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