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Introduction:

Despite numerous studies comparing different two and three drug combinations, efficacy levels for chemotherapy for patients with advanced non-small cell lung cancer( NSCLC) have reached a plateau and there is a need for new treatment strategies. Botanical drug products (BDPs) containing phenolic compounds with strong antioxidant property are presently undergoing evaluation as potential anti-neoplastic agents. We have elucidated the apoptotic effect of novel BDPs on several human lung cancer cell lines.

Methods

Lethal dose, 50% (LD 50) was determined by trypan blue viability assay from single exposure of the cells to each of the drugs for 48 hrs. Apoptosis induced by combinations of BDPs with conventional drugs, platinum and taxol (P/T) was examined. Percent apoptosis was measured by Flow Cytometry (FACS) using annexin-FITC assay. Effect on cell cycle was determined using propidium iodide (PI) staining analyzed by FACS. Proteins harvested from cancer cells were analyzed by Western Blot (WB) using anti caspase 3, caspase 8, caspase 9 and DR-4.

Results

Of the six BDPs that were tested, Imotin A and Phytoban were more cytotoxic to five lung cancer cell lines ( NCIH-1299, NCIH-23, MDR,NCIH-522 and BBM). Imotin A and Phytoban acted synergistically in inducing apoptosis. Western blot analysis demonstrated the increase in caspase 3 split products, up-regulation of caspase 8 and DR-4 in NCIH-23 and up-regulation of caspase 8 and caspase 9 in NCIH-1299. Pretreatment of NCIH-1299 cells with pan-caspase inhibitor (z-VAD-fmk) blocked the phytoban induced apoptosis by 30% suggesting that phytoban-induced apoptosis in NCIH-1299 cells is mediated by caspase -3-dependent pathway and possibly through a caspase-independent pathway as well. Combination of Imotin A alone and with platinum and taxol based chemotherapies showed an increase in apoptosis to about 15% and 36% Annexin V positive cells respectively. Imotin A combined with one half LD50 of P/T maintained similar cytotoxic effects on all tested cell lines as initial LD50 levels. PI staining followed by FACS analysis of cell cycle status showed cell accumulation in the G2-S phases in the presence of phytoban. The potentiation effects of the extracts when added to the P/T based chemotherapy combination appears to be due to cell cycle arrest in the G2-S phase, possibly through a mechanism other than the M-phase or G2 arrest brought about by Taxols and platinum drugs respectively.

Conclusions

Present results show that the BDPs can induce caspase dependent and caspase independent apoptosis in human lung cancer cell lines in-vitro. BDPs may potentially enhance the effects of conventional chemotherapy, which may be administered at lower dosages allowing for longer treatment periods and significantly reduce dosage-related side effects. These studies have identified drug combinations that may be used as adjuvants to conventional chemotherapeutic regimens.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA