Miapaca2 cells were transfected with Beclin1-siRNA or NC-siRNA for 48 h. They were then treated as described previously. For these experiments, I used two antibodies to detect LC3: an anti-rabbit IgG antibody purchased from Santa Cruz Biotechnology and an anti-LC3 antibody from MBL International.
Beclin1 inhibits cell proliferation and promotes apoptosis in vitro and vivo. The gene may also participate in the process of autophagy, which is an essential step in cancer biology. Beclin1 can be used as a target for gene therapy for LC.
The expression of beclin 1 in cancer cells is associated with chemosensitivity. However, it remains unclear how this protein affects chemosensitivity in cancer cells. In one study, researchers found that overexpression of beclin 1 increased the chemosensitivity of CaSki cells while increasing their apoptosis rate. In another study, researchers created an expression vector for 1 in HeLa cells, which increased 1 expression at the protein and mRNA levels.
Miapaca2 and Beclin1 are both genes that are expressed in Hep-2 cells. The expression of Beclin1 in these cells influences the growth of the cells. However, their effects on Hep-2 cell growth are not well understood. Therefore, the authors used a cell line expressing both genes and then examined their effects in Hep-2 cells.
The results showed that the expression of Beclin1 inhibits the proliferation of Hep-2 cells. The cells were stained with DAPI to visualize their punctate pattern of LC3 localization. Western blot analysis demonstrated that Beclin1 inhibition increased the expression of LC3-II protein and enhanced autophagy.
The Beclin1 gene is known to promote apoptosis, which may be why it inhibits the proliferation of Hep-2 cells. Its role in autophagy also makes it an exciting target for gene therapy for LC. However, further studies are required to identify the precise mechanism of Beclin1 inhibition.
Autophagy is an evolutionarily conserved process in which cells remove unwanted proteins and organelles. Disorders in autophagy are associated with a wide variety of human diseases, such as cancer. Beclin1 encodes a protein associated with autophagy, a component of the PI3K complex and is involved in vesicular transport.
Effect of Beclin1 inhibition on apoptosis
In this study, we tested whether Beclin1 inhibition reduces the apoptotic process in Miapaca2 cells. To perform this, we transfected the cells with Beclin1-siRNA and used the flow cytometry method. We used a dual labelling method that includes PI and annexin-V-FITC. We also assessed cell viability using Cell counting Kit-8.
Beclin1 is an essential regulator of autophagy and a tumour suppressor. It is often down-regulated or has monoallelic deletions in various cancers. In this study, we used a novel technique to block the expression of Beclin1 in pancreatic cancer cells. To test the mechanism of inhibition, we inhibited the Beclin1 gene by siRNA transduction. We also measured gene expression using Real-time PCR and Western blot. We also treated the cells with Gemcitabine to induce apoptosis and autophagy.
Effect of Beclin1 inhibition on cell cycle
Inhibition of Beclin1 increased LC3-II synthesis, which is a marker for autophagy. It also promoted the progression of Miapaca2 cells through the S/G2 phase boundary. Inhibition of Beclin1 also induced an increase in autophagosome formation. Furthermore, it inhibited Gemcitabine-induced cell apoptosis. These results suggest that Beclin1 inhibition can interfere with autophagy and decrease the therapeutic effect of Gemcitabine on cancer cells.
Triptolide induces autophagy in S2-013 cells and S2-VP10 cells, suggesting that autophagy and apoptosis are not mutually exclusive. However, the knockdown of caspase-3 prevents triptolide-induced cell death in MiaPaCa-2 cells. Triptolide also rescues the apoptosis pathway in these cells, which is inconsistent with earlier reports.