Epibrassinolide (EBR), a member of brassinostreoids plant hormones with cell proliferation promoting role in plants, is a natural polyhydroxysteroid with structural similarity to steroid hormones of vertebrates. EBR has antiproliferative and apoptosis-inducing effect in various cancer cells. Although EBR has been shown to affect survival and mitochondria-mediated apoptosis pathways in a p53-independent manner, the exact molecular targets of EBR are still under investigation. Our recent SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) data showed that the most significantly altered protein after EBR treatment was calreticulin (CALR). CALR, a chaperone localized in endoplasmic reticulum (ER) lumen, plays role in protein folding and buffering Ca ions. The alteration of CALR may cause ER stress and unfolded protein response correspondingly the induction of apoptosis. Unfolded proteins are conducted to 26S proteasomal degradation following ubiquitination. Our study revealed that EBR treatment caused ER stress and UPR by altering CALR expression causing caspase-dependent apoptosis in HCT 116, HT29, DLD-1, and SW480 colon cancer cells. Furthermore, 48 h EBR treatment did not caused UPR in Fetal Human Colon cells (FHC) and Mouse Embryonic Fibroblast cells (MEF). In addition our findings showed that HCT 116 colon cancer cells lacking Bax and Puma expression still undergo UPR and related apoptosis. CALR silencing and rapamycin co-treatment prevented EBR-induced UPR and apoptosis, whereas 26S proteasome inhibition further increased the effect of EBR in colon cancer cells. All these findings showed that EBR is an ER stress and apoptotic inducer in colon cancer cells without affecting non-malignant cells.
Epibrassinolide (EBR), a plant-derived polyhydroxylated derivative of 5α-cholestane, structurally shows similarities to animal steroid hormones. According to the present study, EBR treatment triggered a significant stress response via activating ER stress, autophagy, and apoptosis in cancer cells. EBR could also increase Akt phosphorylation in vitro. While the activation of Akt resulted in cellular metabolic activation in normal cells to proceed with cell survival, a rapid stress response was induced in cancer cells to reduce survival. Therefore, Akt as a mediator of cellular survival and death decision pathways is a crucial target in cancer cells. In this study, we determined that EBR induces stress responses through activating Akt, which reduced the mTOR complex I (mTORC1) activation in SW480 and DLD-1 colon cancer cells. As a consequence, EBR triggered macroautophagy and led to lipidation of LC3 most efficiently in SW480 cells. The cotreatment of spermidine (Spd) with EBR increased lipidation of LC3 synergistically in both cell lines. We also found that EBR promoted polyamine catabolism in SW480 cells. The retention of polyamine biosynthesis was remarkable following EBR treatment. We suggested that EBR-mediated Akt activation might determine the downstream cellular stress responses to induce autophagy related to polyamines.
Epibrassinolide (EBR) is a natural plant polyhydroxysteroid with structural similarity to steroid hormones. Lately we showed by SILAC assay that EBR treatment induces apoptosis by significantly altering the expressions of proteins having role in unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. ER stress level has been proposed as a critical step in cancer chemotherapy. Moderate ER stress was shown as an inducer of pro-survival machinery via autophagy, which is an important catabolic process that delivers cytoplasmic material to the lysosome for degradation. However chronic ER stress lead cells to apoptosis. EBR treatment induced ER stress in a time-dependent manner within 48 h in SW480 and DLD1 colon cancer cells. Downregulation of p62, LC3 and increase in ATGs expressions indicated that autophagy is induced in these cell lines after 12 h EBR exposure. The mammalian target of rapamycin (mTOR), a coordinator between nutritional stress and cellular growth machinery, is associated with ER stress. Our results indicated that short time EBR exposure induced mTOR expression accompanied by Ser2448 dephosphorylation. After 48 h EBR treatment, with prolonged ER stress, both cell lines undergo apoptosis. Therefore we conclude that time-dependent EBR treatment caused autophagy or apoptosis decision through ER stress in colon cancer cells.
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