5-azacytidine inhibits Sox2 promoter methylation during the induction of Thy-1+Lin- cells into hepatocytes.

OBJECTIVE: To investigate the changes and functions of Sox2 gene expression and promoter methylation during induced differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocytes (HCs). METHODS: Rat bone marrow Thy-1+Lin- cells were prepared and divided into control group (directed induction of differentiation into HCs) and experimental group (5-azacytidine intervention induced differentiation). The mRNA expression levels of ALB and Sox2 were detected by fluorescence quantitative polymerase chain reaction (PCR), and the Sox2 gene promoter methylation level was determined by Bisulfite sequencing PCR (BSP). RESULTS: Sox mRNA expression level was significantly increased in experimental group compared to the control group at 0, 7, and 14 days, respectively (all P<0.05). The Sox2 promoter methylation level was gradually increased after 0, 7 and 14 days induction in both groups, accompanied by an increase in methylated loci (all P<0.05). Statistical significance was present in CpG methylated loci between groups (all P<0.05). CONCLUSIONS: The expression of Sox2 gene increased first and then decreased in the process of inducing rat BMSCs into stem cells, and the methylation level of CpG loci in the promoter region changed dynamically, with an increased overall methylation level. After 5-aza treatment, the Sox2 promoter was in a non-methylated state, and its mRNA expression increased, which hindered the cell differentiation.

Evaluation of Neutrophil-Lymphocyte Ratio and Platelet-Lymphocyte Ratio on Predicting Responsiveness to Neoadjuvant Chemoradiotherapy in Locally Advanced Rectal Cancer Patients.

Objective: Neutrophil-lymphocyte ratio (NLR) and Platelet-lymphocyte ratio (PLR) have been proposed as prognostic biomarkers in multiple cancers. However, the implications of NLR and PLR in the responsiveness to neoadjuvant chemoradiotherapy (nCRT) remain to be clarified in locally advanced rectal cancer (LARC) patients. This retrospective study investigated the prognostic value of NLR and PLR in nCRT responsiveness of LARC patients. Methods: A total number of 86 patients diagnosed with LARC and treated with nCRT and total mesorectal excision were retrospectively followed from 2013 to 2016. Receiver operating characteristic (ROC) curve was used to determine the cutoff values of NLR and PLR, and the patients were divided into NLR elevation and NLR decrease groups, or PLR elevation and PLR decrease groups. The correlation between NLR and PLR changes, and clinicopathological factors were analyzed. The relationship between NLR and PLR changes and the curative responsiveness towards nCRT were further evaluated. Results: NLR and PLR changes after nCRT were significantly correlated with the distance of tumors to the anus and BMI (body mass index) (P < 0.05). The clinical remission rate of patients with NLR reduction was 72.09% (31/43), which was significantly higher than that in patients with NLR increment (22/43, 51.16%). There was no significant difference in the clinic remission rate between the patients with PLR reduction and those with PLR increment (P > 0.05). However, the pathological responsiveness rate was significantly higher in patients with PLR reduction (21/43, 48.84%) when compared to the ones with PLR increment (9/43, 20.9%) (P = 0.036). Conclusion: Our data indicate that in LARC patients with nCRT, the reduction of NLR and the reduction of PLR could serves as predictors for the clinic remission rate and pathological responsiveness rate, respectively. The combination of NLR and PLR changes may be employed as a simple and effective prognostic parameter to predict the treatment outcome of nCRT in LARC.

High Expression of PhospholipaseD2 Induced by Hypoxia Promotes Proliferation of Colon Cancer Cells through Activating NF- κ Bp65 Signaling Pathway.

Hypoxia is a typical feature of colon cancer occurrence and progression. We have reported that high expression and activity of PhospholipaseD2 (PLD2) induced by hypoxia in colon cancer cells. In order to further investigate the role of PLD2 in colon cancer under hypoxic conditions. MTT assay was used to detect the proliferation of human colon cancer cells (SW480 and SW620) under hypoxic conditions by decrease the PLD2 gene expression or inhibit the activity of PLD2. Expression level of p-P65/T-P65 and Cyclin D1 were detected in those cells treated as above through using western blot and RT-PCR analysis. Effect of NF-Bp65 inhibitor (BAY-117082) on the proliferation and expression level of Cyclin D1 and PLD2 of colon cancer cells under hypoxic conditions were further analysised. As a result, decreased the expression of PLD2 or inhibited the activity of PLD2 leaded to the proliferation of hypoxia colon cancer cells reduced, and along with the expression level of p-P65/T-P65 and Cyclin D1 reduced. However, inhibition the expression level of p-P65/T-P65 lead to the proliferation and expression of Cyclin D1 in those hypoxia colon cancer cells also reduced. In vivo growth decreased in response to PLD2 and NF-Bp65 inhibition. Our study indicates that high expression of PLD2 induced by hypoxia promotes the proliferation of colon cancer cells, and it may elevate the expression level of Cyclin D1 through activating NF-Bp65 signaling pathway. Inhibition of the PLD2 expression may provide a new clue for treatment for colon cancer.

Peroxiredoxin 2 is essential for maintaining cancer stem cell-like phenotype through activation of Hedgehog signaling pathway in colon cancer.

Cancer stem cells (CSCs) are a key target for reducing tumor growth, metastasis, and recurrence. Redox status is a critical factor in the maintenance of CSCs, and the antioxidant enzyme Peroxiredoxin 2 (Prdx2) plays an important role in the development of colon cancer. Therefore, we investigated the contribution of Prdx2 to the maintenance of stemness of colon CSCs. Here, we used short-hairpin RNAs and a Prdx2-overexpression vector to determine the effects of Prdx2. We demonstrated that knockdown of Prdx2 reduced the self-renewal and sphere formation and resulted in increased 5-FU-induced apoptosis in human colon CSCs. Prdx2 overexpression induced reversion of the self-renewal and sphere formation. Furthermore, the effects of Prdx2 resulted in an altered expression of stemness associated with the Hh/Gli1 signaling pathway. Finally, knockdown of Prdx2 in CD133+ cells reduced the volume of xenograft tumors in BALB/c-nu mice. Taken together, colon CSCs overexpress Prdx2, which promotes their stem cell properties via the Hh/Gli1 signaling pathway. The results suggest that Prdx2 may be an effective therapeutic target for the elimination of CSCs in colorectal cancer.

Inhibition of phospholipaseD2 increases hypoxia-induced human colon cancer cell apoptosis through inactivating of the PI3K/AKT signaling pathway.

Hypoxia is a common feature of solid tumor, and is a direct stress that triggers apoptosis in many human cell types. As one of solid cancer, hypoxia exists in the whole course of colon cancer occurrence and progression. Our previous studies shown that hypoxia induce high expression of phospholipase D2 (PLD2) and survivin in colon cancer cells. However, the correlation between PLD2 and survivin in hypoxic colon cancer cells remains unknown. In this study, we observed significantly elevated PLD2 and survivin expression levels in colon cancer tissues and cells. This is a positive correlation between of them, and co-expression of PLD2 and survivin has a positive correlation with the clinicpatholic features including tumor size, TNM stage, and lymph node metastasis. We also found that hypoxia induced the activity of PLD increased significant mainly caused by PLD2 in colon cancer cells. However, inhibition the activity of PLD2 induced by hypoxia promotes the apoptosis of human colon cancer cells, as well as decreased the expression of apoptosis markers including survivin and bcl2. Moreover, the pharmacological inhibition of PI3K/AKT supported the hypothesis that promotes the apoptosis of hypoxic colon cancer cells by PLD2 activity inhibition may through inactivation of the PI3K/AKT signaling pathway. Furthermore, interference the PLD2 gene expression leaded to the apoptosis of hypoxic colon cancer cells increased and also decreased the expression level of survivin and bcl2 may through inactivation of PI3K/AKT signaling pathway. These results indicated that PLD2 play antiapoptotic role in colon cancer under hypoxic conditions, inhibition of the activity, or interference of PLD2 gene expression will benefit for the treatment of colon cancer patients.

Peroxiredoxin 2 is involved in vasculogenic mimicry formation by targeting VEGFR2 activation in colorectal cancer.

The mammalian peroxiredoxin 2 (Prdx2) is a member of thiol-dependent antioxidant proteins and plays an important role in the progression of colorectal cancer (CRC). The aim of this study was to confirm the role of Prdx2 in formation of VM and progression of CRC. Immunohistochemistry and CD34/periodic acid Schiff double staining were performed to explore the expression of Prdx2 and VM formation in 70 CRC tissues, and there was a positive correlation between Prdx2 expression and VM formation by the Pearson correlation coefficient (r = 0.282, p < 0.05). Prdx2 was suppressed in poorly differentiated HCT116 cells by Prdx2-siRNA-LV transduction. The expression of Prdx2 at both mRNA and protein levels in HCT116 cells transfected with the Prdx2 siRNA was significantly lower than that of negative control siRNA as confirmed by quantitative real-time PCR and Western blotting analysis, respectively (p < 0.05). The well-established in vitro 3D culture model was chosen to investigate the VM formation of HCT116 cells. The numbers of the tubular structures were significantly fewer in Prdx2 siRNA explants than those of negative control siRNA explants after VEGF induction (p < 0.05). Although VEGFR2 expressions had no change after VEGF induction, we found that VEGFR2 phosphorylation levels were markedly reduced in cells of siPrdx2 over time compared with those of negative control siRNA by Western blotting analysis (p < 0.05, p < 0.01). The effects of Prdx2 siRNA on the invasive capabilities of HCT116 cells with VEGF induction were examined by using Matrigel invasion chamber assay. The invasive capabilities of HCT116 cells were significantly declined in Prdx2 siRNA explants than those of negative control siRNA explants (p < 0.05). The effects of Prdx2 siRNA on pathological tumor growth were examined by using a tumor xenograft model in vivo. After implant of HCT116 cells that transduced with Prdx2 siRNA and negative control siRNA as xenografts into nude mice, the growth of xenograft tumors with Prdx2 siRNA was much slower than that of negative control siRNA, and the volumes of tumor xenografts with Prdx2 siRNA were smaller than those of negative control siRNA after 5 weeks (p < 0.05). Further conclusion showed that Prdx2 regulates VM formation by targeting VEGFR2 activation, which now represents as a therapeutic target for RC.

Hypoxia-inducible factor 1-alpha up-regulates the expression of phospholipase D2 in colon cancer cells under hypoxic conditions.

Hypoxia is a common characteristic of solid tumors. Recent studies confirmed that phospholipase D2 (PLD2) plays significant roles in cancer progression. In this study, correlation between the expression of PLD2 and the change in the protein level of hypoxia-inducible factor 1-alpha (HIF1-α) was studied. Thirty human colon cancer tissues were examined for the expression of HIF1-α and PLD2 protein, and mRNA levels. SW480 and SW620 cells were exposed to normoxia (20 %) or hypoxia (<1 %). HIF1-α and PLD2 protein, and mRNA levels were analyzed by Western blot and qRT-PCR, respectively. Growth studies were conducted on cells with HIF1-α inhibition through xenograft tumor model. Finally, PLD2 protein was detected by Western blot analysis in vivo. There was a positive correlation between HIF1-α and PLD2 in colon cancer tissues. Hypoxic stress induced PLD2 mRNA and protein expression in SW480 and SW620 cells. Cells transfected with HIF1-α siRNA showed attenuation of hypoxia stress-induced PLD2 expression. In vivo growth decreased in response to HIF1-α and PLD2 inhibition. These results suggest that PLD2 expression in colon cancer cells is up-regulated via HIF1-α in response to hypoxic stress and underscores the crucial role of HIF1-α-induced PLD2 in tumor growth.