[Effect of Sunitinib on Pyroptosis of Drug-Resistant Leukemia K562/ADR Cells and Its Pathway].

OBJECTIVE: To investigate the inducing effect of sunitinib on the death of drug-resistant leukemia K562/ADR cells and the related signaling pathway. METHODS: K562/ADR cells were treated with different concentrations of sunitinib, and the cells were collected at 24, 48, 72, and 96 hours, respectively. MTS assay was used to detect the effect of sunitinib on the proliferation of K562/ADR cells, and the appropriate sunitinib intervention time and concentration were determined. QPCR and Western blot were used to detect the mRNA and protein expression levels of apoptosis-related genes in K562/ADR cells treated with sunitinib. Four different cell death inhibitors Nec-1, VX-765, CQ and Fer-1 were used to detect the death mode of K562/ADR cells treated with sunitinib. QPCR and Western blot were used to detect the mRNA and protein expression levels of pyroptosis-related genes in K562/ADR cells treated with sunitinib. RESULTS: Sunitinib significantly inhibited the proliferation of K562/ADR cells in a time - and concentration-dependent manner(R48 H=0.9579, r4 µg/ml=0.9740). The IC50 of sunitinib was (3.96±0.14) µg/ml at 48 hours. The mRNA and protein expression levels of apoptosis-related genes Bax, BCL-2 , Caspase-3 and Caspase-9 in K562/ADR cells treated with sunitinib did not change significantly. After treatment with four different cell death inhibitors, only the pyroptosis inhibitor VX-765 could significantly reverse the inhibitory effect of sunitinib on the proliferation of K562/ADR cells (P<0.01). The mRNA and protein expression levels of pyroptosis-related genes Caspase-1, Caspase-4, Caspase-5, NLRP3, GSDMD and IL-1ß in K562/ADR cells treated with sunitinib were significantly increased (P<0.01). CONCLUSION: Sunitinib can induce pyroptosis in drug-resistant leukemia K562/ADR cells. Further study of the signaling pathways related to pyroptosis may provide experimental basis for the treatment of drug-resistant leukemia.

miR-29b-3p suppresses the malignant biological behaviors of AML cells via inhibiting NF-κB and JAK/STAT signaling pathways by targeting HuR.

BACKGROUND: HuR/ELAVL1 (embryonic lethal abnormal vision 1) was a downstream target of miR-29b in some cancer cells. HuR protein exerts important prognostic effects of involving in the pathogenesis and development of acute myeloid leukemia (AML). This study aims to investigate the role of miR-29b-3p in biological behaviors of AML cells by targeting HuR and the involvement of the NF-κB and JAK/STAT signaling pathways. METHODS: The expressions of HuR and miR-29b-3p in AML cells were determined using RT-qPCR and Western blot, and the association between them was analyzed using the Spearman method. Next, the target relationship between HuR and miR-29b-3p was predicted by biological information databases and verified by the dual-luciferase reporter gene assay. MTS, methyl cellulose, flow cytometry and transwell assay were employed to detect the cell proliferation, clone formation, cell cycle and apoptosis, invasion and migration respectively, the effect of miR-29b-3p targeted HuR on the biological behaviors of AML cells was explored after over- /down-expression of miR-29b-3p and rescue experiment. Then, immunofluorescence assay and western blot were employed to detect location expression and phosphorylation levels of NF-κB and JAK/STAT signaling pathways related molecules respectively. RESULTS: HuR was negatively correlated with miR-29b-3p, and was the downstream target of miR-29b-3p in AML cells. When miR-29b-3p was overexpressed in AML cells, HuR was down-regulated, accompanied by cell viability decreased, cell cycle arrest, apoptosis increased, invasion and migration weakened. Moreover, the opposite result appeared after miR-29b-3p was down-regulated. The rescue experiment showed that miR-29b-3p inhibitor could reverse the biological effect of HuR down-regulation in AML cells. Molecular pathway results showed that miR-29b-3p could inhibit p65 expression in nucleus and phosphorylation levels of p65, IκBα, STAT1, STAT3 and STAT5. CONCLUSION: miR-29b-3p can inhibit malignant biological behaviors of AML cells via the inactivation of the NF-κB and JAK/STAT signaling pathways by targeting HuR. miR-29b-3p and its target HuR can be used as a new potential molecular for AML treatment.

[Effect of γδ T cells on the Proliferation, Apoptosis and Autophagy of Multiple Myeloma Cells].

AbstractObjective: To investigate the effect of γδ T cells on the proliferation, apoptosis and autophagy of multiple myeloma cells. METHODS: Peripheral blood mononuclear cells (PBMNC) were isolated from healthy volunteers, and stimulated with zoledronic acid (Zol) in combination with rhIL-2. Flow cytometry analysis was used to detected the purity of γδ T cells. γδ T cells were collected and co-cultured with RPMI-8226 or U-266 cells at different effector target ratios. The proliferation of RPMI-8226 or U-266 cell lines were detected by CCK-8. Cell cycle and cell apoptosis were detected by flow cytometry and Western blot．The expressions of autophagy-related proteins were detected by Western blot. RESULTS: γδ T cells can be expanded in vitro. γδ T cells could inhibit the proliferation of RPMI-8226 or U-266 cells, induced cell cycle arrest and promoted apoptosis in an effector target-dependent manner. In addition, γδ T cells could induce autophagy of myeloma cells, inhibited the expression of autophagy-related PI3K, P-AKT and P-mTOR, while increased the expression of AMPK and Beclin-1. CONCLUSION: γδ T cells can inhibit the proliferation of RPMI-8226 and U-266 myeloma cells, induce cell cycle arrest, promote apoptosis, and enhance autophagy in vitro. The mechanism may be related to inhibition of PI3K/AKT/mTOR signaling pathway and/or activation of AMPK/Beclin-1 signaling pathway.

Silencing of the CrkL gene reverses the doxorubicin resistance of K562/ADR cells through regulating PI3K/Akt/MRP1 signaling.

BACKGROUND: Doxorubicin is a first-line chemotherapy agent on human myelogenous leukemia clinical treatment, but the development of chemoresistance has largely limited curative effect. In this study, we aimed to evaluate the biological function and molecular mechanisms of CrkL to Doxorubicin resistance. METHODS: Quantitative reverse transcription-PCR (qRT-PCR) assay was performed to examine the expression of CrkL in K562 and K562/ADR cells. The expression of CrkL was silenced through RNA interference technology. MTT assay and flow cytometry were performed to detect the proliferation inhibition and apoptosis rate after CrkL siRNA transfection. The protein expression changes of PI3K/AKT/MRP1 pathway induced by CrkL siRNA were observed by Western Blot assay. Xenograft tumor model was carried out to observe tumor growth in vivo. RESULTS: We observed that silencing of CrkL could effectively increase apoptosis rate induced by doxorubicin and dramatically reversed doxorubicin resistance in K562/ADR cells. Further studies revealed knockdown CrkL expression suppressed PI3K/Akt/MRP1 signaling, which indicated CrkL siRNA reversed doxorubicin effect through regulating PI3K/Akt/MRP1 pathway. In addition, overexpression of MRP1 could evidently reduce apoptosis rate and reversed the inhibitory effects of doxorubicin resistance caused by CrkL siRNA on K562/ADR cells. Finally, in vivo experiments revealed that CrkL silencing acted a tumor-suppressing role in myelogenous leukemia via regulating PI3K/Akt/MRP1 signaling. CONCLUSION: Together, we indicated that CrkL is up-regulated in myelogenous leukemia cells and silencing of CrkL could reverse Doxorubicin resistance effectively. These results show a potential novel strategy for intervention chemoresistance in myelogenous leukemia during chemotherapy.

[Effect of MiR-29b-3p Targeting STAT3 on Proliferation and Apoptosis of Acute Myeloid Leukemia Cells].

OBJECTIVE: To investigate the effect of miR-29b-3p on apoptosis and proliferation of acute myeloid leukemia (AML) cells by targeting signal transducer and activator of transcription 3 (STAT3). METHODS: TargetScan and miRanda online databases were used to predict the binding sites of miR-29b-3p and STAT3 3'UTR. The targeting relationship between them was estimated by Dual-Luciferase reporter assay experiment. After miR-29b-3p over-expression, qPCR and Western blot were used to detect the expression of STAT3 mRNA and proteins, flow cytometry to determine the apoptosis of AML cells, and MTS to detect the changes of cell proliferation in each group. RESULTS: Dual-Luciferase reporter assay confirmed that STAT3 was the target gene of miR-29b-3p. After miR-29b-3p overexpression, the expression of STAT3 mRNA and protein decreased. Compared with the control groups, the proliferation of AML cells in the overexpression group decreased and the apoptosis increased (P<0.05). CONCLUSION: MiR-29b-3p can inhibit the proliferation and induce apoptosis of AML cells by down-regulating STAT3.

[Effect of SARI Overexpression on Apoptosis of CBFL Cells and Its Mechanism].

OBJECTIVE: To investigate the effect of SARI overexpression on the proliferation and apoptosis of core binding factor leukemia (CBFL) cells and explore the potential molecular mechanisms. METHODS: C-KIT N822K mutation status in Kasumi-1 cell line was detected by exon 17 sequencing. Then the SARI lentiviral vector (pGC-FU-SARI) was constructed, meanwhile Kasumi-1 cells were transfected with the SARI lentiviral vector. Quantitative PCR and Western blot were employed to identify efficacy of SARI overexpression after the transfection of cells. Cells were divided into three groups, including the cells infected with pGC-FU-SARI (OE group), the cells infected with pGC-FU-GFP (NC group) and the untreated cells (blank control group). Cell proliferative activity was tested by MTT assay, cell apoptosis was measured by flow cytometry (FCM) and the expression of apoptosis-related proteins: BCL-2,BAX,Cyto C,Caspase 9,Caspase 3,cleaved-Caspase 3,PARP and cleaved-PARP as well as PI3K/Akt pathway proteins: PI3K（p85）,p-PI3K（p85）,Akt and p-Akt were detected by Western blot. RESULTS: The Kasumi-1 cells were detected to bear c-KIT N822K (T>A) mutation. The Kasumi-1 cells with SARI was overexpression were construeted successfully. Compared with NC group, the cell proliferation was decreased and cell apoptosis was increased; BCL-2 expression was reduced, BAX expression was enharued; cyto C expression appeared; the expression of Caspase 9 and Caspase 3 was down-regulated, the expression of cleaved Caspase 3 was up-regulated; the PARP expression was decreased, cleaved PARP expression was increased; the phosphorylation level of PI3K/Akt pathway proteins: p-PI3K/PI3K, p-Akt/Akt was down-regulated in OE group (P<0.05). CONCLUSION: SARI gene may suppress the proliferation of CBFL cells, and induce their apoptosis through the mitochondrial pathway, which may be related with the inhibition of PI3K/Akt pathway.

[Cytocompatibility of Co-Cr ceramic alloys after recasting].

PURPOSE: To evaluate the cytocompatibility of Co-Cr ceramic alloys after recasting. METHODS: Cobalt-chromium (Co-Cr) ceramic alloys were recasted for 1-3 times. Samples of Co-Cr ceramic alloys were immersed in DMEM culture medium. The concentration of ions of leaching liquor was determined by ICP-MS total quantization method. L-929 cells were cultured with leaching liquor, flow cytometry was used to test cell cycle progression, Annexin-V-FITC/PI apoptosis staining kit was used to quantitatively detect cell death patterns. SPSS 13.0 software package was used for statistical analysis. RESULTS: After casting for 1-3 times, no significant increase in the concentration of ions of Co-Cr ceramic alloys was noted (P>0.05) except Ga (P<0.05) . No significant differences in L-929 cell cycle were found in Co-Cr ceramic alloys between the samples cast for 1 time and for 2~3 times (P>0.05). No significant difference in the rates of apoptosis and necrosis was found (P>0.05). CONCLUSIONS: The biocompatibility of Co-Cr ceramic alloys casted for 1-3 times is not changed.

Genotype GG of rs895819 Functional Polymorphism Within miR-27a Might Increase Genetic Susceptibility to Colorectal Cancer in Han Chinese Population.

BACKGROUND: MicroRNA-27a (miR-27a) is supposed to be an oncogene in various types of cancers, and genetic variation of miR-27a might result in aberrant expression and abnormal second structure of mature-miR-27a, contributing to elevated genetic risk and poor prognosis for colorectal cancer (CRC). METHODS: In order to explore the possible association between rs895819 within miR-27a and CRC in Han Chinese population, we investigated the genotype distributions of rs895819 in 508 CRC cases and 562 healthy check-up controls using TaqMan genotype discrimination system, and analyzed the possible association between them. Odds ratio (OR) and 95% confidential interval (95% CI) were used to assess the strength between allele and genotype of the locus and risk of CRC. RESULTS: In our study, we found that genotype GG of rs895819 was significantly associated with an increased risk for CRC (17.1% vs. 11.6%, adjusted OR = 1.546, 95% CI = 1.070-2.236), and allele A carrier (AA/AG) was significantly associated with a decreased risk for CRC (82.9% vs. 89.4%, adjusted OR = 0.63, 95% CI = 0.446-0.893). In addition, a significant association was observed between genotype GG and larger tumor size (>5 cm; P < 0.001), and allele G was significantly associated with higher pathological stage (TNM-III) (P = 0.008). CONCLUSION: These results indicated that miR-27a might be involved in the development and progression of CRC, genotype GG within rs895819 might be a genetic susceptible factor for CRC. Further multicentral, large sample size, and well-designed epidemiological study as well as functional study are warrant to verify our findings.

Diagnostic Accuracies of the TUNEL, SCD, and Comet Based Sperm DNA Fragmentation Assays for Male Infertility: a Meta-analysis Study.

BACKGROUND: Recent studies have provided new insights into the diagnostic value of sperm DNA fragmentation (SDF) for male factor sterility. This study aimed to systematically evaluate the diagnostic accuracy of the SDF test for male infertility. METHODS: Eligible studies were retrieved by searching electronic databases. The quality of the studies was assessed on the basis of quality assessment for studies of diagnostic accuracy (QUADAS) criteria tool. The bivariate metaanalysis model was employed to summarize the diagnostic indices and plot the summary receiver operator characteristic (SROC) curve by using Meta-disc 1.4 software. Influence analysis, meta-regression, and publication bias assay were all conducted through Stata 12.0 software. RESULTS: Our bivariate random effect meta-analysis yielded an AUC (area under curve) value of 0.9211 with a sensitivity (95% confidence interval) of 0.80 (0.78 - 0.82) and specificity of 0.83 (0.80 - 0.86) for the use of the SDF test in differentiating infertile males from normal fertile controls. Moreover, our subgroup analysis suggested that SDF analysis with a single TUNEL test resulted in an AUC value of 0.9506, with a pooled sensitivity of 0.77 (0.74 - 0.80) and specificity of 0.91 (0.87 - 0.94), while SCD and Comet assays displayed a combined sensitivity of 0.77 (0.67 - 0.81) or 0.91 (0.88 - 0.94), and specificity of 0.84 (0.75 - 0.91) or 0.63 (0.54 - 0.70), accompanied by an AUC value of 0.8408 or 0.9473. CONCLUSIONS: The SDF assay confers a relatively high diagnostic accuracy for infertility detection, among which the TUNEL based methodology seems to achieve higher accuracy than the SCD and Comet assays.

[Effect of SU11248 on leukemia cell line K562 and its molecular mechanisms].

This study was aimed to investigate the effect of SU11248 on proliferation and apoptosis of leukemia cell line K562 in vitro and its mechanism. The inhibitory effect of 3.2 µg/ml SU11248 on K562 proliferation was tested by MTT assay. The ability of SU11248 to induce apoptosis of K562 cells was examined by TUNEL and DNA ladder. The expression of C-MYC, hTERT and BCR-ABL mRNA in K562 cells was detected by RT-PCR. The protein expression of Akt and p-Akt in K562 cells was detected by Western blot. The results showed that the proliferation of K562 cells was obviously inhibited by 3.2 µg/ml SU11248 in a time-dependent manner. SU11248 could induce K562 cells apoptosis in dose-and time-dependent manner. The mRNA expression of C-MYC, hTERT and BCR-ABL was reduced significantly by SU11248 in a time-dependent manner (P < 0.05). Western blot detection showed that the expression of p-Akt protein in K562 cells decreased in dose-and time-dependent manner after SU11248 treatment, but the expression of Akt was not significantly changed. It is concluded that SU11248 can inhibit the growth of K562 cells efficiently through inducing apoptosis, its mechanism may be closely relate with the expression down-regulation of C-MYC, hTERT, BCR-ABL and the inhibition of Akt phosphorylation.

Thrombolytic effects in vivo of nattokinase in a carrageenan-induced rat model of thrombosis.

BACKGROUND/AIMS: Nattokinase is a serine protease produced by Bacillus subtilis during the fermentation of the soybean product natto. The fibrinolytic activity and thrombolytic effects of nattokinase have been observed in vitro, but the effect in vivo has still to be researched. The objective of this study was to demonstrate the activity of nattokinase in vivo. METHODS: To establish a rat model of thrombosis, κ-carrageenan was injected subcutaneously into the toes of Sprague-Dawley (SD) rats. Histological examination confirmed thrombosis. The rats were then treated with varying doses of nattokinase and the resulting thrombolysis was histologically assessed. ELISA was used to determine the levels of the fibrin/fibrinogen degradation products (FDPs) and D-dimer, which are sensitive indices of fibrinolytic activity. Vermis kinase, a known thrombolytic agent, was used as a positive control. RESULTS: Biopsy results revealed partial thrombolysis in the tail vessels of the rats treated with nattokinase or vermis kinase. FDP and D-dimer levels were higher in rats treated with high-dose nattokinase than in those treated with saline. No difference in FDP or D-dimer levels was observed between rats treated with high-dose nattokinase and those treated with vermis kinase. CONCLUSIONS: Both the histological and physiological evidence from this study indicate that nattokinase exerts thrombolytic effects in vivo.

Effects of recasting on the biocompatibility of a Ni-Cr alloy.

OBJECTIVE: To evaluate the effects of recasting on the biocompatibility of a commercially available Ni-Cr alloy. METHODS: The alloy tested was cast and subsequently recast four more times. For each cast condition, 24 disk shaped specimens were fabricated (5 mm in diameter, 0.5 mm in thickness). All the recasting was performed without adding new alloy. After the first cast and following each recast, the surface composition and microstructure of the alloy were determined using an X-ray fluorescence spectrometer and optical microscope, respectively. The in vitro cytotoxicity and in vivo mucous irritation potential of the cast and recast Ni-Cr alloy were investigated. The results were statistically analysed at the significance level of 0.05. RESULTS: Recasting neither yielded to cytotoxicity or to changes in the surface composition of the Ni-Cr alloy tested. However, an increase in impurities and porosity of the surface structure was observed with recasting. Also, the segregation of the impurities to grain boundaries was evident after multiple castings. After the fourth recast, the alloys showed significantly greater mucosal irritation than the control. CONCLUSION: After fourth recast, the alloy of this type may contribute to mucosal inflammation. Furthermore, there is a need for diverse methods addressing different biological endpoints for the evaluation of dental alloys.

[Expression of P27(kip1) and cyclin G in patients with acute leukemia and its correlation].

This study was purposed to explore the expression of P27(kip1)and cyclin G in patients with acute leukemia (AL) and its correlation. The reverse polymerase chain reaction (RT-PCR) was used to analyse the expression of P27(kip1) and cyclin G mRNA in 89 AL patients and 10 normal persons; Western blot was used to analyze the expression of P27(kip1) and cyclin G protein in 39 AL patients and 10 normal persons. The results showed that the cyclin G mRNA and protein expressions in new diagnosed/relapsed cases of AL were significantly higher than those in patients with remission and normal controls (p < 0.05 and p < 0.01), but there was no difference between remission cases and normal controls (p > 0.05). The expression of P27(kip1) mRNA in newly diagnosed/relapsed patients with AL was not significantly different from patients with remission and normal controls (p > 0.05), while the P27(kip1) protein expression in remission cases of AL and normal controls was significantly higher than that in new diagnosed/relapsed cases (p < 0.05), but there was no difference between remission cases and normal controls (p > 0.05). The expression of P27(kip1) negatively and lowly correlated with the expression of cyclin G in patients with AL. It is concluded that the low expression of P27(kip1) and the high expression of cyclin G in patients with AL may have some correlation with genesis and development of AL and may be an indication for poor prognosis of AL.

[Killing effect of ZnPcH(1)-PDT on lymphoma cells].

The objective of this study was to investigate the effect of ZnPcH(1)-PDT on the lymphoma cells and its mechanism. Human Burkitt's lymphoma cell line CA46 and mouse lymphoma cell line P388 were selected as objects for study. The killing effect of ZnPcH(1)-PDT on cells were assessed by MTT method and colony formation assay; the cell death patterns were analyzed by AO/EB fluorescence stain, TdT-mediated dUTP nick end labeling (TUNEL), DNA ladder assay; and the different proportions of each death pattern were determined by Annexin-V(-FITC)/PI double stains. The results showed that ZnPcH(1)-PDT displayed anti-proliferation effect on both CA46 cells and P388 cells in dose-dependent manner. CA46 cells were less sensitive to PDT than P388 cells (p < 0.05). Furthermore, PDT could induce cell apoptosis in time-dependent manner. The rate of cell apoptosis increased in the PDT-treated cells. The results of Annexin-V(-FITC)/PI stain indicated that early apoptosis was the main death pattern in the PDT-treated CA46 cells, while early apoptosis and necrosis were the main death model in the PDT-treated P388 cells. It is concluded that ZnPcH(1)-PDT can effectively inhibit lymphoma cell proliferation and induce cell apoptosis.