withanolides and Colorectal-Neoplasms

Withaferin A (WFA), a withanolide, is isolated from plants of Withania somnifera (L.) Dual (Solanaceae), known as Indian ginseng, Indian winter cherry or Ashwagandha. It has been reported to exert multifaceted anti-neoplastic effects. Here, we analyzed the impact of WFA on apoptosis and autophagy activation in different human colorectal cancer cell lines. We observed that WFA exposure caused an increased aggregation of cells in the subG1 arrest in cell cycle, and increased the number of late apoptotic cells. WFA also induced the apoptosis via PARP and caspase-3 cleavage accompanied with suppression of levels of anti-apoptotic proteins like Bcl-2 and Bcl-xl. The influence of WFA on autophagy was validated by acridine orange, MDC staining, and immunocytochemistry of LC3. It was found that 24 h treatment of WFA increased the acridine and MDC stained autophagosome with induced the LC3 and other autophagy markers Atg7 and beclin-1 activation. We used Z-DEVD-FMK, a caspase-3 blocker, and 3-MA, an autophagy inhibitor, to confirm whether these effects were specific to apoptosis and autophagy, and observed the recovery of both these processes upon exposure to WFA. Moreover, the activation of β-catenin protein was attenuated by WFA. Interestingly, small interfering RNA (siRNA)-promoted β-catenin knockdown augmented the WFA-induced active form of p-GSK-3β, and stimulated autophagy and apoptosis through PARP and LC3 activation. These findings suggested that WFA could stimulate activation of both apoptosis and autophagy process via modulating β-catenin pathway.

Topics: Apoptosis; Autophagy; beta Catenin; Caspase 3; Cell Line; Cell Line, Tumor; Colorectal Neoplasms; Glycogen Synthase Kinase 3 beta; Humans; Poly(ADP-ribose) Polymerase Inhibitors; Withanolides

Withaferin A (WA) is an active steroidal lactone derived from the herbal plant Withania somnifera, which exhibits antitumor activity with reactive oxygen species (ROS) modulating in a variety of cancer models, such as breast cancer, lung cancer and pancreatic cancer. However, to the best of our knowledge, the direct effect and mechanism of WA on CRC cells has not been previously determined. The present study investigated the anti-tumor effects of WA on CRC cells in vitro, and explored the mechanisms of action. The flow cytometry was applied for detecting the accumulation of ROS with the treatment of withaferin A. We performed the flow cytometry and western blot to evaluate the withaferin A induced apoptosis and cell cycle arrest in human colon cancer cells. And to verify the ROS accumulation induced mitochondrial dysfunction after the treatment of withaferin A, fluorescence microscope and western blot were applied. WA exerted a dose-dependent cytotoxic effect on HCT-116 and RKO cells. The effect was associated with ROS-mediated cell cycle arrest and the expression of apoptotic proteins. In addition, WA promoted ROS production and decreased mitochondrial membrane potential accompanying with mitochondrial dysfunction. Taken together, these results strongly indicated that WA directly inhibits cell growth and induces apoptosis in CRC cells through ROS-mediated mitochondrial dysfunction and JNKs pathway, and WA may be a promising potential candidate for therapeutic application of CRC.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Line, Tumor; Colorectal Neoplasms; Humans; Membrane Potential, Mitochondrial; Mitochondria; Oxidative Stress; Reactive Oxygen Species; Withania; Withanolides

There are increasing evidences of proinflammatory cytokine involvement in cancer development. Here, we found that two cytokines, IL-6 and TNF-

Topics: Blotting, Western; Chromatin Immunoprecipitation; Colorectal Neoplasms; HT29 Cells; Humans; Immunoprecipitation; Interleukin-6; NF-kappa B; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Telomerase; Tumor Necrosis Factor-alpha; Withanolides

The oncogenic activation of AKT gene has emerged as a key determinant of the aggressiveness of colorectal cancer (CRC); hence, research has focused on targeting AKT signaling for the treatment of advanced stages of CRC. In this study, we explored the anti-tumorigenic effects of withaferin A (WA) on CRC cells overexpressing AKT in preclinical (in vitro and in vivo) models. Our results indicated that WA, a natural compound, resulted in significant inhibition of AKT activity and led to the inhibition of cell proliferation, migration and invasion by downregulating the epithelial to mesenchymal transition (EMT) markers in CRC cells overexpressing AKT. The oral administration of WA significantly suppressed AKT-induced aggressive tumor growth in a xenograft model. Molecular analysis revealed that the decreased expression of AKT and its downstream pro-survival signaling molecules may be responsible for tumor inhibition. Further, significant inhibition of some important EMT markers, i.e., Snail, Slug, β-catenin and vimentin, was observed in WA-treated human CRC cells overexpressing AKT. Significant inhibition of micro-vessel formation and the length of vessels were evident in WA-treated tumors, which correlated with a low expression of the angiogenic marker RETIC. In conclusion, the present study emphasizes the crucial role of AKT activation in inducing cell proliferation, angiogenesis and EMT in CRC cells and suggests that WA may overcome AKT-induced cell proliferation and tumor growth in CRC.

Topics: Animals; Apoptosis; Cell Proliferation; Colorectal Neoplasms; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Cells, Cultured; Withanolides; Xenograft Model Antitumor Assays

Withania somnifera L. Dunal (Ashwagandha) is used over centuries in the ayurvedic medicines in India. Withaferin A, a withanolide, is the major compound present in leaf extract of the plant which shows anticancer activity against leukemia, breast cancer and colorectal cancer. It arrests the ovarian cancer cells in the G2/M phase in dose dependent manner. In the current study we show the effect of Withaferin A on cell cycle regulation of colorectal cancer cell lines HCT116 and SW480 and its effect on cell fate. Treatment of these cells with this compound leads to apoptosis in a dose dependent manner. It causes the G2/M arrest in both the cell lines. We show that Withaferin A (WA) causes mitotic delay by blocking Spindle assembly checkpoint (SAC) function. Apoptosis induced by Withaferin A is associated with proteasomal degradation of Mad2 and Cdc20, an important constituent of the Spindle Checkpoint Complex. Further overexpression of Mad2 partially rescues the deleterious effect of WA by restoring proper anaphase initiation and keeping more number of cells viable. We hypothesize that Withaferin A kills cancer cells by delaying the mitotic exit followed by inducing chromosome instability.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cdc20 Proteins; Cell Line, Tumor; Chromosome Aberrations; Colorectal Neoplasms; G2 Phase Cell Cycle Checkpoints; HCT116 Cells; Humans; M Phase Cell Cycle Checkpoints; Mad2 Proteins; Proteasome Endopeptidase Complex; Spindle Apparatus; Withanolides