Abstract

1. BT474 MCF10A UT A3 BN108 UT A3 BN108 BT474 MCF10A Hrs: 0 1 2 4 8 16 0 1 2 4 8 16 REDD1 Hrs: 0 4 8 16 C-myc MYC 231-vec TspA3 BN108 pAKT 231-REDD1 KD(D-1) Id-1 Hrs: 0 1 2 4 8 16 1 2 4 8 16 AKT Phospho eIF2a pS6 Results S6 P-4eBP1 eIF2a 4eBP1 Bim Id-1 GAPDH GAPDH Timosaponin A3 is a steroidal saponin from Anemarrhena asphodeloides that has a selective cytotoxic activity towards cancer cellsFrank King, Sylvia Fong, Isaac Cohen, Emma ShtivelmanBioNovo, Inc, Emeryville, CA Abstract BN108 and TspA3 produce similar changes in expression and activity of certain cellular proteins Inhibition of caspase 4 partially protects from BN108-induced cell death Silencing of REDD1 has no effect on inhibition of mTORC by TspA3 and does not protect cells from death induction by TspA3 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. Breast cancer cells sensitive to BN108 undergo apoptotic death confirmed by Annexin V staining, caspase activation, cleavage of PARP and DNA fragmentation. Caspases 4 and 9 which are involved in apoptosis induced by endoplasmic reticulum stress are activated by BN108. Inhibition of caspase 4 significantly inhibits BN108 induced death. Timosaponin A3, a component of BN108 recapitulates the cytotoxicity and other effects of BN108 TspA3 (A3) and BN108 induce same changes in expression/activation of a proteins in cancer but do not induce these changes in normal cells. Stress-response protein REDD1 and pro-apoptotic protein Bim are induced; expression of MYC is suppressed. Activity of Akt and mTORC are inhibited in breast cancer cells. None of these proteins are affected in normal immortalized MCF10A cells. REDD1 is a negative regulator of mTORC; it appeared to be a good target to silence in order to prevent inhibition of mTORC by A3 and BN108. REDD1 was targeted in BT474 and MDA MB 231 cells with REDD1 siRNA lentiviruses. Inhibition of REDD1 expression did not prevent inactivation of mTORC In TspA3 treated cells (not shown). Treatment of BT474 or MM231 cells with knockdown of REDD1 did not protect them from TspA3 cytotoxicity Timosaponin A3 is a steroidal saponin found in BN108 extract. TspA3 appears to be the compound responsible for the cytotoxic effect of BN108 based on the following: TspA3 is selectively cytotoxic to same cancer cell lines as BN108 and is not cytotoxic to normal cells; expression array analysis of gene expression changes induced by BN108 and TspA3 largely overlap; cytotoxicity of TSpA3 could be partially inhibited in presence of caspase 4 inhibitor; time course of changes in expression of important signaling proteins is similar in cells treated withTspA3 and BN108 Summary • BN108 induces cell death selectively in cancer cells but not in normal cells. • Timosaponin A3 (TspA3) was identified as an active compound from BN108 responsible for the selective cytotoxic activity of BN108. • BN108/TspA3 do not induce generation of reactive oxygen species, DNA damage or mitochondrial disfunction. • Induction of ER stress is a likely mechanism of cytotoxicity of BN108/TspA3 as seen from the involvement of caspase 4 and inactivation of eIF2a kinase, as well as induction of some genes implicated in ER stress (GRP78, ATF4 and phosphorylation of peIF2a). • BN108/TspA3 induce rapid inactivation of AKT and mTORC selectively in breast cancer cells, which is most likely contibutes to cell death. • However, silencing of REDD1, an inhibitor of mTORC activity, does not protect cells from TspA3/BN108 induced death • TspA3 is active against breast cancer cells in low micromolar range. This, and its selectivity towards cancer cells make it a promising candidate drug BN108 induces apoptosis selectively in cancer cell lines Genes induced or repressed in both MDA MB231 and BT474 but not in MCF10A by BN108 and TspA3: Antiproliferative and pro-apoptotic, induced: -REDD1 (mTORC inhibitory), p21CIP, stratifin, cyclin G2, GDF15 ER stress response, induced: LRF, HEPRUD/HERP, TRIB3, GRP78 Pro-proliferative and/or anti-apoptotic, downregulated: -Id1, Id3, Myc, Snai3, CYR61 Enzymes in cholesterol biosynthesis pathway (particularly in BT474) Pathways affected: Akt, mTORC (inhibited), SREBP2 and cholesterol biosynthesis (moderately activated) Time course of changes induced by TspA3 versus BN108 in BT474 cells: practically identical BN108 induces apoptotic death in cancer cell lines but not in non-transformed cell lines and cells. Tumor and non-transformed cell lines and cells were treated with BN108 at 0.5 mg/ml for 24 hours. The chart shows percentage of cells that were binding annexin V. TspA3 inhibits expression C-myc and Id-1 proteins in cancer cells but not in normal cells. Printed by