ITCH facilitates proteasomal degradation of TXNIP in hypoxia- induced lung cancer cells.

BACKGROUND: Lung cancer (LC) is one of the most common cancers and a leading cause of cancer-related deaths worldwide. In many pathological conditions, particularly in the tumor microenvironment, cells and tissues frequently exist in a hypoxic state. Here, we evaluated Itchy E3 ubiquitin protein ligase (ITCH) expression in LC cells following hypoxia treatment. METHODS: LC cell lines were treated with hypoxic condition. Cell migration, invasion, inflammation, reactive oxygen species (ROS) production, and apoptosis of LC cells were determined by wound healing assay, Transwell invasive assay, ELISA, DCFH-DA staining, and flow cytometry, respectively. qPCR and WB were used to determine the expression of ITCH and TXNIP. Co-IP was performed to assess the interaction between ITCH and TXNIP. RESULTS: ITCH expression was downregulated in LC cells under hypoxic conditions. Next, LC cells were subjected to hypoxic conditions and changes in cell viability and metastasis were determined. Hypoxic conditions resulted in increased migration and invasion abilities of LC cells. Intracellular reactive oxygen species (ROS) production, inflammation, and apoptosis were also promoted by hypoxia. We found that ITCH overexpression led to the proteasomal degradation of thioredoxin-interacting protein (TXNIP), whereas the expression of the ITCH C830A mutant did not affect TXNIP levels in LC cells. The gain-of-function experiment demonstrated that migration, invasion, ROS generation, inflammation, and apoptosis of hypoxia-conditioned LC cells were ameliorated by ITCH overexpression, whereas the ITCH C830A mutant did not cause any changes in these phenotypes. Furthermore, the contribution of TXNIP knockdown and ITCH overexpression to the hypoxia-induced features in LC cells with ITCH C830A was found to be similar. CONCLUSION: Our results suggest a novel mechanism underlying the changes in ITCH-mediated malignant phenotypes of hypoxia-conditioned LC cells via TXNIP.

Clinical outcomes and prognostic factors of stereotactic body radiation therapy combined with gemcitabine plus capecitabine for locally advanced unresectable pancreatic cancer.

PURPOSE: This study aimed to evaluate the clinical outcomes, toxicity, and prognostic factors of SBRT combined with gemcitabine plus capecitabine (GEM-CAP) in treating locally advanced pancreatic cancer (LAPC). METHODS: A total of 56 patients with LAPC treated with SBRT combined with GEM-CAP were reviewed from October 2010 to October 2016. The median total prescription dose at five fractions was 40 Gy (30-50 Gy). The patients were subjected to two cycles of GEM-CAP before SBRT. GEM-CAP chemotherapy was then offered for four cycles or until disease tolerance or progression. The primary endpoints included overall survival (OS) and progression-free survival (PFS). RESULTS: The median OS and PFS from the date of diagnosis was 19 (95% CI 14.6-23.4) and 12 months (95% CI 8.34-15.66), respectively. The 1-year and 2-year survival rates were 82.1% and 35.7%, whereas the 1-year and 2-year PFS rates were 48.2% and 14.3%, respectively. The median carbohydrate antigen 19-9-determined PFS time was 11 months (95% CI 5.77-16.24). Multivariate analysis demonstrated that tumor diameter, lymph node metastasis, pre-treatment CA19-9 level, and post-treatment CA19-9 decline were independent prognostic factors (p < 0.05). Acute toxicity was minimal, with two cases (3.6%) experiencing grade 3 duodenal obstruction. No adverse events greater than grade 3 occurred. In late toxicity, three patients (5.4%) developed grade 3 gastrointestinal toxicity and two (3.6%) suffered from perforation caused by grade 4 radiation enteritis and intestinal fistula. CONCLUSIONS: The combination of Cyberknife SBRT and GEM-CAP achieved excellent efficacy with acceptable toxicity for LAPC.

Aurora-a confers radioresistance in human hepatocellular carcinoma by activating NF-κB signaling pathway.

BACKGROUND: Radiotherapy failure is a significant clinical challenge due to the development of resistance in the course of treatment. Therefore, it is necessary to further study the radiation resistance mechanism of HCC. In our early study, we have showed that the expression of Aurora-A mRNA was upregulated in HCC tissue samples or cells, and Aurora-A promoted the malignant phenotype of HCC cells. However, the effect of Aurora-A on the development of HCC radioresistance is not well known. METHODS: In this study, colony formation assay, MTT assays, flow cytometry assays, RT-PCR assays, Western blot, and tumor xenografts experiments were used to identify Aurora-A promotes the radioresistance of HCC cells by decreasing IR-induced apoptosis in vitro and in vivo. Dual-luciferase reporter assay, MTT assays, flow cytometry assays, and Western blot assay were performed to show the interactions of Aurora-A and NF-κB. RESULTS: We established radioresistance HCC cell lines (HepG2-R) and found that Aurora-A was significantly upregulated in those radioresistant HCC cells in comparison with their parental HCC cells. Knockdown of Aurora-A increased radiosensitivity of radioresistant HCC cells both in vivo and in vitro by enhancing irradiation-induced apoptosis, while upregulation of Aurora-A decreased radiosensitivity by reducing irradiation-induced apoptosis of parental cells. In addition, we have showed that Aurora-A could promote the expression of nuclear IkappaB-alpha (IκBα) protein while enhancing the activity of NF-kappaB (κB), thereby promoted expression of NF-κB pathway downstream effectors, including proteins (Mcl-1, Bcl-2, PARP, and caspase-3), all of which are associated with apoptosis. CONCLUSIONS: Aurora-A reduces radiotherapy-induced apoptosis by activating NF-κB signaling, thereby contributing to HCC radioresistance. Our results provided the first evidence that Aurora-A was essential for radioresistance in HCC and targeting this molecular would be a potential strategy for radiosensitization in HCC.

Expression of monocyte chemotactic protein-1/CCL2 in gastric cancer and its relationship with tumor hypoxia.

AIM: To investigate the expression and prognostic value of CCL2 in gastric cancer, as well as its relationship with tumor hypoxia. METHODS: Tumor tissues from 68 gastric cancer patients (GC) were analyzed, and the expression of CCL2 and hypoxia-inducible factor 1 alpha (HIF-1α) in tumor tissues was detected by immunohistochemistry. Statistical evaluations that were used included univariate log-rank tests of Kaplan-Meier curves and multivariate Cox regression model analysis. RESULTS: CCL2 was highly expressed in 66.2% (45/68) of gastric cancer specimens. The distribution of CCL2 expression in tumor tissue was consistent with that of HIF-1α. Patients with high CCL2 expression in GC had a lower overall survival rate [50.6 mo (95%CI: 44.44-56.93) vs 64.6 mo (95%CI: 60.27-68.94), P = 0.013]. CONCLUSION: CCL2 expression correlates closely with HIF-1α expression in gastric cancer. CCL2 may be an independent prognostic marker for GC.

MicroRNA-650 was a prognostic factor in human lung adenocarcinoma and confers the docetaxel chemoresistance of lung adenocarcinoma cells via regulating Bcl-2/Bax expression.

Increasing evidence shows that dysregulation of microRNAs (miRNAs) is involved in malignant transformation. We investigated the clinical significance of miR-650 and its involvement in chemoresistance to docetaxel. Our results showed that the relative expression level of miR-650 was significantly higher in LAD tissues than in corresponding nontumor tissues and high level of miR-650 expression was found to be significantly associated with high incidence of lymph node metastasis, advanced clinical stage and poor prognosis of LAD patients. Univariate and multivariate analyses indicated that high miR-650 expression was an independent prognostic factor for survival. Also, we found that the level of miR-650 in LAD tissues was correlated with the response of patients to docetaxel-based chemotherapy. Silencing of miR-650 could increase the in vitro sensitivity of docetaxel-resistant LAD cells to docetaxel, while upregulation of miR-650 decreased the sensitivity of parental LAD cells to docetaxel both in vitro and in vivo. Additionally, silencing of miR-650 could enhance the caspase-3-dependent apoptosis, which might be correlated with the decreased ratio of Bcl-2/Bax. Further researches suggested that inhibitor of growth 4 (ING4) was a direct target of miR-650. Downregulated or upregulated ING4 expression could partially rescue the effects of miR-650 inhibitor or mimics in docetaxel-resistant or parental LAD cells. Furthermore, we found that ING4 was upregulated in docetaxel-responding LAD tissues, and its expression was inversely correlated with miR-650. Thus, miR-650 is a novel prognostic marker in LAD and its expression is a potential indicator of chemosensitivity to docetaxel-based chemotherapy regimen.

The role of Aurora A in hypoxia-inducible factor 1α-promoting malignant phenotypes of hepatocelluar carcinoma.

Overexpression of both hypoxia-inducible factor 1α (HIF-1α) and Aurora A has been found in hepatocellular carcinoma (HCC). However, whether HIF-1α and Aurora A synergistically promote malignant phenotypes of HCC cells is unknown. The purpose of this study was to investigate the roles and functional correlation of HIF-1α and Aurora A in HCC progression. Immunohistochemistry was performed to detect HIF-1α and Aurora A protein expression in 55 primary HCC and corresponding non-tumor tissues and their clinical significance. Gene knockout technology using short hairpin RNA (shRNA) was used to knockdown expression of HIF-1α or Aurora A and analyze their effects on malignant phenotypes of HCC cells. The transcriptional regulation of Aurora A by HIF-1α and the possible downstream molecular signaling pathways were also determined. Results showed that hypoxia could induce the increased expression of HIF-1α and Aurora A in HCC cells. Also, shRNA-mediated HIF-1α downregulation could lead to the decreased Aurora A expression and inhibition of growth or invasion in HCC cells. Moreover, HIF-1α could transcriptionally regulate Aurora A expression by binding to hypoxia-responsive elements in the Aurora A promoter and recruiting the coactivator-p300/CBP. Additionally, shRNA-mediated Aurora A knockdown could mimic the effects of HIF-1α downregulation on phenotypes of HCC cells, and overexpression of Aurora A could partially rescue the phenotypical changes of HCC cells induced by HIF-1α downregulation. Further research indicated that activation of Akt and p38-MAPK signaling pathways mediated the downstream effects of HIF-1α and Aurora A in HCC cells under hypoxic condition. Taken together, our findings indicated that Aurora A might be a key regulator of HIF-1α-promoting malignant phenotypes of HCC by activation of Akt and p38-MAPK signaling pathways.

Let-7c governs the acquisition of chemo- or radioresistance and epithelial-to-mesenchymal transition phenotypes in docetaxel-resistant lung adenocarcinoma.

MicroRNA (miRNA) expression and functions have been reported to contribute to phenotypic features of tumor cells. Although targets and functional roles for many miRNAs have been described in lung adenocarcinoma (LAD), their pathophysiologic roles in phenotypes of chemoresistant LAD cells are still largely unclear. Previously, docetaxel (DTX)-resistant LAD cell lines (SPC-A1/DTX and H1299/DTX) were established by our laboratory and displayed chemo- or radioresistance and mesenchymal features with enhanced invasiveness and motility. Unbiased miRNA profiling indicated that let-7c (MIRLET7C) was significantly downregulated in SPC-A1/DTX cells. Ectopic let-7c expression increased the in vitro and in vivo chemo- or radiosensitivity of DTX-resistant LAD cells through enhanced apoptosis, reversal of epithelial-to-mesenchymal phenotypes, and inhibition of in vivo metastatic potential via inactivation of Akt phosphorylation, whereas a let-7c inhibitor decreased the chemo- or radiosensitivity of parental cells. Further investigation suggested that let-7c significantly reduced the luciferase activity of a Bcl-xL 3'-UTR-based reporter, concordant with reduced Bcl-xL protein levels. Additionally, siRNA-mediated Bcl-xL knockdown mimicked the same effects of let-7c precursor, and enforced Bcl-xL expression partially rescued the effects of let-7c precursor in DTX-resistant LAD cells. Furthermore, we found that Bcl-xL was significantly upregulated in DTX-nonresponding LAD tissues, and its expression was inversely correlated with let-7c expression. This study suggests an important role for let-7c in the molecular etiology of chemoresistant lung adenocarcinoma.

Cisplatin pretreatment enhances anti-tumor activity of cytokine-induced killer cells.

AIM: To investigate whether cisplatin (DDP) enhances the anti-tumor activity of cytokine- induced killer (CIK) cells in a murine colon adenocarcinoma model. METHODS: Tumor size and weight served as indicators of therapeutic response. Immunohistochemistry was performed to observe intratumoral lymphocyte infiltration and tumor microvessel density. Changes in the percentage of regulatory T (Treg) cells within the spleens of tumor-bearing mice preconditioned with DDP were monitored using flow cytometry. RESULTS: A marked T cell-dependent, synergistic anti-tumor effect of the combined therapy was observed (1968 ± 491 mm³ vs 3872 ± 216 mm³; P = 0.003). Preconditioning chemotherapy with DDP augmented the infiltration of CD3+ T lymphocytes into the tumor mass and reduced the percentage of both intratumoral and splenic Treg cells. CONCLUSION: Preconditioning with DDP markedly enhances the efficacy of adoptively transferred CIK cells, providing a potential clinical modality for the treatment of patients with colorectal cancer.

XRCC1 genetic polymorphism Arg399Gln and gastric cancer risk: A meta-analysis.

AIM: To evaluate the association between X-ray cross-complementing gene 1 (XRCC1) genetic polymorphism Arg399Gln and gastric cancer risk by means of meta-analysis. METHODS: We searched PubMed and NCBI up to June 1, 2008. A total of 16 clinical trials and reports were identified, but only 8 trials qualified under our selection criteria. Statistical analysis was performed with the software program Review Manage, version 4.2.8. RESULTS: Of the 8 case-control studies selected for this meta-analysis, a total of 1334 gastric cancer cases and 2194 controls were included. For Arg399Gln, the Gln/Gln genotype carriers did not have a decreased cancer risk compared with those individuals with the Arg/Arg genotype (OR = 0.92, 95% CI, 0.71-1.19; P = 0.51). Similarly, no associations were found in the recessive and dominant modeling (Gln/Gln vs Arg/Gln + Arg/Arg: OR = 0.96; 95% CI, 0.77-1.19; P = 0.70 and Gln/Gln + Arg/Gln vs Arg/Arg: OR = 0.90, 95% CI, 0.77-1.05; P = 0.18). CONCLUSION: No association is found between the XRCC1 polymorphism Arg399Gln and gastric cancer risk.