CLEC19A overexpression inhibits tumor cell proliferation/migration and promotes apoptosis concomitant suppression of PI3K/AKT/NF-κB signaling pathway in glioblastoma multiforme.

BACKGROUND: GBM is the most frequent malignant primary brain tumor in humans. The CLEC19A is a member of the C-type lectin family, which has a high expression in brain tissue. Herein, we sought to carry out an in-depth analysis to pinpoint the role of CLEC19A expression in GBM. METHODS: To determine the localization of CLEC19A, this protein was detected using Western blot, Immunocytochemistry/Immunofluorescence, and confocal microscopy imaging. CLEC19A expression in glioma cells and tissues was evaluated by qRT-PCR. Cell viability, proliferation, migration, and apoptosis were examined through MTT assay, CFSE assay, colony formation, wound healing assay, transwell test, and flow cytometry respectively after CLEC19A overexpression. The effect of CLEC19A overexpression on the PI3K/AKT/NF-κB signaling pathway was investigated using Western blot. An in vivo experiment substantiated the in vitro results using the glioblastoma rat models. RESULTS: Our in-silico analysis using TCGA data and measuring CLEC19A expression level by qRT-PCR determined significantly lower expression of CLEC19A in human glioma tissues compared to healthy brain tissues. By employment of ICC/IF, confocal microscopy imaging, and Western blot we could show that CLEC19A is plausibly a secreted protein. Results obtained from several in vitro readouts showed that CLEC19A overexpression in U87 and C6 glioma cell lines is associated with the inhibition of cell proliferation, viability, and migration. Further, qRT-PCR and Western blot analysis showed CLEC19A overexpression could reduce the expression levels of PI3K, VEGFα, MMP2, and NF-κB and increase PTEN, TIMP3, RECK, and PDCD4 expression levels in glioma cell lines. Furthermore, flow cytometry results revealed that CLEC19A overexpression was associated with significant cell cycle arrest and promotion of apoptosis in glioma cell lines. Interestingly, using a glioma rat model we could substantiate that CLEC19A overexpression suppresses glioma tumor growth. CONCLUSIONS: To our knowledge, this is the first report providing in-silico, molecular, cellular, and in vivo evidences on the role of CLEC19A as a putative tumor suppressor gene in GBM. These results enhance our understanding of the role of CLEC19A in glioma and warrant further exploration of CLEC19A as a potential therapeutic target for GBM.

Milk thistle nano-micelle formulation promotes cell cycle arrest and apoptosis in hepatocellular carcinoma cells through modulating miR-155-3p /SOCS2 /PHLDA1 signaling axis.

BACKGROUND: Hepatocellular Carcinoma (HCC) is a prevalent form of liver cancer that causes significant mortality in numerous individuals worldwide. This study compared the effects of milk thistle (MT) and nano-milk thistle (N-MT) on the expression of the genes that participate in apoptosis and cell cycle pathways in Huh-7 and HepG2 cells. METHODS: IC50 values of MT and N-MT were determined using the MTT assay. Huh-7 and HepG2 cell lines (containing mutant and wild-type TP53 gene, respectively) were incubated with MT and N-MT for 24h and 48h and the impact of MT and N-MT on the proliferation of these cell lines was evaluated through a comparative analysis. Cell cycle and apoptosis were assessed by flow cytometry after 24h and 48h treatment in the cell lines mentioned. Real-time PCR was used to analyze miR-155-3p, PHLDA1, SOCS2, TP53, P21, BAX, and BCL-2 expression in the cell lines that were being treated. RESULTS: N-MT reduces cancer cell growth in a time and concentration-dependent manner, which is more toxic compared to MT. Huh-7 was observed to have IC50 values of 2.35 and 1.7 µg/ml at 24h and 48h, and HepG2 was observed to have IC50 values of 3.4 and 2.6 µg/ml at 24 and 48h, respectively. N-MT arrested Huh-7 and HepG2 cells in the Sub-G1 phase and induced apoptosis. N-MT led to a marked reduction in the expression of miR-155-3p and BCL-2 after 24h and 48h treatments. Conversely, PHLDA1, SOCS2, BAX, and P21 were upregulated in the treated cells compared to untreated cells, which suggests that milk thistle has the potential to regulate these genes. N-MT reduced the expression of TP53 in Huh-7 cells after mentioned time points, while there was a significant increase in the expression of the TP53 gene in HepG2 cells. No gene expression changes were observed in MT-treated cells after 24h and 48h. CONCLUSION: N-MT can regulate cancer cell death by arresting cell cycle and inducing apoptosis. This occurs through the alteration of apoptotic genes expression. A reduction in the expression of miR-155-3p and increase in the expression of SOCS2 and PHLDA1 after N-MT treatment showed the correlation between miR-155-3p and PHLDA1/SOCS2 found in bioinformatics analysis. While N-MT increased TP53 expression in HepG2, reduced it in Huh-7. The findings indicate that N-MT can function intelligently in cancer cells and can be a helpful complement to cancer treatment.