Blockade of Autophagy Aggravates Endoplasmic Reticulum Stress and Improves Paclitaxel Cytotoxicity in Human Cervical Cancer Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Autophagy is one of the ways to degrade unfolded proteins after endoplasmic reticulum (ER) stress. The purpose of this study is to determine whether a blockade of autophagy leads to aggravated endoplasmic reticulum stress, which then induces cells apoptosis in HeLa cells treated with paclitaxel. MATERIALS AND METHODS: Autophagy activation and the proapoptotic effects were characterized using monodansylcadaverine labeling and Hoechest staining, respectively. A Western blot analysis was used to detect the expression of apoptotic and autophagy-related genes. A flow cytometry was used to assess the cell apoptosis ratio. RESULTS: Paclitaxel exposure induced the aggregation of autophagosomes in the cytoplasms of cervical cancer HeLa cells. The expression of Beclin 1 and LC3 II were upregulated, but p62 was downregulated, which suggests that autophagy was promoted by paclitaxel. On the other hand, the expression of GRP78 obviously increased, suggesting that ER stress was induced after paclitaxel treatment. The cell proliferation assay indicated that a knockdown of Beclin 1 sensitized HeLa cells to paclitaxel. Furthermore, paclitaxel-mediated apoptotic cell death was further potentiated by the pretreatment with autophagy inhibitor chloroquine or small interfering RNA against Beclin 1. These results suggest that an induction of autophagy by paclitaxel may induce cell survival rather than cell death in HeLa cells; moreover, inhibition of autophagy led to an aggravated ER stress and an induction of downstream apoptosis. CONCLUSION: Our results reveal autophagy induced by paclitaxel conferred protection of tumor cells against apoptosis, and blockade of autophagy subsequently aggravated ER stress, enhancing the apoptosis associated with paclitaxel treatment in HeLa cells.

Effect of autophagy gene Beclin 1 on the growth of cervical cancer HeLa cells in vitro and vivo / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of autophagy gene Beclin 1 on growth of cervical cancer HeLa cells in vitro and vivo.</p><p><b>METHODS</b>The eukaryotic expression vector of Beclin1 was constructed and transfected via lipofectamine into HeLa cells. The experimental cells were classified into 3 groups: pcDNA3.1(+)-Beclin1 group,pcDNA3.1(+) group and HeLa group. Real time-ploymerase chain reaction and Western blot were used for detecting expression of Beclin1 mRNA and protein in the transfected cells. Flow cytometry (FCM) was employed to observe the effect of transfection on the apoptosis of HeLa cells, and proliferation was analyzed by MTT assay. The formation of autophagic vacuoles was measured by MDC staining. HeLa cells transfected with plasmid pcDNA3.1(+)-Beclin1 and pcDNA3.1(+) were inoculated subcutaneously in nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed.</p><p><b>RESULTS</b>Eukaryotic expression vector pcDNA3.1(+)-Beclin1 was constructed successfully. It significantly improved the expression of Beclin1 mRNA and protein in HeLa cells. The proliferation of HeLa cells was inhibited, and the inhibition rate was 58.7%. FCM investigation showed that the apoptotic rate was (28.22 ± 2.34)% of pcDNA3.1(+)-Beclin1 group, significantly higher than the (14.6 ± 4.6)% in the pcDNA3.1(+) group and (11.2 ± 3.0)% in the HeLa group (P < 0.05). The monodansylcadaverin (MDC) staining showed significantly more autophagic vacuoles in the pcDNA3.1(+)-Beclin1 group (10.9%) than that in the pcDNA3.1(+) group (3.1%) and HeLa group (2.5%) (P < 0.05). After transfected with vector pcDNA3.1(+)-Beclin1, the carcinogenic activity of HeLa cells was decreased in nude mice, and the inhibition rate of tumor growth was 52.2%.</p><p><b>CONCLUSIONS</b>Autophagy gene Beclin 1 overexpression can inhibit the proliferation and growth of HeLa cells in vitro and vivo,while promote autophagy and apoptosis of HeLa cells. So it might be one of new gene therapy strategies for cervical carcinoma.</p>

Growth inhibitory effects of Beclin1 gene on epithelial ovarian cancer SKOV3 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effect of Beclin1 overexpression on the growth of ovarian carcinoma cell line SKOV3 in vitro and in vivo.</p><p><b>METHODS</b>The recombinant plasmid pcDNA3.1/Beclin1 was constructed and transfected into SKOV3 cells via lipofectamine 2000. MTT assay was used to evaluate the effect of Beclin1 overexpression on the proliferation and growth of the transfected cells, whose apoptosis and autophagy were analyzed by flow cytometry. SKOV3 cells transfected with the plasmids pcDNA3.1/Beclin1 or pcDNA3.1 were inoculated subcutaneously in nude mice, and their carcinogenic and growth activities in vivo were evaluated.</p><p><b>RESULTS</b>MTT assay showed that transfection with pcDNA3.1/Beclin1 significantly inhibited the proliferations of SKOV3 cells, with a cell inhibition rate of 58.68% (P<0.05). The transfection also resulted in a cell apoptosis rate of (21.26-/+3.89)%, significantly higher than that of pcDNA3.1 trasnfection (P<0.05). Flow cytomerty showed that pcDNA3.1/Beclin1 transfection of SKOV3 cells produced a significantly higher MDC fluorescent intensity than pcDNA3.1 transfection. The SKOV3 cells transfected with vector pcDNA3.1/Beclin1 also showed decreased carcinogenic activity in nude mice, with a growth inhibition rate of 50.27%.</p><p><b>CONCLUSION</b>Beclin1 overexpression can inhibit the proliferation and growth of SKOV3 cells in vitro and vivo, suggesting its potential role in gene therapy of ovarian carcinoma.</p>