WIPI2 enhances the vulnerability of colorectal cancer cells to erastin via bioinformatics analysis and experimental verification.

Introduction: WD Repeat Domain Phosphoinositide Interacting 2 (WIPI2) is a WD repeat protein that interacts with phosphatidylinositol and regulates multiprotein complexes by providing a b-propeller platform for synchronous and reversible protein-protein interactions assembled proteins. Ferroptosis is a novel iron-dependent form of cell death. It is usually accompanied with the accumulation of membrane lipid peroxides. Our study is to focus on investigating the effect of WIPI2 on the growth and ferroptosis of colorectal cancer (CRC) cells and its potential mechanism. Methods: We analyzed the expression of WIPI2 in colorectal cancer versus normal tissues through The Cancer Genome Atlas (TCGA), and the relationship between clinical traits and WIPI2 expression and prognosis was assessed by univariate and multifactorial cox analysis. Next, we constructed the siRNAs targeting the WIPI2 sequence si-WIPI2 to further investigate the mechanism of WIPI2 in CRC cells through vitro experiments. Results: Public data from the TCGA platform showed that WIPI2 expression was significantly elevated in colorectal cancer tissues compared to paracancerous tissues, and high WIPI2 expressionpredicted poor prognosis for CRC patients. Moreover, we found that the knockdown of WIPI2 expression could inhibit the growth and proliferation of HCT116 and HT29 cells. Furthermore, we found that the expression level of ACSL4 decreased and that of GPX4 increased when WIPI2 was knocked down, suggesting that WIPI2 can potentially positively regulate CRC ferroptosis. Meanwhile, both NC and si groups were able to further inhibit cell growth activity, as well as increase WIPI2 and decrease GPX4 expression when treated with Erastin, but the rate of cell viability inhibition and the trend of protein changes were more significantly in the NC group than si groups, which indicated that Erastin induced CRC ferroptosis through the WIPI2/GPX4 pathway thereby enhancing the sensitivity of colorectal cancer cells to Erastin. Conclusions: Our study suggested that WIPI2 had a promotional effect on the growth of colorectal cancer cells, and it also played an important role in the ferroptosis pathway.

Autophagy blockage and lysosomal dysfunction are involved in diallyl sulfide-induced inhibition of malignant growth in hepatocellular carcinoma cells.

Diallyl sulfide (DAS), as a major component of garlic extracts, has been shown to inhibit growth of hepatocellular carcinoma cells (HCC), but the underlying mechanism is still elusive. In this study, we aimed to explore the involvement of autophagy in DAS-induced growth inhibition of HepG2 and Huh7 hepatocellular carcinoma cells. We studied growth of DAS-treated HepG2 and Huh7 cells using the MTS and clonogenic assays. Autophagic flux was examined by immunofluorescence and confocal microscopy. The expression levels of autophagy-related proteins AMPK, mTOR, p62, LC3-II, LAMP1, and cathepsin D in the HepG2 and Huh7 cells treated with DAS as well as the tumors formed by HepG2 cells in the nude mice in the presence or absence of DAS were examined using western blotting and immunohistochemistry analysis. We found that DAS treatment induced activation of AMPK/mTOR, and accumulation of LC3-II and p62 both in vivo and in vitro. DAS inhibited autophagic flux through blocking the fusion of autophagosomes with lysosomes. Furthermore, DAS induced an increase in lysosomal pH and inhibition of Cathepsin D maturation. Co-treatment with an autophagy inhibitor (Chloroquine, CQ) further enhanced the growth inhibitory activity of DAS in HCC cells. Thus, our findings indicate that autophagy is involved in DAS-mediated growth inhibition of HCC cells both in vitro and in vivo.

Estrogen receptor-α36 is involved in diallyl sulfide-induced inhibition of malignant growth of HepG2 and Huh7 hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a highly malignant disease that currently lacks effective treatment. Epidemiological studies have suggested the preventive role of raw garlic intake in different tumors, such as HCC. Although diallyl sulfide (DAS), the main component of garlic extracts, has been reported to inhibit the growth of HCC cells, the underlying mechanism remains elusive. This study aimed to investigate the inhibitory effect of DAS on the growth of HepG2 and Huh7 hepatocellular carcinoma cells and its underlying mechanism. HepG2 and Huh7 cells were treated with DAS and nude mice were intrahepatically injected with human HCC HepG2 cells and maintained with or without DAS administration for 28 days. MTS and clonogenic assays revealed that DAS inhibited the growth and clonogenicity of HepG2 and Huh7 hepatocellular carcinoma cells. Furthermore, DAS inhibited the growth of xenograft tumors accompanied by a decreased rate of pathological karyomitosis as observed by H&E staining. The expression levels of estrogen receptor-α36 (ER-α36) and epidermal growth factor receptor (EGFR) in HepG2 and Huh7 cells and in xenograft tumors derived from HepG2 cells after DAS treatment were detected by immunohistochemistry and western blotting. We found that DAS disrupted the positive regulatory loop between ER-α36 and EGFR, and decreased the phosphorylation of AKT at Ser 473 both in vivo and in vitro. DAS also induced cell apoptosis, as evidenced by Hoechst and TUNEL staining. Western blotting revealed activation of caspase3, increased BAX and decreased Bcl-2 expression. However, the ER-α36 expression knockdown attenuated DAS-induced ERK and AKT phosphorylation in HCC cells. DAS was also able to inhibit ER-α36-mediated activation of the MAPK/ERK signaling induced by estrogen. Thus, our results indicate that ER-α36 signaling is involved in DAS-induced inhibition of HCC cell growth both in vitro and in vivo.

Diagnostic significance of CK19, galectin-3, CD56, TPO and Ki67 expression and BRAF mutation in papillary thyroid carcinoma.

The aim of the present study was to examine the rate of BRAF mutation and the expression profiles of CK19, galectin-3, CD56, thyroid peroxidase (TPO) and Ki67 in papillary thyroid carcinoma (PTC) and papillary thyroid micro-carcinoma (PTMC). A total of 246 cases of thyroid disease were collected, including PTC, PTMC, nodular goiter (NG) and Hashimoto thyroiditis (HT). The results revealed that CK19 expression was 116/120 in PTC, 61/64 in PTMC, 2/34 in NG and 1/28 in HT. Galectin-3 positive expression was 115/120 in PTC, 60/64 in PTMC, 6/34 in NG and 4/28 in HT. TPO positive expression was 8/120 in PTC, 1/64 in PTMC, 30/34 in NG and 25/28 in HT. CD56-positive expression was 12/120 in PTC, 3/64 in PTMC, 33/34 in NG and 26/28 in HT. Ki67 labeling index was 2.52±0.46% in PTC (120 cases), 2.62±0.52% in PTMC (64 cases), 2.55±0.44% in NG (34 cases) and 2.58±0.48% in HT (28 cases). BRAF mutation rate was 93/120 in PTC, 47/64 in PTMC, 3/34 in NG and 2/28 in HT. These results suggested that expression patterns of CK19, galectin-3, CD56 and TPO and BRAF mutation exhibit diagnosis value in thyroid disease. However, Ki67-positive rate exhibits no notable diagnosis value in thyroid disease.

Protective effect of granulocyte colony-stimulating factor on intracerebral hemorrhage in rat.

Granulocyte colony-stimulating factor (G-CSF) is a member of the cytokine family of growth factors that can protect the neurons from focal cerebral ischemia-induced injuries. The intracerebral hemorrhage (ICH) has been widely observed in the clinic; however, the protective effect of G-CSF on ICH is still elusive. We found in the present study that the intraperitoneal injection of G-CSF for 5 days could improve the ICH-induced neuronal behavioral impairment measured by limb placement assay. We also observed that injection of G-CSF could increase the number of stem cells in the specific zone of the hemorrhagic areas, demonstrated by the enhanced expression of nestin. Additionally, G-CSF could also promote the mobilization of circulating hemopoietic stem cells (HSCs) to the damaged brain areas and activate the astrocytes. Our results reveal that G-CSF is also protective for the ICH with the mechanisms involving proliferation of neural stem cells, the migration of HSCs and the activation of astrocytes.