Apoptosis In Colorectal HCT-116 Cells Research Articles

[Phosphorylation status of ASPP2 modulates p53 apoptotic function in oxaliplatin-induced apoptosis of colorectal cancer HCT116 cells].

To investigate the role of apoptosis stimulating protein 2 of p53 (ASPP2) phosphorylation status in the regulation of ASPP2-p53 apoptotic pathway activity. Cells were individually transfected with green fluorescent protein (GFP)-encoding vector, constitutively non-phosphorylatable ASPP2 mutant-ASPP2 (Am)-encoding vector, and wild type ASPP2 (Aw)-encoding vector) plasmids, respectively, to make them overexpressing phosphorylated and non-phosphorylated ASPP2 proteins, respectively. Cell apoptosis was induced by oxaliplatin. The apoptosis rate of cells was determined by flow cytometry after staining with FITC-conjugated annexin V and PI. ASPP2 protein level and its phosphorylation status were observed by Western blot. The interaction between ASPP2 and p53 was observed by immunoprecipitation assay. Oxaliplatin induced cell apoptosis and caused phosphorylation of ASPP2 at ser92/ser361 in the HCT116 cells. The apoptosis rate of Aw and Am plasmids-transfected cells were (3.8 ± 1.0)% and (3.9 ± 1.2)% respectively, statistically with a non-significant difference (P > 0.05) in comparison with that of the GFP plasmid-transfected cells [(4.0 ± 0.8)%]. After oxaliplatin treatment, the apoptosis rate of Aw plasmid-transfected cells was (46.7 ± 3.9)%, significantly higher than that of the Am and GFP plasmid-transfected cells [(40.1 ± 10.2)% and (37.1 ± 6.9)%, respectively, P < 0.05], however, there was no statistically significant difference (P > 0.05) between Am and GFP plasmid-transfected cells. These results indicate that phosphorylated ASPP2 promoted the oxaliplatin-induced apoptosis of HCT116 cells through a p53-dependent pathway. Phosphorylation status of ASPP2 influenced its binding activity to p53. Phosphorylation status of ASPP2 modulates p53 apoptotic function in oxaliplatin-induced apoptosis of colorectal cancer HCT116 cells.

ASK1-JNK signaling cascade mediates Ad-ST13-induced apoptosis in colorectal HCT116 cells

ST13, a co-factor of heat shock protein, has shown potential antitumor efficacy for colorectal cancer in our previous study. However, the molecular mechanisms governing ST13-induced apoptosis are poorly understood. Here, we demonstrate that Ad-ST13 (ST13 mediated by adenovirus) activates apoptosis signal-regulated kinase (ASK1) and c-Jun N-terminal kinase (JNK) but not p38 (mitogen-activated protein kinase) in human colorectal HCT116 cells. Ad-ST13 also increases extracellular-regulated kinase (ERK) phosphorylation levels, but the change is due to adenovirus replication. Overexpression of ST13 also increases the transcription activity of AP-1. Blocking ASK1-JNK pathway affects Ad-ST13-mediated colorectal cell apoptosis, decreases the release of cytochrome c in cytoplasm and caspase activation. Because ASK1 is known to contain a tetratricopeptide repeat (TPR)-acceptor site and ST13 has TPR domain, we found the interaction between ST13 and ASK1. These results strongly indicate Ad-ST13 triggers colorectal cell apoptosis via ASK1-JNK signaling cascade.

Moscatilin Induces Apoptosis in Human Colorectal Cancer Cells: A Crucial Role of c-Jun NH2-Terminal Protein Kinase Activation Caused by Tubulin Depolymerization and DNA Damage

To study the effect of moscatilin (purified from the stem of orchid Dendrobrium loddigesii) on the proliferation of human colorectal cancer HCT-116 cells in vitro and in vivo. The growth inhibition of moscatilin was screened on several human cancer cell lines. The effect of moscatilin on tubulin was detected in vitro. Following moscatilin treatment on HCT-116 cells, c-Jun NH(2)-terminal protein kinase (JNK) and caspase activation was studied by Western blot analysis, and DNA damage was done by Comet assay. Specific JNK inhibitor SP600125 was cotreated to reverse moscatilin-induced apoptosis. Tumor growth inhibition of moscatilin was done on HCT-116 xenograft models. Moscatilin induced a time-dependent arrest of the cell cycle at G(2)-M, with an increase of cells at sub-G(1). Moscatilin inhibited tubulin polymerization, suggesting that it might bind to tubulins. Moscatilin also induced the phosphorylation of JNK1/2. SP600125 significantly inhibited the activation of caspase-9 and caspase-3 and the subsequent moscatilin-induced apoptosis. The data suggest that JNK activation may contribute to moscatilin-mediated apoptosis signaling. A parallel experiment showed that SP600125 significantly inhibits Taxol- and vincristine-induced HCT-116 cell apoptosis. This suggests that the JNK activation may be a common mechanism for tubulin-binding agents. Moreover, moscatilin induces DNA damage, phosphorylation of H2AX and p53, and up-regulation of p21. Our HCT-116 xenograft models show the in vivo efficacy of moscatilin. In summary, our results suggest that moscatilin induces apoptosis of colorectal HCT-116 cells via tubulin depolymerization and DNA damage stress and that this leads to the activation of JNK and mitochondria-involved intrinsic apoptosis pathway.