BACKGROUND:The pro-apoptotic protein CC3/TIP30 has an unusual cellular function as an inhibitor of nucleocytoplasmic transport. This function is likely to be activated under conditions of stress. A number of studies support the notion that CC3 acts as a tumor and metastasis suppressor in various types of cancer. The yeast homolog of CC3 is likely to be involved in responses to DNA damage. Here we examined the potential role of CC3 in regulation of cellular responses to genotoxic stress.RESULTS:We found that forced expression of CC3 in CC3-negative cells strongly delays the repair of UV-induced DNA damage. Exogenously introduced CC3 negatively affects expression levels of DDB2/XPE and p21CIP1, and inhibits induction of c-FOS after UV exposure. In addition, exogenous CC3 prevents the nuclear accumulation of P21CIP in response to UV. These changes in the levels/localization of relevant proteins resulting from the enforced expression of CC3 are likely to contribute to the observed delay in DNA damage repair. Silencing of CC3 in CC3-positive cells has a modest delaying effect on repair of the UV induced damage, but has a much more significant negative affect on the translesion DNA synthesis after UV exposure. This could be related to the higher expression levels and increased nuclear localization of p21CIP1 in cells where expression of CC3 is silenced. Expression of CC3 also inhibits repair of oxidative DNA damage and leads to a decrease in levels of nucleoredoxin, that could contribute to the reduced viability of CC3 expressing cells after oxidative insult.CONCLUSIONS:Manipulation of the cellular levels of CC3 alters expression levels and/or subcellular localization of proteins that exhibit nucleocytoplasmic shuttling. This results in altered responses to genotoxic stress and adversely affects DNA damage repair by affecting the recruitment of adequate amounts of required proteins to proper cellular compartments. Excess of cellular CC3 has a significant negative effect on DNA repair after UV and oxidant exposure, while silencing of endogenous CC3 slightly delays repair of UV-induced damage.

Botanical medicine is one of the most popular complementary medical approaches, and herbal therapies are frequently sought and used by breast cancer patients. However, the molecular mechanisms through which certain herbal extracts exert growth inhibitory activity on breast cancer cells remain largely unknown. BN108, aqueous extract of Anemarrhena asphodeloides Bunge, induces cell death selectively in breast cancer lines and tumor cells of various origins but not in normal mammary epithelial cells and fibroblasts. Breast cancer cells sensitive to BN108 undergo apoptotic death, confirmed by Annexin V staining, caspase activation, cleavage of PARP and DNA fragmentation. In particular, caspases 4 and 9, whose activation is observed during endoplasmic reticulum (ER) stress induced apoptosis, are proteolytically activated. Inhibition of caspase 4 partially protects breast cancer cells from death induced by BN108. Expression array analysis of cells treated with BN108 shows induction of expression of several known pro-apoptotic and anti-proliferative genes such as REDD1, p21CIP, cyclin G2, stratifin and more. None are affected in normal mammary cells. BN108 induces rapid inactivation of AKT and mTOR kinases in breast cancer but not in non-transformed cells. The well-defined targets of mTORC1, S6kinase, S6 ribosomal protein and 4eBP1 are inactivated in BN108 treated cells. Expression array analysis also shows the induction of numerous genes encoding enzymes within the cholesterol synthesis pathway by BN108. They are induced to various degrees in all cell lines examined. However, the changes in total cholesterol levels are relatively minor in treated cells. In addition, BN108 also induces pattern of gene expression consistent with ER stress. We have identified timosaponin A3 (TspA3) as an active compound from BN108 that is responsible for the selective cytotoxicity for the whole extract. TspA3 is a steroidal saponin whose activity against cancer cells remained unexplored until now. Treatment with purified TspA3 at concentrations similar to those in the BN108 extract induces apoptosis in breast cancer cells but not in normal cells. TspA3 and BN108 induce largely overlapping transcriptional changes in cells. Similar to BN108, TspA3 inactivates major signaling pathways for growth and survival selectively in cancer cells (Akt and mTORC) and induces expression of proteins involved in cholesterol biosynthesis pathway and ER stress response. In conclusion, a component of BN108 extract, TspA3 is selectively cytotoxic for cancer versus normal cells. The selective cytotoxic properties of TspA3 could be related to the inhibition of major oncogenic pathways and induction of ER stress. Future studies will be aimed at understanding the relationship between the effect of TspA3 on these pathways and induction of apoptosis, which may give rise to a unique pathway for targeting tumor cells. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2666.

Abstract Abstract #3018 We present preclinical data on the potential mechanisms of the cytotoxic effect of the herbal extract BN108 towards cancer cells. Botanical medicine is one of the most popular complementary medical approaches, and herbal therapies are frequently sought and used by breast cancer patients. However, the molecular mechanisms through which certain herbal extracts exert growth inhibitory activity on breast cancer cells remain largely unknown. Treatment of a panel of breast cancer cell lines and normal mammary cells with BN108 induced cell death selectively in breast cancer lines. Normal mammary epithelial cells and fibroblasts were resistant to the cytotoxic effects of BN108. Breast cancer cells sensitive to BN108 underwent apoptotic death confirmed by Annexin V staining, caspase activation, cleavage of PARP and DNA fragmentation. In particular, caspases 4 and 9, whose activation is observed during apoptosis induced by endoplasmic reticulum stress, were proteolytically activated. Inhibition of caspase 4 partially protected breast cancer cells from cell death induced by BN108. Expression array analysis of cells treated with BN108 showed induction of expression of several known pro-apoptotic and anti-proliferative genes such as REDD1, p21CIP, cyclin G2, stratifin and more. BN108 induced rapid inactivation of AKT and mTOR kinases in breast cancer but not in non-transformed cells. Moreover, the well-defined targets of mTORC1, S6kinase and 4eBP1 were inactivated in BN108 treated cells. The expression array analysis also showed the induction by BN108 of numerous genes whose products code for enzymes within the cholesterol synthesis pathway. Interestingly, a transient increase in cholesterol synthesis was seen in breast cancer cells but not in normal cells following BN108 treatment. In general, cholesterol levels are higher in breast cancer cells compared to normal cells. Steroidal saponins are major components of BN108 extract, and they are known to deplete cell membrane cholesterol. Similar to BN108 extract, treatment with purified timosaponin AIII (a component of BN108 extract) also induced apoptosis and changes in cholesterol production in breast cancer cells but not in normal cells. It is possible that steroidal saponins induce a very transient depletion of cholesterol, followed by inactivation of AKT and mTOR, and negative feedback inhibition of the cholesterol synthesis pathway that is, in the end, is futile. None of these events are observed in normal cells. In conclusion, BN108 extract contains cytotoxic activity selective for transformed versus normal cells. These selective cytotoxic properties of BN108 could be related to its differential effects on cholesterol synthesis in breast cancer cells versus normal cells, as well as inhibition of major oncogenic pathways. Future studies will be aimed at understanding the molecular relationship between the BN1008 effect on cholesterol synthesis and induction of apoptosis, which may give rise to a unique pathway for targeting tumor cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3018.