M.W. Roomi, V. Ivanov, T. Kalinovsky, A. Niedzwiecki, M. Rath
Oncology Reports 2006; 16(5): 943-947.
Five-year survival is limited to 60% in renal cancer patients at diagnosis. Due to the cancer’s resistance to conventional treatments and associated high morbidity, we investigated the antimetastatic effects of a specific nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on human renal adenocarcinoma cell line 786-0 by measuring: cell proliferation, modulation of MMP-2 and –9 secretion, and cancer cell invasive potential.
Human renal cancer cell line 786-0 (ATCC) was grown in RPMI medium in 24-well tissue culture plates. At near confluence, the cells were treated with NM, dissolved in media, and tested at 0, 10, 50, 100, 500, and 1000 mg/ml in triplicate at each dose. Cells were also treated with PMA 200 ng/ml to study enhanced MMP-9 activity. Cell proliferation was evaluated by MTT assay, MMP secretion by gelatinase zymography, and invasion through Matrigel.
Zymography demonstrated MMP-2 and MMP-9 secretion by uninduced renal cancer cells with enhanced MMP-9 induced by PMA (200 ng/ml) treatment. NM inhibited the secretion of both MMPs in a dose-dependent fashion with virtual total inhibition of MMP-2 at 500-µg/ml concentration and MMP-9 at 100 µg/ml. The invasion of renal cancer cells through Matrigel was totally inhibited (p=0.0001) by NM at 1000 µg/ml concentration.
Our results support a potential role for the nutrient mixture tested in the treatment of renal cell carcinoma, by inhibition of MMP-2 and MMP-9 secretion and invasion.
M.W. Roomi, V. Ivanonv, T. Kalinovsky, A. Niedzwiecki, M. Rath
International Journal of Urology 2006, 13(4): 415-419.
Bladder cancer, the fourth incident cancer in men and tenth in women, is associated with a high rate of recurrence, even when treated in situ, and prognosis is poor once the cancer metastasizes to distant sites. Based on anticancer properties, we investigated the effect of a mixture of lysine, proline, arginine, ascorbic acid, and green tea extract on human bladder cancer cells T-24 by measuring: proliferation, MMP expression, and cancer cell invasive potential.
Human bladder cancer cells T-24 (ATCC) were grown in McCoy medium supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 mg/ml) in 24-well tissue culture plates. At near confluence, the cells were treated with the nutrient mixture (NM) dissolved in media and tested at 0, 10, 50, 100, 500, and 1000 mg/ml in triplicate at each dose. Cells were also treated with PMA 200 ng/ml to study enhanced MMP-9 activity. Cell proliferation was evaluated by MTT assay, MMP activity by gelatinase zymography, and invasion through Matrigel.
NM inhibited the T-24 cell secretion of MMP-2 and 9, with virtual total inhibition of MMP-2 at 500 mg/ml and MMP-9 at 100 µg/ml concentration. The nutrient mixture significantly reduced the invasion of human bladder cancer cells T-24 through Matrigel in a dose-dependent fashion, with 95% inhibition at 500 µg/ml and 100% at 1000 µg/ml NM (p<0.001).
Our results suggest that NM is an excellent candidate for therapeutic use in the treatment of bladder cancer, by inhibiting critical steps in cancer development and spread, such as MMP secretion and invasion.
M.W. Roomi, N. Roomi, V. Ivanov,T. Kalinovsky, A. Niedzwiecki, M. Rath
Medical Oncology 2006, 23(2): 245-250
A hallmark of renal cell carcinoma RCC) invasion is its ability to degrade ECM by local production of gelatinase enzymes. Although many studies on RCC have demonstrated the importance of MMPs, very little information is currently known regarding the effect of inducers and inhibitors. We therefore investigated the effect of inducers and inhibitors on RCC 786-0 in vitro.
Human RCC 786-0 ATCC) was grown in RPMI medium supplemented with 10% FBS, penicillin, and streptomycin in 24-well tissue plates. At near confluence, the cells were washed with PBS; the serum free medium was incubated with various inducers: phorbol ester (PMA; tumor necrosis factor alpha (TNF?); interleukin 1-beta ( IL-1ß); and lipopolysaccharides (LPS). Cells were also incubated with inhibitors: EGCG; doxycycline; a nutrient mixture with and without PMA; retinoic acid; dexamethasone; H-7; actinomcin D; and cyclohexamide. After 24 hours, the medium was removed and analyzed for MMP-2 and MMP-9 by gelatinase zymography.
RCC 786-0 secreted two bands, a major band corresponding to MMP-2 and a faint band corresponding to MMP-9. PMA and TNF-?, with increased concentration, increased MMP-9 secretion, while IL-1ß and LPS did not significantly modify MMP-9 activity. MMP-2 secretion was not affected by any of the inducers. All the inhibitors tested without and with PMA showed a dose-dependent decrease in both MMP-2 and -9 expression.
In conclusion, this study has shown that exposure of the highly metastatic renal cell carcinoma cell line 786-0 to different growth factors increases secretion of MMP-9 but not MMP-2; MMP-2 and-9 activity was decreased by exposure to inhibitors. Further studies are in progress to confirm the role of MMP-9 on Matrigel invasion using PMA, cytokines and LPS.
M.W. Roomi, T. Kalinovsky, A. Niedzwiecki and M. Rath
Bladder Cancer: Etymology, Diagnosis and Treatments, ed. W.E. Nilsson, Nova Science Publishers, Inc, 2010, Ch. 12, pp. 229-243
Consumption of a plant-based diet has been associated with prevention of the development and progression of cancer. We have developed strategies to inhibit cancer by increasing the stability and integrity of connective tissue as the common mechanisms used by all types of cancer cells for their development and spread. This can be achieved naturally through the synergistic effects of selected nutrients, such as lysine, proline, ascorbic acid and green tea extract (NM). This micronutrient mixture has exhibited anticancer activity in vivo and in vitro in a large variety of cancer cell lines by simultaneously affecting several key mechanisms involved in cancer. Among them it was effective in inhibition of cancer cell growth, MMP secretion, invasion and metastasis. It inhibited cellular MMP secretion and had anti-angiogenic and pro-apoptotic effects. We investigated the effect of NM on bladder cancer, which is associated with a high rate of recurrence, even when treated in situ, and poor prognosis once the cancer has metastasized. The effect of NM on human bladder cancer cells T-24 was studied in vitro by measuring: cell proliferation, MMP expression, Matrigel invasion, cell migration, apoptosis, and inflammatory protein expression Cox-2 and iNOS. Human bladder cancer cells T-24 (ATCC) were grown in McCoy medium supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 mg/ml) in 24-well tissue culture plates. At near confluence, the cells were treated with NM dissolved in media and tested at 0, 10, 100, 500, and 1000 µg/ml in triplicate at each dose. Cells were also treated with PMA 200 ng/ml to study enhanced expression of MMP-9. Cell proliferation was evaluated by MTT assay, MMP expression by gelatinase zymography, migration by scratch test, invasion through Matrigel, morphology by H&E staining, apoptosis by live-green caspase, and Cox-2 and iNOS by Western blot. NM showed no significant antiproliferative effect on human bladder cancer cell growth but induced apoptosis in a dose-dependent manner. NM inhibited the T-24 cell expression of MMP-2 and –9 in a dose-dependent fashion, with virtual total inhibition of MMP-2 at 500 µg/ml and MMP-9 at 100 µg/ml concentration. The nutrient mixture significantly reduced cell migration and the invasion of human bladder cancer cells T-24 through Matrigel in a dose-dependent fashion, with 95% inhibition at 100 µg/ml and 100% at 500 µg/ml NM (p<0.001). Cox-2 and iNOS expression by T-24 cells were also inhibited in a dose-dependent manner. Our results suggest that NM is an excellent candidate for therapeutic use in the treatment of bladder cancer, by inhibiting critical steps in cancer development and spread, such as MMP expression, cell migration, and invasion and by inducing apoptosis.
bladder cancer cell line T-24; nutrient mixture; MMP expression; Matrigel invasion; apoptosis; Cox-2
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