[Expression of Apoptosis-related Protein Livin and Caspase-3 in Diffuse Large B Cell Lymphoma and Its Clinical Significance].

OBJECTIVE: To detect the expression levels of Livin and Caspase-3 proteins in diffuse large B cell lymphoma (DLBCL), and to explore its correlation with clinicopathological characteristics, so as to provide a reference for clinical treatment and evaluation prognosis of DLBCL. METHODS: Immunohisto-chemical staining was performed to detect the expression of Livin and Caspase-3 in the samples from 51 DLBCL patients and 20 patients with reactive hyperplasia of lymph node (RHL). The relation between Livin and Caspase-3 with the factors influencing the prognosis of DLBCL was analyzed. RESULTS: Livin+ was not expressed in the tissues of RHL, but its expression rates reached to 58.82%(30/51) in 51 cases of DLBCL(P<0.05). The expression rats of Caspase-3 in DLBCL and RHL were 25.49%(13/51) and 85.0%(17/20) respectively, and the difference was statistically significant(P<0.05). The expression level of Livin in DLBCL tissues was related with clinical stage and pathological type(P<0.01, P<0.01). The expression of Livin was not obviously related with sex, age and extranodal involvement. The expression level of Caspase-3 in DLBCL tissues was related with clinical stage (P<0.05). The expression of Caspase-3 was not obviously related with sex, age, pathological type and extranodal involvement. There was a negative correlation between Livin and Caspase-3 in DLBCL(γs=-0.333,P=0.017). Kaplan-meier survival analysis revealed that the expressions of Livin and Caspase-3 were related stalistically significantly with the patients prognosis (P<0.01, P<0.05). CONCLUSION: Over-expression of Livin in DLBCL and DLBCL of non-GCB subtypes, and low-expression of Caspase-3 in DLBCL may play a significant role in clinical prognosis of DLBCL.

Circadian Gene CLOCK Affects Drug-Resistant Gene Expression and Cell Proliferation in Ovarian Cancer SKOV3/DDP Cell Lines Through Autophagy.

Abnormal autophagy regulation affects the chemoresistance of ovarian cancer, during which the circadian gene clock may play a major role. In this study, RNA interference plasmid pSUPER-Clock and overexpression plasmid pcDNA3.1-Clock of CLOCK were used to stably transfect the SKOV3/DDP cells by lipofection. Upon screening, the in vitro transfected cell lines with pSUPER-Clock, the autophagy level, and G0/G1 phase cells were significantly reduced, and the expression levels of Clock, LC3, P-gp, and MRP2 were inhibited. In contrast, the autophagy level and G0/G1 phase cells in cell lines transfected with pcDNA3.1-Clock were significantly increased, and the expressions of Clock, LC3, P-gp, and MRP2 were enhanced. In comparison with the untransfected control group showed the percentage of apoptotic cells in SKOV3/DDP cell lines of Clock interfering expression group after cisplatin treatment was significantly increased while the survival was substantially reduced. These results indicated that inhibiting the circadian gene Clock expression can reverse the cisplatin resistance of ovarian cancer SKOV3/DDP cell lines by affecting the protein expression of drug resistance genes during which autophagy plays an important role. The CLOCK gene may be designated as a novel candidate for targeted gene therapy in drug-resistant ovarian cancer.

Interfering EZH2 Expression Reverses the Cisplatin Resistance in Human Ovarian Cancer by Inhibiting Autophagy.

We aimed to determine the effects of the inhibition of enhancer of zeste homolog 2 (EZH2) gene expression on the cisplatin resistance of the human ovarian cancer cell line, SKOV3/DDP, and to identify the underlying mechanisms. SKOV3/DDP cells were stably transfected with pSUPER-EZH2 (EZH2 RNA interference plasmid) or pcDNA3.1-EZH2 (EZH2 gene overexpression plasmid) using the lipofection method. Real-time fluorescence quantitative reverse transcription polymerase chain reaction and western blotting confirmed that EZH2 expression was downregulated in pSUPER-EZH2-transfected cells. Flow cytometry revealed that EZH2 inhibition did not induce apoptosis, but significantly inhibited autophagy. In addition, it significantly increased the expression of the cellular senescence-signaling proteins p14(ARF), p16(INK4a), p53, pRb, and p21, and significantly decreased the expression of cyclin-dependent kinase (CDK)1, CDK2, and H3K27me3. Cellular senescence was characterized by a significant increase in the G0/G1 ratio and the restoration of sensitivity to cisplatin in the drug-resistant cells. These findings suggest that interfering with EZH2 expression can inhibit SKOV3/DDP cell autophagy and reverse resistance to cisplatin. The underlying mechanisms could be associated with the regulation of the cellular senescence-signaling pathway.

Effect of autophagy-related beclin1 on sensitivity of cisplatin-resistant ovarian cancer cells to chemotherapeutic agents.

The purpose of the study was to determine the effects of autophagy related gene Beclin1 at different levels of expression on the sensitivity of cisplatin-resistant ovarian cancer cells (SKOV3/DDP) to different chemotherapeutics. In pSUPER-Beclin1 transfected cells, real-time fluorescence quantitative RT-PCR and Western blot analysis showed that expression was significantly inhibited. Flow cytometry revealed that the mean fluorescence intensity (MDC), reflecting autophagy, and cells in the G0/G1 phase were markedly reduced. When compared with the blank control group, inhibition of Beclin1 expression in SKOV3/DDP cells not only increased the rate of apoptosis following treatment with chemotherapeutics, but also increased the sensitivity. These findings suggest that Beclin1 expression plays an important role in chemotherapeutic agent-induced death of SKOV3/DDP cells. Inhibition of autophagy related gene Beclin1 expression in SKOV3/DDP cells may increase the rate of apoptosis and elevate the sensitivity to chemotherapeutics.

Inhibition of Beclin 1 expression enhances cisplatin-induced apoptosis through a mitochondrial-dependent pathway in human ovarian cancer SKOV3/DDP cells.

The purpose of this study was to determine the influence of autophagy on cisplatin-induced ovarian cancer SKOV3/DDP cell line death through regulation of the expression of the autophagy gene, Beclin 1, and to explore the potential mechanism underlying the relationship between autophagy and apoptosis. When compared with a blank control group, the proportion of apoptotic cells undergoing Beclin 1 interfering increased significantly after cisplatin treatment, accompanied by reduction in mitochondrial membrane potential, increase in activities of caspase-9/3 and cytoplasmic cytochrome C, elevation of Bax expression, and reduction in Bcl-2 expression. However, the proportion of apoptotic cells with Beclin 1 overexpression reduced. These findings suggest that Beclin 1 plays an important role in the regulation of potent antitumor activity through a mitochondrial-dependent pathway in SKOV3/DDP cell line, and inhibition of Beclin 1 expression may become a new target for the sensitization therapy of ovarian cancer with cisplatin.

Beclin 1 influences cisplatin-induced apoptosis in cervical cancer CaSki cells by mitochondrial dependent pathway.

PURPOSE: To investigate the role of Beclin 1 expression on the cisplatin-induced apoptosis in cervical cancer CaSki cells and to explore the potential mechanism underlying this effect. MATERIALS AND METHODS: After overexpression or partial silencing of Beclin 1 in cervical cancer CaSki cells, the transfected group and the control group were treated with cisplatin for 24 hours. The percentage of apoptotic cells were assessed by flow cytometry. The mitochondrial membrane potential and activities of caspase-8/9/3 were detected by JC-1 fluorescence staining and colorimetry. The expression of cytochrome c was measured using a Western blot. The messenger RNA expression of Bax and Bcl-2 were detected by real-time quantitative reverse transcription polymerase chain reaction. RESULTS: Expression of Beclin 1 protein was up-regulated in overexpressed transfectants of CaSki cells. After treatment with cisplatin, the Beclin 1 overexpression group led to the decrease of mitochondrial membrane potential and increase of activities of caspase-9 and caspase-3, and showed a greater increase in apoptosis than did the nontransfected group. Furthermore, Beclin 1 overexpression resulted in increased cytoplasmic cytochrome c and Bax expression and decreased mitochondrial cytochrome c and Bcl-2 expression. CONCLUSION: Overexpression of Beclin 1 in CaSki cells may influence cisplatin-induced apoptosis by mitochondrial dependent pathway.

Beclin1 overexpression inhibitis proliferation, invasion and migration of CaSki cervical cancer cells.

The influence of the autophagy-related gene Beclin1 on proliferation, invasion and metastasis of the cervical cancer CaSki cells and its possible mechanism in vitro were here targeted. After the overexpression vector pcDNA3.1-Beclin1 and RNA interference vector pSUPER-Beclin1 were transfected into CaSki cells in vitro, stable expression cell lines demonstration Beclin1 expression was upregulated, and VEGF and MMP-9 expression were decreased, leading to cell arrest in the G0/G1 phase of the cell cycle. MTT assays further revealed proliferation of cells was significantly inhibited in Beclin1-overexpressing transfectant cells, with invasion and metastasis also being inhibited in Transwell chamber assays. The present results suggest that Beclin1 inhibits invasion and metastasis of cervical cancer CaSki cells in vitro. Mechanisms probably involve Beclin1 inhibition of cell proliferation, and decreased expression of VEGF and MMP-9 proteins.

Modulatory effects of Beclin 1 on expression of angiopoietin and Tie-2 receptor in human cervical cancer cells.

AIM: To investigate the effect of Beclin 1, an autophagy gene, on the expression of angiopoietin (Ang) protein and the Tie-2 receptor in CaSki human cervical cancer cells. MATERIALS AND METHODS: Beclin 1 overexpression (pcDNA3.1-Beclin1) and knockdown (pSUPER-Beclin1) plasmids were independently transfected into CaSki cells, and stably transfected cells were selected. Real-time fluorescence quantitative PCR and Western blot analyses were performed to detect the mRNA and protein expression of Beclin 1, Ang-1, Ang-2, and Tie-2. MTT assays were employed to determine cell proliferation rates. RESULTS: In the cells transfected with pcDNA3.1-Beclin1, the expression of Beclin 1, Ang-2, and Tie-2 was markedly increased, but expression of Ang-1 was dramatically reduced. MTT assays revealed that the proliferation of these cells was also significantly suppressed. In the CaSki cells transfected with pSUPER-Beclin1, the expression of Beclin 1, Ang-2, and Tie-2 was inhibited. CONCLUSION: Overexpression of Beclin 1 can inhibit the proliferation of CaSki cells, which may be attributed to an imbalance among the expression of Ang-1, Ang-2 and Tie-2.

Over-expression of the Beclin1 gene upregulates chemosensitivity to anti-cancer drugs by enhancing therapy-induced apoptosis in cervix squamous carcinoma CaSki cells.

The purpose of this study was to investigate whether the autophagy-related gene, Beclin1, plays a role in the regulation of chemosensitivity to anti-cancer drugs in cervical cancer CaSki cells. Expression of the Beclin1 protein was up-regulated in pcDNA3.1-Bec transfectants and led to cell arrest in the G(0)/G(1) phase of the cell cycle. The MTT assay indicated that over-expression of Beclin1 sensitized CaSki cells to chemotherapeutic drugs (cisplatin, paclitaxel, 5-fluorouracil, and epirubicin) and induced greater degrees of cytotoxicity than vector-only controls. After treatment with anti-cancer drugs, flow cytometric analysis indicated that the Beclin1-transfected group showed a greater increase in apoptosis than did the non-transfected group. Furthermore, pSUPER-Bec transfectants did not lead to a significant increase of resistance to each of these anti-cancer drugs. These results suggest that Beclin1 plays an important role in the regulation of potent anti-tumor activity, and over-expression of Beclin1 in CaSki cells may enhance apoptosis signaling induced by anti-cancer drugs.

[Effects of combined use of total alkaloids of Uncaria rhynchophylla and Coryadlis ambailis migo on cerebral ischemia-reperfusion injury in rats].

OBJECTIVE: To study the effects of combined use of total alkaloids (TA) of Uncaria rhynchophylla (UR) and Coryadlis ambailis migo (CAM) on cerebral ischemia/reperfusion injury in rats. METHODS: Rat model of middle cerebral artery ischemia/reperfusion was established, the changes of neurological state was scored before and after treatment with the two kinds of TA, single or combined, and the changes of cerebral infarcted volume, cerebral water content, activities of NOS and SOD and content of MDA in rats' brain were estimated as well. RESULTS: After being treated with the combination of both TA, the average neurological score, cerebral infracted volume, cerebral water content, activity of NOS and content of MDA in the model rats significantly decreased, and the activity of SOD was significantly increased (all P < 0.05). The effect of combined use of the two TA was higher than that of use TA of UR or CAM alone (P <0.05). Moreover, the central nervous system inhibitory effect induced by combined TA was significantly weaker than that of UR. CONCLUSION: Combined use of TA of UR and CAM may facilitate the protection against cerebral ischemia/reperfusion damage, the action mechanism might be relevant to reducing the lipid peroxidation injury of brain cells through inhibiting the NOS activity and increasing the SOD activity.