Advances in the functions of CTRP6 in the development and progression of the malignancy.

CTRP6, a member of the C1q/TNF-related protein (CTRP) family, has gained increasing scientific interest because of its regulatory role in tumor progression. Previous studies have shown that CTRP6 is closely involved in regulating various pathophysiological processes, including glucose and lipid metabolism, cell proliferation, apoptosis, and inflammation. To date, CTRP6 has been identified as related to eight different malignancies, including lung cancer, oral cancer, gastric cancer, colon cancer, liver cancer, bladder cancer, renal cancer, and ovarian cancer. CTRP6 is reported to be associated with tumor progression by activating a series of related signal networks. This review article mainly discusses the biochemistry and pleiotropic pathophysiological functions of CTRP6 as a new molecular mediator in carcinogenesis, hoping that the information summarized herein could make a modest contribution to the development of novel cancer treatments in the future.

Transcriptomics-based analysis of co-exposure of cadmium (Cd) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) indicates mitochondrial dysfunction induces NLRP3 inflammasome and inflammatory cell death in renal tubular epithelial cells.

Environmental pollution often releases multiple contaminants resulting in as yet largely uncharacterized additive toxicities. Cadmium (Cd) is a widespread pollutant that induces nephrotoxicity in animal models and humans. However, the combined effect of Cd in causing nephrotoxicity with 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a typical congener of polybrominated diphenyl ethers (PBDEs), has not been evaluated and mechanisms are not completely clear. Here, we applied transcriptome sequencing analysis to investigate the combined toxicity of Cd and BDE-47 in the renal tubular epithelial cell lines HKCs. Cd or BDE-47 exposure decreased cell viability in a dose-dependent manner, and exhibited cell swelling and rounding similar to necrosis, which was exacerbated by co-exposure. Transcriptomic analysis revealed 2191, 1331 and 3787 differentially-expressed genes following treatment with Cd, BDE-47 and co-exposure, respectively. Interestingly, functional annotation and enrichment analyses showed involvement of pathways for oxidative stress, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and inflammatory cell death for all three treatments. Examination of indices of mitochondrial function and oxidative stress in HKC cells showed that the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and intracellular calcium ion concentration [Ca2+]i were elevated, while superoxide dismutase (SOD) and mitochondrial membrane potential (MMP) were decreased. The ratio of apoptotic and necrotic cells and intracellular lactate dehydrogenase (LDH) release were increased by Cd or BDE-47 exposure, and was aggravated by co-exposure, and was attenuated by ROS scavenger N-Acetyl-L-cysteine (NAC). NLRP3 inflammasome and pyroptosis pathway-related genes of NLRP3, adaptor molecule apoptosis-associated speck-like protein (ASC), caspase-1, interleukin-18 (IL-18) and IL-1ß were elevated, while gasdermin D (GSDMD) was down-regulated, and protein levels of NLRP3, cleaved caspase-1 and cleaved GSDMD were increased, most of which were relieved by NAC. Our data demonstrate that exposure to Cd and BDE-47 induces mitochondrial dysfunction and triggers NLRP3 inflammasome and GSDMD-dependent pyroptosis leading to nephrotoxicity, and co-exposure exacerbates this effect, which could be attenuated by inhibiting ROS. This study provides a further mechanistic understanding of kidney damage, and co-exposure impact is worthy of concern and should be considered to improve the accuracy of environmental health assessment.

Inhibition of cytoplasmic EZH2 induces antitumor activity through stabilization of the DLC1 tumor suppressor protein.

mRNA expression of the DLC1 tumor suppressor gene is downregulated in many lung cancers and their derived cell lines, with DLC1 protein levels being low or absent. Although the role of increased EZH2 methyltransferase in cancer is usually attributed to its histone methylation, we unexpectedly observed that post-translational destabilization of DLC1 protein is common and attributable to its methylation by cytoplasmic EZH2, leading to CUL-4A ubiquitin-dependent proteasomal degradation of DLC1. Furthermore, siRNA knockdown of KRAS in several lines increases DLC1 protein, associated with a drastic reduction in cytoplasmic EZH2. Pharmacologic inhibition of EZH2, CUL-4A, or the proteasome can increase the steady-state level of DLC1 protein, whose tumor suppressor activity is further increased by AKT and/or SRC kinase inhibitors, which reverse the direct phosphorylation of DLC1 by these kinases. These rational drug combinations induce potent tumor growth inhibition, with markers of apoptosis and senescence, that is highly dependent on DLC1 protein.

Factors Associated with Cranial Nerve Injury after Radiotherapy for Large Brain Metastases.

This paper mainly studied the correlation factors of cranial nerve injury after radiotherapy for large brain metastases by investigating the influencing factors and predictors of cranial nerve injury, which can provide a good reference and idea for radiotherapy. Through a large number of experiments, it is proved that the research idea proposed in this paper is reasonable and correct.

Novel Prognostic Predictor for Primary Pulmonary Hypertension: Focus on Blood Urea Nitrogen.

Background: Primary pulmonary hypertension (PPH) is a life-threatening disease associated with increased mortality. The urea cycle pathway plays a major role in PPH severity and treatment response. Little is known about the association of the blood urea nitrogen (BUN) and PPH prognosis. Methods: Clinical data were extracted from the Medical Information Mart for Intensive Care III (MIMIC-III) database. Adult patients (≥18 years) patients with primary pulmonary hypertension (PPH) in the database were enrolled. Spearman correlation was used to analyze the association of BUN with length of hospital and intensive care unit (ICU) stays. The chi-square test was used to analyze the association of BUN with mortality rate. Survival curves were estimated using the Kaplan-Meier method and compared by the log-rank test. Multivariable logistic regression was used to identify the BUN as an independent prognostic factor of mortality. Receiver operating characteristic (ROC) curves and the area under the curve (AUC) were used to analyze the sensitivity and specificity for mortality. Results: In total, 263 patients who met the selection criteria were enrolled. BUN was significantly positively associated with length of hospital stay and ICU stay (hospital stay: ρ = 0.282, ICU stay: ρ = 0.276; all P < 0.001). Higher hospital, 90-day and 4-year mortality rates were observed in the higher BUN quartile of PPH patients (hospital: P = 0.002; 90-day: P = 0.025; 4-year: P < 0.001). The Kaplan-Meier survival curves showed that patients in higher BUN quartile tended to have lower 4-year survival (Q1:7.65%, Q2: 10.71%; Q3: 14.80%, Q4: 16.84%; P < 0.0001). Logistic regression analyses found a significant association of BUN and mortality (hospital: OR = 1.05, 95% CI = 1.02-1.08, P = 0.001; 90-day: OR = 1.02, 95% CI = 1.00-1.05, P = 0.027; 4-year: OR = 1.05, 95% CI = 1.02-1.08, P = 0.001). Results of ROC and AUC showed that the diagnostic performance of BUN for mortality was moderately good. Conclusion: BUN was positively correlated with the length of hospital stay and ICU stay of PPH patients. Higher BUN was associated with higher hospital, 90-day and 4-year mortality and lower 4-year survival of PPH patients. These findings indicate that BUN can be a novel potential prognostic predictor for PPH.

The tumor suppressor activity of DLC1 requires the interaction of its START domain with Phosphatidylserine, PLCD1, and Caveolin-1.

BACKGROUND: DLC1, a tumor suppressor gene that is downregulated in many cancer types by genetic and nongenetic mechanisms, encodes a protein whose RhoGAP and scaffolding activities contribute to its tumor suppressor functions. The role of the DLC1 START (StAR-related lipid transfer; DLC1-START) domain, other than its binding to Caveolin-1, is poorly understood. In other START domains, a key function is that they bind lipids, but the putative lipid ligand for DLC1-START is unknown. METHODS: Lipid overlay assays and Phosphatidylserine (PS)-pull down assays confirmed the binding of DLC1-START to PS. Co-immunoprecipitation studies demonstrated the interaction between DLC1-START and Phospholipase C delta 1 (PLCD1) or Caveolin-1, and the contribution of PS to those interactions. Rho-GTP, cell proliferation, cell migration, and/or anchorage-independent growth assays were used to investigate the contribution of PS and PLCD1, or the implications of TCGA cancer-associated DLC1-START mutants, to DLC1 functions. Co-immunoprecipitations and PS-pull down assays were used to investigate the molecular mechanisms underlying the impaired functions of DLC1-START mutants. A structural model of DLC1-START was also built to better understand the structural implications of the cancer-associated mutations in DLC1-START. RESULTS: We identified PS as the lipid ligand for DLC1-START and determined that DLC1-START also binds PLCD1 protein in addition to Caveolin-1. PS binding contributes to the interaction of DLC1 with Caveolin-1 and with PLCD1. The importance of these activities for tumorigenesis is supported by our analysis of 7 cancer-associated DLC1-START mutants, each of which has reduced tumor suppressor function but retains wildtype RhoGAP activity. Our structural model of DLC1-START indicates the mutants perturb different elements within the structure, which is correlated with our experimental findings that the mutants are heterogenous with regard to the deficiency of their binding properties. Some have reduced PS binding, others reduced PLCD1 and Caveolin-1 binding, and others are deficient for all of these properties. CONCLUSION: These observations highlight the importance of DLC1-START for the tumor suppressor function of DLC1 that is RhoGAP-independent. They also expand the versatility of START domains, as DLC1-START is the first found to bind PS, which promotes the binding to other proteins.

2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) activates Aryl hydrocarbon receptor (AhR) mediated ROS and NLRP3 inflammasome/p38 MAPK pathway inducing necrosis in cochlear hair cells.

Tetrabromodiphenyl ether (BDE-47) is widely used as commercial flame retardants that can be released into the environment and finally enter human body through the food chain. It has been identified to generate neurotoxicity, but little is known about auditory damage and the underlying mechanism following BDE-47 exposure. This study aimed to assess the cell viability with BDE-47 concentration ranging from 0 to 150 µM in mouse organ of Corti-derived cell lines (HEI-OC1). Aryl hydrocarbon receptor (AhR) as an environmental sensor, reactive oxygen species (ROS), NLRP3 inflammasome and p38 MAPK pathways were detected. Results: (1) BDE-47 inhibited the viability in a time- and dose-dependent way in HEI-OC1 cells. Cell cycle was arrested in G1 phase by BDE-47; (2) Elevated intracellular ROS, LDH levels and necrosis were found, which was alleviated by pretreatment with ROS scavenger N-acetylcysteine (NAC); (3) AhR plays an essential role in ligand-regulated transcription factor activation by exogenous environmental compounds. We found increased expression of AhR and decreased downstream targets of CYP 1A1 and CYP 1B1 in BDE-47-treated HEI-OC1 cells, which was reversed by the AhR antagonist CH-223191 for 2 h before BDE-47 exposure. No significant change was detected in CYP 2B; (4) Enhanced expressions of NLRP3 and caspase-1 were induced by BDE-47, with up-regulations of both pro-inflammatory factors for IL-1ß, IL-6 and TNF-α, and anti-inflammatory factors for IL-4, IL-10 and IL-13, but down-regulation for IL-1α; (5) Additionally, the p38 MAPK signaling pathway was activated with increased phosphorylation levels of MKK/3/6, p38 MAPK and NF-kB. Overall, our findings illustrate a role of AhR in ROS-induced necrosis of cochlear hair cells by BDE-47 exposure, in which NLRP3 inflammasome and p38 MAPK signaling pathways are activated. The current study first elucidates the sense of hearing damage induced by BDE-47, and cell-specific or mixture exposures in vivo or human studies are needed to confirm this association.

MiR-454-3p Promotes Oxaliplatin Resistance by Targeting PTEN in Colorectal Cancer.

Colorectal cancer is one of the most common malignancies worldwide. Oxaliplatin is the first-line chemotherapeutic agent for the treatment of advanced colorectal cancer. However, acquired resistance to oxaliplatin limits its therapeutic efficacy, and the underlying mechanism remains largely unclear. In this study, we compared the expression of a panel of microRNAs (miRNAs) between oxaliplatin-sensitive and -resistant HCT-116 colorectal cancer cells. We found that miR-454-3p was significantly up-regulated in oxaliplatin-resistant cells and was the most differently expressed miRNA. Interestingly, we observed that inhibition of miR-454-3p resensitized resistant cells to oxaliplatin and enhanced oxaliplatin-induced cellular apoptosis. Moreover, we determined that miR-454-3p promoted oxaliplatin resistance through targeting PTEN and activating the AKT signaling pathway. In vivo study revealed that overexpression of miR-454-3p decreased the sensitivity of HCT-116 xenograft tumors to oxaliplatin treatment in a mouse model. Clinically, overexpression of miR-454-3p was associated with decreased responsiveness to oxaliplatin-based chemotherapy, as well as a short progression-free survival. Taken together, our study indicated that the expression of miR-454-3p could be used to predict oxaliplatin sensitivity, and targeting miR-454-3p could overcome oxaliplatin resistance in colorectal cancer.

Establishment of Rat Model of Insulin Resistance Exposed to Chronic Renal Allograft Dysfunction.

BACKGROUND: The main cause of chronic renal allograft dysfunction (CRAD) still remains unclear. Insulin resistance (IR) may be a potential inducement, but there is insufficient evidence about this association. We aimed to establish a rat model of CRAD complicated with IR and to explore the function and pathologic changes of the renal allograft induced by IR. METHODS: F344-to-Lewis rats of CRAD were fed a high-fat diet to induce IR. They were divided into 3 groups: IR (CRAD+IR), CRAD, and control (CTL). Serum levels of blood urea nitrogen (BUN) and serum creatinine (Scr) were measured to evaluate the renal function. The Homeostasis Model Assessment (HOMA)-IR index was detected by comparing the values of fasting serum insulin levels (FINS) with fasting blood glucose levels (FBG). The pathologic analysis was conducted by the degree of renal lesions including glomerular lesions, renal tubular lesions, hemorrhage, inflammatory cell infiltration, fibrillation, and hyperplasia of the renal interstitium. RESULTS: In the second, third, and fourth month after surgery, serum levels of Scr and BUN in the IR group were reduced more than those in the CRAD group, while they were both higher compared to the CTL group, suggesting that renal function in the CRAD group was declined. The HOMA-IR in the IR group was greater than that in the CRAD and CTL groups, showing that simple high-fat diet feeding significantly and steadily increased FINS and FBG in CRAD complicated with IR rats. Pathologic changes indicated that the CRAD rat model was successfully constructed and was still in the early-middle stages of renal lesions 4 months after surgery, yet IR presented a significant effect on CRAD. CONCLUSION: These results indicate that the stable CRAD complicated with IR rat model can be established through a high-fat diet in CRAD rats in 4 months, and IR could be an influencing factor.

Cancer-Associated Point Mutations in the DLC1 Tumor Suppressor and Other Rho-GAPs Occur Frequently and Are Associated with Decreased Function.

In advanced cancer, the RHOA GTPase is often active together with reduced expression of genes encoding Rho-specific GTPase-accelerating proteins (Rho-GAP), which negatively regulate RHOA and related GTPases. Here we used the The Cancer Genome Atlas dataset to examine 12 tumor types (including colon, breast, prostate, pancreas, lung adenocarcinoma, and squamous cell carcinoma) for the frequency of codon mutations of 10 Rho-GAP and experimentally tested biochemical and biological consequences for cancer-associated mutants that arose in the DLC1 tumor suppressor gene. DLC1 was the Rho-GAP gene mutated most frequently, with 5%-8% of tumors in five of the tumor types evaluated having DLC1 missense mutations. Furthermore, 20%-26% of the tumors in four of these five tumor types harbored missense mutations in at least one of the 10 Rho-GAPs. Experimental analysis of the DLC1 mutants indicated 7 of 9 mutants whose lesions were located in the Rho-GAP domain were deficient for Rho-GAP activity and for suppressing cell migration and anchorage-independent growth. Analysis of a DLC1 linker region mutant and a START domain mutant showed each was deficient for suppressing migration and growth in agar, but their Rho-GAP activity was similar to that of wild-type DLC1. Compared with the wild-type, the linker region mutant bound 14-3-3 proteins less efficiently, while the START domain mutant displayed reduced binding to Caveolin-1. Thus, mutation of Rho-GAP genes occurs frequently in some cancer types and the majority of cancer-associated DLC1 mutants evaluated were deficient biologically, with various mechanisms contributing to their reduced activity. SIGNIFICANCE: These findings indicate that point mutation of Rho-GAP genes is unexpectedly frequent in several cancer types, with DLC1 mutants exhibiting reduced function by various mechanisms.

Responses of metastatic primary fallopian tube carcinoma to pembrolizumab and nab-paclitaxel: A case report.

RATIONALE: Primary fallopian tube carcinoma (PFTC) is an extremely rare but invasive malignancy with a dismal prognosis. Very few data exist on the salvage treatment for patients with PFTC. Here we report a case showing an impressive response to immunotherapy combined with chemotherapy, which have never been reported before on patients with metastatic PFTC. PATIENT CONCERNS: A 42-year-old woman, who was diagnosed with PFTC in 2010, had been failed of multiple systemic therapies and antiangiogenic therapy because of the disease recurrence and progression. DIAGNOSIS: Metastatic primary fallopian tube carcinoma. INTERVENTIONS: The patient underwent surgery in May 2010 and had multi-line chemotherapies plus an anti-vascular endothelial growth factor (anti-VEGF) monoclonal antibody for about 9 years. Due to treatment failure the patient accepted the immunotherapy with the checkpoint inhibitor, pembrolizumab, combined with nab-paclitaxel from December 2018 to April 2019. OUTCOMES: The patient showed a complete response after 6 cycles treatment. Thus far, the patient is taking pembrolizumab as maintenance and remains in good health. LESSONS: Pembrolizumab combined with chemotherapy for treatment of PFTC may provide a positive antitumor effect in multiple metastatic lesions, but more clinical evidence is needed to confirm the efficacy and safety.

SRC and ERK cooperatively phosphorylate DLC1 and attenuate its Rho-GAP and tumor suppressor functions.

SRC and ERK kinases control many cell biological processes that promote tumorigenesis by altering the activity of oncogenic and tumor suppressor proteins. We identify here a physiological interaction between DLC1, a focal adhesion protein and tumor suppressor, with SRC and ERK. The tumor suppressor function of DLC1 is attenuated by phosphorylation of tyrosines Y451 and Y701 by SRC, which down-regulates DLC1's tensin-binding and Rho-GAP activities. ERK1/2 phosphorylate DLC1 on serine S129, which increases both the binding of SRC to DLC1 and SRC-dependent phosphorylation of DLC1. SRC inhibitors exhibit potent antitumor activity in a DLC1-positive transgenic cancer model and a DLC1-positive tumor xenograft model, due to reactivation of the tumor suppressor activities of DLC1. Combined treatment of DLC1-positive tumors with SRC plus AKT inhibitors has even greater antitumor activity. Together, these findings indicate cooperation between the SRC, ERK1/2, and AKT kinases to reduce DLC1 Rho-GAP and tumor suppressor activities in cancer cells, which can be reactivated by the kinase inhibitors.

Inhibition of HHIP Promoter Methylation Suppresses Human Gastric Cancer Cell Proliferation and Migration.

BACKGROUND/AIMS: Human hedgehog-interacting protein (HHIP) is a negative regulator of the hedgehog (HH) signaling pathway. It is deregulated in gastric cancer. The underlying molecular mechanism of HHIP-induced inhibition of HH signaling remains to be determined. METHODS: A lentiviral HHIP expression vector ("LV-HHIP") was established to exogenously over-express HHIP in gastric cancer cells. HHIP protein and mRNA were tested by Western blotting assay and quantitative real-time PCR assay, respectively. Cell survival was tested by the Cell Counting Kit-8 (CCK-8) assay. Cell proliferation was examined by the BrdU ELISA assay and [H3] Thymidine DNA incorporation assay. Cell invasion and migration were tested by the phagokinetic track assay and the "Transwell" assay. The bisulfite-sequencing PCR was applied to test HHIP promoter methylation. RESULTS: In the established (AGS cell line) and primary human gastric cancer cells, LV-HHIP transfection increased HHIP expression and inhibited cancer cell survival and proliferation as well as cell migration and invasion. Furthermore, LV-HHIP significantly attenuated promoter methylation of the endogenous HHIP gene in AGS cells, causing it upregulation. Inhibition of methylation by 5-aza-dc similarly induced HHIP expression in gastric cancer cells, which inhibited cancer cell proliferation and migration. CONCLUSIONS: Our results suggest that inhibition of HHIP promoter methylation can efficiently inhibit human gastric cancer cell proliferation and migration.

Effects of DLC1 Deficiency on Endothelial Cell Contact Growth Inhibition and Angiosarcoma Progression.

Background: Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene frequently deleted in cancer. However, DLC1 is not known to be deleted in angiosarcoma, an aggressive malignancy of endothelial cell derivation. Additionally, the physiologic functions of DLC1 protein in endothelial cells are poorly defined. Methods: We investigated the effects of shRNA-induced DLC1 depletion in endothelial cells. Cell growth was measured by 3H thymidine incorporation, IncuCyte imaging, and population doublings; cell death by cell cycle analysis; gene expression by Affimetrix arrays and quantitative polymerase chain reaction; NF-κB activity by reporter assays; and protein levels by immunoblotting and immunofluorescence staining. We tested Tanespimycin/17-AAG and Fasudil treatment in groups of nine to 10 mice bearing ISOS-1 angiosarcoma. All statistical tests were two-sided. Results: We discovered that DLC1 is a critical regulator of cell contact inhibition of proliferation in endothelial cells, promoting statistically significant (P < .001) cell death when cells are confluent (mean [SD] % viability: control DLC1 = 15.6 [19.3]; shDLC1 = 73.4 [13.1]). This prosurvival phenotype of DLC1-depleted confluent endothelial cells is attributable to a statistically significant and sustained increase of NF-κB activity (day 5, P = .001; day 8, P = .03) associated with increased tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) signaling. Consistently, we found that DLC1 is statistically significantly reduced (P < .001 in 5 of 6) and TNFAIP3/A20 is statistically significantly increased (P < .001 in 2 of 3 and P = 0.02 in 1 of 3) in human angiosarcoma compared with normal adjacent endothelium. Treatment with the NF-κB inhibitor Tanespimycin/17-AAG statistically significantly reduced angiosarcoma tumor growth in mice (treatment tumor weight vs control, 0.50 [0.19] g vs 0.91 [0.21] g, P = .001 experiment 1; 0.66 [0.26] g vs 1.10 [0.31] g, P = .01 experiment 2). Conclusions: These results identify DLC1 as a previously unrecognized regulator of endothelial cell contact inhibition of proliferation that is depleted in angiosarcoma and support NF-κB targeting for the treatment of angiosarcoma where DLC1 is lost.

Receptor tyrosine kinase activation of RhoA is mediated by AKT phosphorylation of DLC1.

We report several receptor tyrosine kinase (RTK) ligands increase RhoA-guanosine triphosphate (GTP) in untransformed and transformed cell lines and determine this phenomenon depends on the RTKs activating the AKT serine/threonine kinase. The increased RhoA-GTP results from AKT phosphorylating three serines (S298, S329, and S567) in the DLC1 tumor suppressor, a Rho GTPase-activating protein (RhoGAP) associated with focal adhesions. Phosphorylation of the serines, located N-terminal to the DLC1 RhoGAP domain, induces strong binding of that N-terminal region to the RhoGAP domain, converting DLC1 from an open, active dimer to a closed, inactive monomer. That binding, which interferes with the interaction of RhoA-GTP with the RhoGAP domain, reduces the hydrolysis of RhoA-GTP, the binding of other DLC1 ligands, and the colocalization of DLC1 with focal adhesions and attenuates tumor suppressor activity. DLC1 is a critical AKT target in DLC1-positive cancer because AKT inhibition has potent antitumor activity in the DLC1-positive transgenic cancer model and in a DLC1-positive cancer cell line but not in an isogenic DLC1-negative cell line.

Inhibition of MicroRNA-21-5p Promotes the Radiation Sensitivity of Non-Small Cell Lung Cancer Through HMSH2.

BACKGROUND: This study aimed to explore the effects of microRNA-21-5p (miR-21-5p) on the radiation sensitivity of non-small cell lung cancer (NSCLC) and the involvement of human MutS homolog 2 (hMSH2) One hundred fourteen NSCLC patients at stage II or III who received surgery and postoperative radiotherapy were enrolled in this study. METHODS: The patients were assigned into radiation-sensitive and -insensitive groups. NSCLC A549 cells were transfected to generate control, Negative control (NC), miR-21-5p inhibitor, miR-21-5p mimic, small interfering hMSH2 (sihMSH2), miR-21-5p inhibitor + sihMSH2 and hMSH2 overexpression groups. Immunohistochemistry was performed to detect the hMSH2 expression in transfected and irradiated cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were performed to evaluate A549 miR-21-5p and hMSH2 expression in transfected and irradiated cells. A colony formation assay was adopted for cell survival analysis. The relationship between miR-21-5p and hMSH2 was verified by a luciferase reporter assay. Cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and apoptosis was assessed by flow cytometry. NSCLC nude mouse models were established, and tumor volumes and tumor weights were recorded. RESULTS: The radiation-sensitive group of patients exhibited lower miR-21-5p but higher hMSH2 expression than the insensitive group. For irradiated A549 cells, lower cell survival, higher apoptosis, increased miR-21-5p expression and decreased hMSH2 expression were observed at 6 and 8 Gy than at 0, 2 and 4 Gy; compared to 6 Gy, cell survival and hMSH2 expression were decreased and apoptosis and miR-21-5p expression were increased at 8 Gy. Additionally, miR-21-5p was found to target hMSH2. Compared with the control group, the cell survival rate was lower and the apoptosis rate higher in the miR-21-5p inhibitor group, whereas the opposite was observed for the miR-21-5p mimic and sihMSH2 groups. For the mouse model, decreased tumor volume and tumor weight and higher hMSH2 expression were found in the miR-21-5p inhibitor, radiation, hMSH2 overexpression, miR-21-5p inhibitor + radiation and hMSH2 overexpression + radiation groups compared with the control group. In addition, tumor volume and tumor weight were decreased and hMSH2 expression increased in the miR-21-5p inhibitor + radiation and hMSH2 overexpression + radiation groups compared with the radiation alone group. CONCLUSION: These findings indicate that inhibition of miR-21 can promote the radiation sensitivity of NSCLC by targeting hMSH2.

Characterization of Microcystin-Induced Dualistic Toxic Effects on Primary Rat Hepatocytes.

Microcystins (MCs) comprise a group of widely characterized cyclic heptapeptides able to induce a series of liver injuries, including acute liver failure and primary liver cancer. Although the dualistic effects of MCs have been postulated, the specific action mode according to the exposure dosage of MCs remains unknown. In the present study, primarily cultured rat hepatocytes were used to systematically investigate hepatotoxic characteristics of MC-LR (one of the most abundant and toxic MCs variants). Results showed that the dualistic toxicity of MC-LR on hepatocytes is dose dependent. Specifically, MC-LR at a high dose (>10-8 mol/L) induced a significant reduction in cell viability, whereas low-dose MC-LR (<10-8 mol/L) was observed to promote cell proliferation. Oxidative stress measurements showed that reactive oxygen species (ROS) levels undergo a massive and rapid increase in high-dose MC-LR-treated hepatocytes and a mild and slow increase in low-dose MC-LR-treated hepatocytes. These in vitro data suggest that MC-LR is able to exert dualistic toxic effects on hepatocytes through the "two-faced" character of ROS, which causes cell death or even necrosis at high concentrations and promotes cell proliferation exclusively at low or transient concentrations.

LD Motif Recognition by Talin: Structure of the Talin-DLC1 Complex.

Cell migration requires coordination between integrin-mediated cell adhesion to the extracellular matrix and force applied to adhesion sites. Talin plays a key role in coupling integrin receptors to the actomyosin contractile machinery, while deleted in liver cancer 1 (DLC1) is a Rho GAP that binds talin and regulates Rho, and therefore actomyosin contractility. We show that the LD motif of DLC1 forms a helix that binds to the four-helix bundle of the talin R8 domain in a canonical triple-helix arrangement. We demonstrate that the same R8 surface interacts with the paxillin LD1 and LD2 motifs. We identify key charged residues that stabilize the R8 interactions with LD motifs and demonstrate their importance in vitro and in cells. Our results suggest a network of competitive interactions in adhesion complexes that involve LD motifs, and identify mutations that can be used to analyze the biological roles of specific protein-protein interactions in cell migration.

DLC1 is the principal biologically-relevant down-regulated DLC family member in several cancers.

The RHO family of RAS-related GTPases in tumors may be activated by reduced levels of RHO GTPase accelerating proteins (GAPs). One common mechanism is decreased expression of one or more members of the Deleted in Liver Cancer (DLC) family of Rho-GAPs, which comprises three closely related genes (DLC1, DLC2, and DLC3) that are down-regulated in a wide range of malignancies. Here we have studied their comparative biological activity in cultured cells and used publicly available datasets to examine their mRNA expression patterns in normal and cancer tissues, and to explore their relationship to cancer phenotypes and survival outcomes. In The Cancer Genome Atlas (TCGA) database, DLC1 expression predominated in normal lung, breast, and liver, but not in colorectum. Conversely, reduced DLC1 expression predominated in lung squamous cell carcinoma (LSC), lung adenocarcinoma (LAD), breast cancer, and hepatocellular carcinoma (HCC), but not in colorectal cancer. Reduced DLC1 expression was frequently associated with promoter methylation in LSC and LAD, while DLC1 copy number loss was frequent in HCC. DLC1 expression was higher in TCGA LAD patients who remained cancer-free, while low DLC1 had a poorer prognosis than low DLC2 or low DLC3 in a more completely annotated database. The poorest prognosis was associated with low expression of both DLC1 and DLC2 (P < 0.0001). In cultured cells, the three genes induced a similar reduction of Rho-GTP and cell migration. We conclude that DLC1 is the predominant family member expressed in several normal tissues, and its expression is preferentially reduced in common cancers at these sites.

The expansion of neighborhood and pattern formation on spatial prisoner's dilemma.

The prisoner's dilemma (PD), in which players can either cooperate or defect, is considered a paradigm for studying the evolution of cooperation in spatially structured populations. There the compact cooperator cluster is identified as a characteristic pattern and the probability of forming such pattern in turn depends on the features of the networks. In this paper, we investigate the influence of expansion of neighborhood on pattern formation by taking a weak PD game with one free parameter T, the temptation to defect. Two different expansion methods of neighborhood are considered. One is based on a square lattice and expanses along four directions generating networks with degree increasing with K=4m. The other is based on a lattice with Moore neighborhood and expanses along eight directions, generating networks with degree of K=8m. Individuals are placed on the nodes of the networks, interact with their neighbors and learn from the better one. We find that cooperator can survive for a broad degree 4≤K≤70 by taking a loose type of cooperator clusters. The former simple corresponding relationship between macroscopic patterns and the microscopic PD interactions is broken. Under a condition that is unfavorable for cooperators such as large T and K, systems prefer to evolve to a loose type of cooperator clusters to support cooperation. However, compared to the well-known compact pattern, it is a suboptimal strategy because it cannot help cooperators dominating the population and always corresponding to a low cooperation level.