 24, 48 and 72 hours of incubation with CF, as compared to untreated cells (Figures 5.3 A, 5.3 B and 5.3 C).

 

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A significant inhibition of leukemic cell proliferation upon CF treatment was observed after each experimental time point. Cell growth reduction was maximum after 72 hours of incubation with CF in which the inhibition reached up 38%, 48% and 36% respectively in Jurkat, U937 and K562 cells, as compared to untreated cells. 71

An example of leukemic cell growth inhibition in CF-treated cells has also been captured by digital camera as reported in Figure 5.4 (A, B, C, D, E, F) (inverted microscope, magnification 10x).

 

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Data obtained evaluating cell viability through WST-1 reagent confirmed the inhibition of leukemic cell proliferation upon CF treatment. Cell growth reduction was significantly observed after each experimental time point and was maximum after 72 hours of incubation with CF. As observed through the trypan blue dye exclusion method, the most sensitive cell line to CF treatment was the U937. Moreover, being the maximum inhibition of leukemic cell proliferation and viability after 72 hours of incubation with CF, this time point was selected to perform some of the subsequent biochemical and molecular evaluations.

To verify the specificity of CF, isolated lymphocytes were employed as a non-tumor cell system and incubated up to 96 hours upon the administration of the same dose of CF (5 μl/ml). As shown in Figure 5.6, no effect was observed on cell growth, indicating that CF is not toxic to healthy cells.

 

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Figure 5.7. shows that CF treatment induced a slightly increased percentage of cells in S phase both after 24 (35% compared to control: 28%) and 48 hours of incubation (28% compared to control: 22%). Therefore CF treatment could induced an arrest in S phase of the cell cycle in U937 cells.

5.4 Effect of CF on apoptosis induction

The capacity of CF to induce apoptosis in the leukemic cells was evaluated studying three features of the apoptotic cell death: morphological changes, caspase-3 activation and nuclear DNA fragmentation.

First of all, cell morphology was studied to identify apoptotic cells on the basis of morphological features commonly recognized as hallmark of apoptosis, such as contracted cell bodies, condensed chromatin and membrane bound apoptotic bodies containing one or more nuclear fragments. The assay was performed in light microscopy through May-Grunwald Giemsa staining. The number of apoptotic cells was determined and results (Figure 5.8 A, 5.8 B and 5.8 C that are representative of three independent experiments) were finally expressed as percentage of apoptotic cells.

 

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As shown in Figure 5.8 C CF induced apoptosis in K562 cells after each experimental time point. The percentages of apoptosis in CF-treated cells were: 15% after 6 hours, 19% after 24 hours, 27% after 48 hours and reached 32% after 72 hours of incubation, compared to untreated cells. Etoposide-treated cells showed 31% of morphologically altered cells.

Here below are reported images of Jurkat cells (Figure 5.9 A, 5.9 B and 5.9 C).

 

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As a second step, the evaluation of the pro-apoptotic protein caspase-3 was assessed. Results show that, after 24, 48 and 72 hours upon CF administration, a significant increment of the enzyme activity was observed in all tested cell lines, as compared to the respective untreated controls, represented in the following figures by the dashed lines (Figure 5.10 A, 5.10 B and 5.10 C).

As shown in Figure 5.10 B, caspases-3 activity was significantly increased after 48 and 72 hours upon CF treatment, as compared to untreated cells. This activity reached the highest value after 72 hours of incubation with CF (2,6 fold higher than untreated cells).

As shown in Figure 5.10 C, caspases-3 activity was significantly increased after each experimental time, even though the raise was moderate after 24 and 48 hours upon CF treatment, as compared to untreated cells. This activity reached the highest value after 72 hours of incubation with CF (0,7 fold higher than untreated cells). Thus, results on caspases-3 activity measurement showed that this protein involved in the apoptotic pathway was considerably increased in particular after 48 and 72 hours after CF treatment in comparison with control.

The increase in caspase-3 activity is generally followed by a full execution of the apoptotic pathway and finally by a pronounced nuclear DNA fragmentation. In this regard, DNA fragmentation was evaluated by agarose gel electrophoresis, as a biochemical marker of late apoptosis. Electrophoretic analysis was first performed in Jurkat cells treated for 6 hours with different concentration of etoposide (25, 50 and 100 μM), which is an apoptosis inducer (Figure 5.11).

 

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The reduction of HIF-1 alpha in Jurkat, U937 and K562 cells, upon CF treatment, reached respectively 20%, 38% and 22% as compared to control, represented in figure by the dashed line.

5.6 Effect of CF on GLUT-1 expression

It is known that GLUT-1 is regulated by the activation of HIF-1 alpha and that this glucose transporter is up-regulated in cancer cells as a consequence of an increased requirement of glucose.

As shown in Figure 5.14, after 72 hours of incubation with CF, a reduction of GLUT-1 expression was observed in the three leukemic cell lines, and in particular in U937, as revealed by the densitometric analysis of the bands in comparison with untreated cells.

The reduction of GLUT-1 expression in Jurkat, U937 and K562 cells, upon CF treatment, reached respectively 17%, 71% and 32% as compared to control.

 

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The reduction of GLUT-1 expression in Jurkat, U937 and K562 cells, upon CF treatment, reached respectively 17%, 71% and 32% as compared to control. 85

Figure 5.15: HK activity in leukemic cells after 24 h of incubation with CF (5 μl/ml) in comparison with untreated cells (CTR) (*p<0.05 vs CTR).

These results are in accordance with GLUT-1 decreased expression after CF treatment, indicating a decreased glucose phosphorylation by HK as a consequence of a decreased glucose uptake.

5.7.2 Effect of CF on G6PDH activity

G6PDH is a key enzyme that regulate carbon flow in the pentose cycle, thus the ribose synthesis and NADPH production for proliferating cells. As shown in Figure 5.16, CF induced a significant reduction of G6PDH activity. The reduction was similar in the three leukemic cells (21% in Jurkat, 20% in U937 and 18% in K562).

 

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7. DISCUSSION Over the last decades, many studies have shown numerous dietary constituents and nutraceuticals as cancer chemopreventive agents (Aggarwal et al, 2009); in fact, it has generally been accepted that they can suppress transformation, hyperproliferation, invasion, angiogenesis and metastasis of various tumors (Shanmugam et al, 2011). Because oxidative and inflammatory stress contributes to malignant transformation, dietary agents with antioxidative, anti-inflammatory and pro-apoptotic properties would be good candidates for preventing human malignancies (Gao et al, 2012; Huang et al, 2012). Taking into consideration that CF is a nutritional supplement whose antioxidant properties have been well documented in vitro (Benedetti et al, 2011), that the marine algae from which the organic and inorganic components of CF are extracted, has demonstrated a growth-inhibitory effect, both in vitro (Aslam et al, 2009) and in vivo (Aslam et al, 2012) and that CF is able to modulate O2 availability improving respiratory metabolism and mitochondrial activity (Ferrero et al, 2011), in the present thesis we wonder if CF could also affect cancer cell growth by inducing metabolism modifications and apoptosis. Initially, we performed assays to evaluate the effect of CF on cell growth and viability to determine the best experimental conditions without interferences caused by cytotoxicity. Hence, once identified the most efficient concentration (5 μl/ml) of CF in inhibiting cancer cell growth, we observed that CF treatment reduced cancer cell proliferation and viability in both leukemic cell lines and in all solid tumor cells tested (mesothelioma, melanoma, lung, breast and colon cancer). Cell growth reduction reached 50% in the U937 108

leukemic cell line as compared to untreated cells and percentages were even higher in some solid cancer cells, such as the mesothelioma MSTO (>80%) and the colon cancer HCT116 cell lines (about 90%), which were selected for further and detailed experiments, given their particularly high sensitivity to CF treatment. To investigate whether CF could affect healthy cell growth, human lymphocytes and fibroblasts were also seeded in the same experimental conditions. Data revealed no significant differences between untreated and treated cells, confirming that CF did not affect healthy cell viability and inhibited selectively only cancer cell growth. This inhibition was firstly revealed by the trypan blue exclusion method and then confirmed by cell viability assays, measuring mitochondrial dehydrogenase activity. Furthermore, the antiproliferative effect of CF was also examined in HFF (fibroblasts), MSTO (mesothelioma) and HCT116 (colon) cell lines by clonogenic assays, evaluating cell colony formation capacity. No visible change in healthy fibroblast growth was noted while no colony was observed in cancer cells, suggesting that CF significantly decreased cell growth only in the above mentioned cancer cell lines.

Moreover, to clarify whether CF was able to reduce cancer cell viability by promoting apoptotic cell death and cell cycle arrest, further experiments were conducted on leukemic cells and on two solid cancer cell lines, representing the most sensitive to CF treatment (the mesothelioma cell line MSTO and the colon carcinoma cell line HCT116). The capacity of CF to induce pro-apoptotic mechanisms was evaluated studying some typical apoptotic markers in the leukemic cells (cell morphological changes, caspase-3 activity and DNA fragmentation) and the expression of numerous proteins (caspase-3, PARP, p53, c-109

myc, p21 and p27, pAKT, AKT and Bcl-2) involved both in apoptotic and survival processes and in the cell cycle control, in the two solid tumor cell lines.

Caspase-3 is considered to be the most important effector of apoptosis and an early marker for both intrinsic and extrinsic pathways (Elmore, 2007) and our results, obtained using a colorimetric kit, show that CF treatment significantly stimulated caspase-3 activity in all leukemic cells at each experimental time point, as compared to untreated cells. In particular, caspases-3 activity was considerably and significantly increased in U937 cells after 72 hours of incubation with CF (2,6 fold higher than untreated cells). Moreover, cells undergoing apoptosis are characterized by the presence of morphological changes, such as contracted cell bodies, condensed chromatin and membrane bound apoptotic bodies (Elmore, 2007). To evaluate these morphological changes, the three leukemic cell lines were stained with MGG and observed using a light microscope at 400x magnification. Our results show that, upon CF treatment, the morphology observed was compatible with that of apoptotic cells. As a common hallmark of late-stage apoptosis, genomic DNA fragmentation was analyzed through gel electrophoresis. In this context, our data suggest that after 72 hours of incubation with CF an internucleosomal DNA cleavage (or DNA laddering) was observed in the leukemic cell lines, as compared to the respective untreated controls. The laddering is particularly evident in the CF-treated U937 cell line.

Taken together, these data lead us to suggest that the observed cancer cell growth inhibition was due to apoptosis induction in the three leukemic cell lines.

 

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8.CONCLUSIONS

Modulation of cell signaling, apoptotic pathways and tumor metabolism by dietary agents and nutraceutical compounds may provide new opportunities in both prevention and treatment of cancer. Herein we supply evidence for a significant anti proliferative effect of the nutritional supplement Cellfood on leukemic cells and on mesothelioma and colon cancer cell lines by inducing cell death through an apoptotic mechanism and by altering cell metabolism through HIF-1 alpha and GLUT-1 regulation. Thanks to its anti oxidative and pro-apoptotic properties, CF might be a good candidate for cancer prevention and of great importance in the development of new and safe strategies to regulate and slow down the proliferation of cancer cells. CF could be an effective nutritional supplement, that may give chemopreventive benefits in human cancers and possibly improve the quality of life of patients undergoing conventional antineoplastic therapy. However, large-scale clinical trials will be needed to validate the usefulness of this agent either alone or in combination with the existing standard care.

 

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This is a 133 page study, so I tried finding the most relevant writings on it. I thought @Further would like to read this, since he's working in a cancer research center.

 

[Denny Crane]

http://www.medscape.com/viewarticle/757713

Over the last years, evidence has accumulated suggesting that by systematically reducing the amount of dietary carbohydrates (CHOs) one could suppress, or at least delay, the emergence of cancer, and that proliferation of already existing tumor cells could be slowed down. This hypothesis is supported by the association between modern chronic diseases like the metabolic syndrome and the risk of developing or dying from cancer. CHOs or glucose, to which more complex carbohydrates are ultimately digested, can have direct and indirect effects on tumor cell proliferation: first, contrary to normal cells, most malignant cells depend on steady glucose availability in the blood for their energy and biomass generating demands and are not able to metabolize significant amounts of fatty acids or ketone bodies due to mitochondrial dysfunction. Second, high insulin and insulin-like growth factor (IGF)-1 levels resulting from chronic ingestion of CHO-rich Western diet meals, can directly promote tumor cell proliferation via the insulin/IGF1 signaling pathway. Third, ketone bodies that are elevated when insulin and blood glucose levels are low, have been found to negatively affect proliferation of different malignant cells in vitro or not to be usable by tumor cells for metabolic demands, and a multitude of mouse models have shown antitumorigenic properties of very low CHO ketogenic diets.

(I have been on a ketogenic diet for over a year and loving it)

 

[Denny Crane]

Also, the weight loss has been pretty spectacular without counting calories.

 

[Further]

Looks very interesting Mags. I'll read more later but a quick thumb-through looks fascinating. I would caution that there are many substances in tissue culture that show markedly differing effects, especially over time, than in live studies. Especially on inhibition of cellular proliferation. Simply put, the food we can provide in vitro are often not the same food available in vivo. But I haven't read much, if you have in vivo studies that becomes more impressive.

By the way Mags, I no longer work in research. I'm in the wine industry now, and going to school for enology and viticulture to hopefully own my own winery and become a winemaker in a few years.

I'm busy at this moment, but I'll read more later. Congrats, this looks like great news for your company and possibly for all of us.