Abstract
The aqueous extract of Anemarrhena asphodeloides (BN108) induces apoptosis in various cancer cell lines but is significantly less cytotoxic in non-transformed cells. Chemical fractionation of BN108 showed that its cytotoxicity is associated with timosaponins, steroidal saponins of coprostane type. Timosaponin BII (TBII) is a major saponin in BN108, but it shows little cytotoxicity. A much less abundant TAIII induces cell death in tumor cells but not in normal cells, reproducing the selectivity of the total extract BN108. Glycosidase treatment, by removing the extra sugar moiety in TBII, converts it to TAIII and confers cytotoxic activity. Analysis of the mechanisms of death induced by TAIII revealed activation of two distinct pro-apoptotic pathways: first, inhibition of mTORC1 manifested in much reduced phosphorylation of mTORC1 targets; second, induction of endoplasmic reticulum stress culminating in phosphorylation of eIF2α and activation of caspase 4. These pro-apoptotic pathways are activated by TAIII selectively in tumor cells but not in normal cells. Both pathways play a causative role in TAIII cytotoxicity, as restoration of either mTOR activity or relief of ER stress alone offer only partial protection from TAIII. Inhibition of mTORC1 and induction of ER stress apparently contribute to the induction of the previously reported autophagic response in TAIII-treated cells. TAIII induced autophagy plays a protective role in TAIII induced death signaling, and failure to mount autophagic response is associated with heightened sensitivity to TAIII induced apoptosis. The multiple death-promoting and apparently tumor-selective responses to TAIII, its ability to inhibit mTORC1, and the possibility of further enhancing its cytotoxicity by pharmacological inhibition of autophagy, make TAIII an attractive candidate for development as a cancer therapeutic agent.
Highlights
This work describes the anti-tumor activity of the aqueous extract from the plant Anemarrhena asphodeloides (BN108), and one of the timosaponins present in the extract, TAIII [1]
To verify the apoptotic nature of cell death induced by BN108, we have examined caspase activation in breast cancer cell line BT474 and primary fibroblasts IMR90 as well as immortalized mammary epithelium MCF10A
This conclusion is based on several lines of evidence: TAIII demonstrates the same selectivity towards breast cancer cell lines as BN108; TAIII induces apoptosis involving caspase-4 activation similar to BN108; TAIII induces transcriptional changes in breast cancer cells that largely overlap with these induced by BN108; both TAIII and BN108 inhibit mTORC1 in cancer cells and induce protective autophagy
Summary
This work describes the anti-tumor activity of the aqueous extract from the plant Anemarrhena asphodeloides (BN108), and one of the timosaponins present in the extract, TAIII [1]. TAIII was recently reported to induce apoptosis and protective autophagy in HeLa cells [4]. The mechanism through which TAIII induces cell death remains unclear. Some of them induce apoptosis through a mitochondrial pathway, but some, most notably the proteasome inhibitors, induce cell death via endoplasmic reticulum (ER) stress mediated apoptotic pathway. ER stress is elicited by a wide variety of conditions including nutrient deprivation, impaired protein degradation or secretion, calcium imbalance and many others. ER stress involves specific transcriptional and translational responses that are largely controlled by three ER resident sensor proteins: IRE1, ATF6, and PERK (reviewed in [5,6]). Activated PERK phosphorylates eukaryotic translation initiation factor eIF2a, resulting in the general inhibition of protein synthesis, but paradoxically induces a specific increase in translation of transcription factor ATF4. Sustained or severe ER stress leads to activation of caspases, in particular caspase-4, followed by apoptosis [7]
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