Source: Experimental Oncology 2011;33(2):71-7.
Affiliation: Dr. Rath's Research Institute, Cancer Division, Santa Clara, California 95050, USA.
Leukemia is characterized by uncontrolled marrow cell proliferation and metastatic foci. The authors investigated the antitumor potential of a nutrient mixture on malignant leukemia P-388 cells. The nutrient mixture containing lysine, proline, ascorbic acid, green tea extract and other nutrients is formulated to target key pathways in cancer progression. The cells were treated with the mixture, and tested at doses 0, 10, 50, 100, 500 and 1000 μg/ml in triplicates. The effects were evaluated by cell proliferation, Matrigel invasion, cell morphology and apoptosis. The in vivo effect was measured in male nude mice (n = 12) inoculated with P-388 cells. After randomly dividing in two groups, each group was fed regular and the nutrient mixture supplemented diet and the mice were sacrificed after four weeks. Results: The nutrient mixture decreased P-388 cell proliferation at 500 and 1000 μg/ml. Only 10% cells were viable at 1000 μg/ml. Matrigel invasion was significantly inhibited in a dose dependent manner with virtually total inhibition at 1000 μg/ml. Cell morphological features notably changed with dose increase to 1000 μg/ml. Analysis of apoptotic cells on live green caspase kit exhibited gradual increase with the increasing dose of the nutrient mixture, and at 1000 μg/ ml 92% of P-388 cells were in late apoptosis. Tumors in the group of mice supplemented with the nutrient mixture had 50% lower weight compared to the tumors in control group (p = 0.0105). Histopathologically, both the groups of tumors were similar, yet size of tumors in the group treated with the nutrient mixture was considerably smaller. Conclusion: These results indicate that the nutrient mixture exhibited significant action against multiple targets in P-388 leukemia and may have potential in human leukemia.
Source: Journal of Clinical Oncology 28:15s, 2010 (suppl; abstr 6522)
Affiliation: Mayo Clinic Rochester, Rochester, MN; Mayo Clinic Arizona, Scottsdale, AZ
Green tea has long been touted as a health promoting substance. In vitro testing and a phase I trial suggest the green tea extract epigallocatechin 3 gallate(EGCG) may have clinical efficacy for chronic lymphocytic leukemia (CLL) patients. To follow-up on these results, the authors performed a phase II trial of daily EGCG in patients with CLL. 42 eligible patients received EGCG 2000 mg twice daily for up to 6 months. Patients received a median of 6 cycles of treatment (range: 1-6). Accrual to the trial is complete. Thirty-one patients have completed study therapy while 11 patients have not yet completed treatment. The most common side effects included transaminitis, abdominal pain and nausea. Only 2/48 (4.8%) patients experienced grade 3 toxicity. One patient experienced an NCI WG* partial remission. Other signs of clinical activity were observed with 13 (31%) patients experiencing a sustained ≥ 20% reduction in ALC and 19 of 29 (66%) patients with palpable adenopathy experiencing at least a 50% reduction in the sum of the products of all nodal areas during treatment. Overall 28 (67%) patients fulfilled the criteria for a biologic response (JCO 27:3808) based on attaining either a sustained ≥ 20% decline in ALC and/or a ≥ 50% reduction in the sum of the products of all nodal areas at some point during the 6 months of active treatment. Conclusions: Daily oral EGCG in the Polyphenon E preparation was well tolerated by CLL patients in this phase II trial. Declines in ALC and/or lymphadenopathy were observed in the majority of patients. EGCG containing green tea extracts may have potential as disease stabilizing agents in patients with early-stage CLL.
*National Cancer Institute (NCI) Working Group (WG) Criteria
Source: Journal of Clinical Oncology 2009; 27(23): 3808–3814.
Affiliation: Department of Internal Medicine, Division of Hematology; Division of Biostatistics, and Departments of Immunology and Oncology, Mayo Clinical, Rochester, MN; and the Department of Chemical Biology, and the Ernest Mario School of Pharmacy Rutgers, the State University of New Jersey, Piscataway, NJ.
This study was done to define the optimal dose of Polyphenon E for chronic daily administration and tolerability in patients with chronic lymphocytic leukemia (CLL). Previously untreated patients with asymptomatic Rai stage 0 to II CLL were eligible for participation. Polyphenon E with a standardized dose of epigallocatechin-3-gallate (EGCG) was administered using the standard phase I design with three to six patients per dose level (range, 400 to 2,000 mg by mouth twice a day). Trough plasma EGCG levels were measured 1 month after initiation of therapy. Response was classified using the National Cancer Institute (NCI) Working Group (WG) Criteria. Thirty-three eligible patients were accrued to dose levels 1 to 8. The maximum-tolerated dose was not reached. The most common adverse effects included transaminitis (33%, all grade 1), abdominal pain (30% grade 1, 0% grade 2, and 3% grade 3), and nausea (39% grade 1 and 9% grade 2). One patient experienced an NCI WG partial remission. Other signs of clinical activity were also observed, with 11 patients (33%) having a sustained ≥ 20% reduction in absolute lymphocyte count (ALC) and 11 (92%) of 12 patients with palpable adenopathy experiencing at least a 50% reduction in the sum of the products of all nodal areas during treatment. Trough plasma EGCG levels after 1 month of treatment ranged from 2.9 to 3,974 ng/mL (median, 40.4 ng/mL). Conclusion: Daily oral EGCG in the Polyphenon E preparation was well tolerated by CLL patients in this phase I trial. Declines in ALC and/or lymphadenopathy were observed in the majority of patients. A phase II trial to evaluate efficacy using 2,000 mg twice a day began in November 2007.
S. Harakeh, M. Diab-Assaf, J.C. Khalife, K.A. Abu-el-Ardat, E. Baydoun, A. Niedzwiecki, M.E. El-Sabban, M. Rath
Anticancer Research 2007; 27: 289-298
Adult T-cell leukemia (ATL) is an acute malignancy of activated T-cells caused by the human T-cell lymphotrophic virus type-1 (HTLV-1). The effects of non-cytotoxic concentrations of ascorbic acid (AA) were evaluated against HTLV-1 positive and negative cells. The effect of AA on apoptosis and proliferation was evaluated by cell cycle analysis. The role of p53, p21 Bax and Bcl-2a on cell cycle modulation and apoptosis was also assessed. The anti-proliferative effects were tested by determining the changes in the expression of transforming growth factors (TGF-?, TGF-?1 and TGF-?2). Ascorbic acid was found to reduce the proliferation of cells and induce apoptosis by the modulation of p53, p21, Bcl-2 and Bax. In conclusion, the results of this study show the anitproliferative effects of AA against leukemic cells.
Apoptosis, HTLV-1, ascorbic acid, Bcl-2a, Bax
S. Harakeh, M. Diab-Assaf, K. Abu-El-Ardat, A. Niedzwiecki, M. Rath
Chemico-Biological Interactions 2006, 164: 102-114
The retrovirus human T-cell lymphotrophic virus type-1 (HTLV-1) causes adult T-cell leukemia (ATL), which remains with no cure. This study evaluates the effects of L-lysine on proliferation and induction of apoptosis using non-cytotoxic concentrations of the test compound against HTLV-1 positive and negative malignant cell lines. The anti-proliferative effect of lysine was established and confirmed by studying the effects of the test compound on the expression of TGF mRNA expression by RT-PCR. To investigate the effect of L-lysine on the induction of apoptosis, DNA flow sytometry analyses was done and the results verified by cell death ELISA. The results indicated that a significant increase in the preG1 phase and a decrease in the s phase of the cell cycle in all of the ATL cells tested. L-lysine up-regulated p53, p21, and Bax protein levels and a down-regulation of Bel-2a in all the cell lines tested. L-lysine was found to exert its effect through the NF-kB pathway by inhibiting the p65 subunit specifically. Also L-lysine caused a decrease in the levels MMP-2 and MMP-9 as well as their enzymatic activity.
Apoptosis, HTLV-1, L-lysine, Adult T-cell leukemia
S. Harakeh, M. Diab-Assaf, A. Niedzwiecki, J. Khalife, K. Abu-El-Ardat, M. Rath
Leukemia Research 2006, 30: 869-881
Human T-cell lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T-cell leukemia, a fatal disease with average survival time of less than one year. A novel nutrient formulation, Nutrient Synergy (NS), which contains lysine, proline, ascorbic acid, and green tea extract, has been shown to be effective in inhibiting cell invasion, proliferation and angiogenesis of various solid cancers.
The effects of Nutrient Synergy were evaluated on proliferation (by MTT assay), expression of TGF mRNA (by RT-PCR) and induction of apoptosis (by flow cytometry and ELISA based apoptosis assays) using non-cytotoxic concentrations against HTLV-1 positive (HuT-102 & C91-PL) and negative (CEM & Jurkat) cells.
NS showed anti-proliferative effect as determined by MTT assay and TGF mRNA protein expression using RT-PCR. NS resulted in the down-regulation of TGF-alpha and an up-regulation in TGF-beta2. NS caused a significant increase in apoptotic cells in the preG(1) phase. These results were confirmed using Cell Death ELISA and Annexin V-FITC. Induction of apoptosis was caused by an up-regulation of p53, p21 and Bax protein levels and a down-regulation of Bcl-2alpha protein expression level.
The relatively non-toxic Nutrient Synergy demonstrated significant anti-proliferative and pro-apoptotic effects in tested HTLV-1 positive and negative adult T-cell leukemia cell lines, warranting further in vivo investigation.
Harakeh S, Abu-El-Ardat K, Diab-Assaf M, Niedzwiecki A, El-Sabban M, Rath M
Medical Oncology 2008; 25 (1): 30-39
The objective of this study is to evaluate the efficacy of epigallocatechin gallate against ATL cells. The anti-proliferative and pro-apoptotic effects of EGCG were evaluated in HTLV-1-positive and -negative cells. EGCG exhibited a marked decrease in proliferation of ATL cells at 96 h of treatment. The results indicated that TGF-alpha was down regulated whereas levels of TGF-beta2 increased. Cell cycle distribution analysis revealed an increase in cells in the pre-G(1) phase which was confirmed by ELISA. The results on proteins showed an up-regulation of p53, Bax and p21 protein levels while the levels of Bcl-2alpha were down regulated.
The full study is available online at:
M.W. Roomi, T. Kalinovsky, N.W. Roomi, M. Rath and A. Niedzwiecki
Dr. Rath Research Institute, Cancer Division, Santa Clara, CA, USA
Experimental Oncology 2013; 35(3): 180-186
A nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract has exhibited anticancer activity in vitro and in vivo in a number of cancer cell lines. We investigated the effect of NM on human leukemic myeloid U-937 cells in vitro by measuring: cell proliferation, MMP expression, invasion, apoptosis, and COX-2 and COX-1 protein expression. Human leukemic cell line U-937 (ATCC) was cultured in RPMI medium supplemented with fetal bovine serum and antibiotics. After 24 h, the cells were treated with NM at 0, 50, 100, 250, 500 and 1000 ?g/ml, in triplicate at each dose. Phorbol 12-myristate 13-acetate (PMA), 100 ng/ml was added to cells to induce MMP-9 secretion. Cell proliferation was evaluated by MTT assay, MMP expression by gelatinase zymography, invasion through Matrigel, apoptosis by using live green caspase detection kit (Molecular Probe), and COX-2 and COX-1 expression by Western blot. NM had no effect on U-937 cell growth at a concentration of 250 ?g/ml and exhibited an antiproliferative effect at 500 ?g/ml concentration. Zymography did not demonstrate MMP-2 or MMP-9 secretion in normal cells; however, PMA strongly induced MMP-9, which was inhibited by NM in a dose-dependent manner. Cell penetration through Matrigel was significantly reduced (by 95%) at 250 ?g/ml NM and completely blocked at 500 ?g/ml NM. NM induced slight apoptosis at 100 ?g/ml and moderate at 500 and 1000 ?g/ml concentration. NM inhibited COX-2 expression in a dose-dependent fashion and had no effect on COX-1 expression. Our results suggest that NM has potent inhibitory effects on U-937 cell growth and expression of inflammatory mediators, significant parameters in AML progression.
Antitumor and anti-inflammatory effect of nutrients on U-937 cells
human leukemic myeloid cell line U-937, nutrient mixture, MMP-9, Matrigel invasion, COX-2, apoptosis