HSP70 protects H9C2 cells from hypoxia and reoxygenation injury through STIM1/IP3R.

Hypoxia/reoxygenation (H/R) is used as an in vivo model of ischemia/reperfusion injury, and myocardial ischemia can lead to heart disease. Calcium overload is an important factor in myocardial ischemia-reperfusion injury and can lead to apoptosis of myocardial cells. Therefore, it is of great clinical importance to find ways to regulate calcium overload and reduce apoptosis of myocardial cells, and thus alleviate myocardial ischemia-reperfusion injury. There is evidence that heat shock protein 70 (HSP70) has a protective effect on the myocardium, but the exact mechanism of this effect is not completely understood. Stromal interaction molecule 1 and inositol 1,4,5-triphosphate receptor (STIM/1IP3R) play an important role in myocardial ischemia-reperfusion injury. Therefore, this study aimed to investigate whether HSP70 plays an anti-apoptotic role in H9C2 cardiomyocytes by regulating the calcium overload pathway through STIM1/IP3R. Rat H9C2 cells were subjected to transient oxygen and glucose deprivation (incubated in glucose-free medium and hypoxia for 6 h) followed by re-exposure to glucose and reoxygenation (incubated in high glucose medium and reoxygenation for 4 h) to simulate myocardial ischemia reperfusion-induced cell injury. H9C2 cell viability was significantly decreased, and lactate dehydrogenase (LDH) release and apoptosis were significantly increased after oxygen and glucose deprivation. Transfection of HSP70 into H9C2 cells could reduce the corresponding effect, increase cell viability and anti-apoptotic signal pathway, and reduce the apoptotic rate and pro-apoptotic signal pathway. After hypoxia and reoxygenation, the expression of STIM1/IP3R and intracellular calcium concentration of HSP70-overexpressed H9C2 cells were significantly lower than those of hypoxia cells. Similarly, direct silencing of STIM1 by siRNA significantly increased cell viability and expression of anti-apoptotic protein Bcl-2 and decreased apoptosis rate and expression of pro-apoptotic protein BAX. These data are consistent with HSP70 overexpression. These results suggest that HSP70 abrogates intracellular calcium overload by inhibiting upregulation of STIM1/IP3R expression, thus reducing apoptosis in H9C2 cells and playing a protective role in cardiomyocytes.

Long Non-Coding RNA NEAT1 Promotes the Proliferation, Migration, and Metastasis of Human Breast-Cancer Cells by Inhibiting miR-146b-5p Expression.

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer in women. Tumor recurrence and metastasis are the key causes of death in BC patients. Long non-coding RNA (lncRNA) is closely associated with BC progression. lncRNA nuclear-enriched abundant transcript (NEAT)1 has been reported to regulate the proliferation and mobility of several types of cancer cells. However, how lncRNA NEAT1 affects the proliferation and invasion of BC cells is not known. METHODS: Quantitative real time-polymerase chain reaction (qRT-PCR) was used to measure expression of lncRNA NEAT1 and microRNA (miR)-146b-5p in BC tissues and cell lines. Cell Counting Kit (CCK)-8, cell colony-formation, wound-healing, and Transwell™ assays were undertaken to determine the effects of lncRNA NEAT1 and miR-146b-5p on progression of BC cells. The interaction between lncRNA NEAT1 and miR-146b-5p was examined by luciferase reporter, RNA-binding protein immunoprecipitation (RIP), and RNA-pulldown assays. RESULTS: Expression of lncRNA NEAT1 was upregulated in BC tissues and cell lines. High expression of lncRNA NEAT1 predicted poor overall survival in BC patients. Silencing of expression of lncRNA NEAT1 inhibited epithelial-mesenchymal transition (EMT) and suppressed the proliferation, migration and invasion of BC cells. Ectopic expression of lncRNA NEAT1 induced EMT and promoted BC progression. Mechanistic investigations revealed that miR-146b-5p was a direct target of lncRNA NEAT1, and its expression was correlated negatively with expression of lncRNA NEAT1 in BC tissues. CONCLUSION: lncRNA NEAT1 could (i) serve as a novel prognostic marker for BC and (ii) be a potential therapeutic target for BC.

Combined thermosensitive in situ gel with AMD3100 in sutureless technique improves the survival and function of kidney transplants in mice.

The mouse is an optimal animal model for kidney transplantation. Recent reports suggest that application of poloxamer 407, a thermosensitive in situ gel, during the sutureless technique significantly increases animal survival, compared to traditional methods. However, further improvement of this technology is greatly needed but remains unexplored. Here, we detected significant inflammation at the region of ureter anastomosis, after kidney transplantation using poloxamer 407. Since chemokines play a pivotal role during inflammation, we implanted an Alzet osmotic pump that gradually releases AMD3100 (a specific inhibitor of the binding of stromal cell-derived factor 1 (SDF-1) to its receptor, CXCR4) at the site of ureter anastomosis in mice that had undergone kidney transplantation. We found that AMD3100 significantly reduced local inflammation, significantly improved animal survival after kidney transplantation, and significantly improved kidney function. Together, these data suggest that inhibition of chemokine signaling at the site of ureter anastomosis may substantially improve animal survival after kidney transplantation through suppression of suturing-related inflammation.

Synergistic antitumor responses by combined GITR activation and sunitinib in metastatic renal cell carcinoma.

Sunitinib, a multitargeted tyrosine kinase inhibitor, is the frontline therapy for renal and gastrointestinal cancers. In view of its well-documented proapoptotic and immunoadjuvant properties, we speculate that combination of Sunitinib and immunotherapy would provide a synergistic antitumor effect. Here, we report that a remarkably synergistic antitumor responses elicited by the combined treatment of Sunitinib and an agonistic antibody against glucocorticoid-induced TNFR related protein (GITR) in a model of metastatic renal cell carcinoma. Sunitinib significantly increased the infiltration, activation, and proliferation and/or cytotoxicity of CD8(+) T cells and NK cells in liver metastatic foci when combined with the anti (α)-GITR agonist, which was associated with treatment-induced prominent upregulation of Th1-biased immune genes in the livers from mice receiving combined therapy versus single treatment. Sunitinib/α-GITR treatment also markedly promoted the maturation, activation and cytokine production of liver-resident macrophages and DCs compared with that achieved by α-GITR or Sunitinib treatment alone in mice. Cell depletion experiments demonstrated that CD8(+) T cells, NK cells and macrophage infiltrating liver metastatic foci all contribute to the antitumor effect induced by combined treatment. Furthermore, mechanistic investigation revealed that Sunitinib treatment reprograms tumor-associated macrophages toward classically activated or "M1" polarization upon GITR stimulation and consequently mounts an antitumor CD8(+) T and NK cell response via inhibiting STAT3 activity. Thus, our findings provide a proof of concept that Sunitinib can synergize with α-GITR treatment to remodel the tumor immune microenvironment to trigger regressions of an established metastatic cancer.

miR-96 suppresses renal cell carcinoma invasion via downregulation of Ezrin expression.

BACKGROUND: The present study examined the role of microRNA (miR)-96 in renal cell carcinoma (RCC) invasion. METHODS: The expression of miR-96 was detected by quantitative reverse transcription-polymerase chain reaction in human RCC cell lines with high (Caki-1) and low (786-O) metastatic potential. Invasive ability and Ezrin expression were assessed in Caki-1 and 786-O cells transfected with a miR-96 mimic or inhibitor using wound healing assays, Transwell assays and western blotting. Expression of miR-96 and Ezrin was also examined in primary RCC samples from 17 patients with metastatic disease and 46 patients who maintained remission during a follow-up period of 37 months. RESULTS: miR-96 expression was significantly lower in Caki-1compared to786-O cells. The invasive ability of Caki-1 and 786-O cells increased following transfection of cells with miR-96 inhibitor, whereas it decreased following transfection with miR-96 mimic. Ezrin levels were negatively correlated with miR-96 in RCC, and inhibition of Ezrin expression suppressed the miR-96-induced change in invasive ability. The negative correlation between miR-96 and metastasis/Ezrin expression was also observed in human RCC specimens. CONCLUSIONS: These results suggest that miR-96 suppresses RCC invasion by modulating Ezrin expression.

Selenocystine-induced cell apoptosis and S-phase arrest inhibit human triple-negative breast cancer cell proliferation.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited effective treatment options. New therapeutic approaches are urgently needed to improve the prognosis of TNBC. Here we demonstrated that a redox modulator, selenocystine (SeC), significantly inhibits TNBC cell proliferation in a dose- and time-dependent manner. Through cell apoptosis assays and cell cycle distribution analyses, we have shown that the in vitro inhibitory effect of SeC on TNBC cells can be attributed to the induction of apoptosis and the S-phase arrest in a dose-dependent manner. Therefore, this finding implies that SeC potentially is a novel therapeutic agent for TNBC.

Mutant prevention concentrations of levofloxacin, pazufloxacin and ciprofloxacin for A. baumannii and mutations in gyrA and parC genes.

Fluoroquinolones are antimicrobial agents that are widely used clinically, but the increasing resistance of Acinetobacter baumannii (A. baumannii) to these agents is a matter of concern. We investigated mutant prevention concentrations (MPCs) of three fluoroquinolones, levofloxacin (LVX), pazufloxacin (PAZ) and ciprofloxacin (CIP). We analyzed an A. baumannii standard strain (ATCC19606) for mutation prevention indices (MPIs), MPCs and mutant selection windows as well as MICs of CIP, PAZ and LVX and compared the derived values with 34 A. baumannii strains collected in hospitals. In addition, A. baumannii standard strain (ATCC19606) fluoroquinolone-resistant mutants were investigated for gyrA and parC gene mutations. MPCs of CIP, prevention antibiotics concentration and LVX for A. baumannii ATCC19606 were 12.8, 5.6 and 2.8 µg ml(-1) and their MPIs were 16, 8 and 4, respectively. Clinically isolated A. baumannii strains had CIP, PAZ and LVX MPC value ranges of 1-8, 1-16 and 0.5-2 µg ml(-1) and their MPIs were 8, 8 and 4 µg ml(-1). Single gyrA mutations (Ser(83)-Leu(83)) occurred in 18 resistant strains (48.7%) and single parC mutations (Ala(79)-Asp(79) or (Ser(80)-Leu(80)) occurred in 8 resistant strains (21.6%), whereas gyrA and parC double mutations occurred in 2 (5.4%) of the resistant strains. MPC and MPI values of LVX were lower than that of CIP and PAZ. Single gyrA and parC mutations accounted for the majority of mutations (n=24), whereas double mutations occurred only in two strains.

New normal values not related to age and sex, of glomerular filtration rate by (99m)Tc-DTPA renal dynamic imaging, for the evaluation of living kidney graft donors.

The aim of this study was to investigate the normal values of glomerular filtration rate (GFR) by technetium-99m diaethylene-triamine-pentaacetic acid ((99m)Tc-DTPA) renal dynamic imaging for living kidney graft donors. In a total of 212 candidate donors, GFR was examined using (99m)Tc-DTPA renal dynamic imaging. Donors with GFR≥80mL/(min×1.73m(2)) and as low as with GFR≥70mL/(min×1.73m(2)) but a normal endogenous creatinine clearance rate (CCr) were quantified for living kidney donation. Differences in GFR levels based on sex and age were analyzed using rank correlation coefficient. Out of the 212 candidates, 161 were finally selected as kidney graft donors. The double kidney total GFR between the male and female donor groups, the GFR levels among differently-aged donor groups, and the GFR levels between the elderly (>55 years) and young- and middle-aged (≤55 years) donor groups did not show any significant difference (P>0.05). After kidney donation, renal function measured by blood urea nitrogen (BUN) and serum creatinine of all donors returned to normal within one week, and no serious complications were noticed. In conclusion, renal dynamic imaging by (99m)Tc-DTPA had a good accuracy and repeatability in GFR evaluation for living kidney donors. Candidate donors with GFR between 70mL/(min×1.73m(2)) and 80mL/(min×1.73m(2)) can be selected as kidney donors after strict screening. In living kidney donors GFR is not significantly correlated with age or sex.