Blockade of Autophagy Aggravates Endoplasmic Reticulum Stress and Improves Paclitaxel Cytotoxicity in Human Cervical Cancer Cells
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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 autophagyleads to aggravated endoplasmic reticulum stress, which then induces cells apoptosis inHeLa cells treated with paclitaxel. Materials and MethodsAutophagy activation and the proapoptotic effects were characterized using monodansylcadaverinelabeling and Hoechest staining, respectively. A Western blot analysis was usedto detect the expression of apoptotic and autophagy-related genes. A flow cytometry wasused to assess the cell apoptosis ratio. ResultsPaclitaxel exposure induced the aggregation of autophagosomes in the cytoplasms ofcervical cancer HeLa cells. The expression of Beclin 1 and LC3 II were upregulated, but p62was downregulated, which suggests that autophagy was promoted by paclitaxel. On theother hand, the expression of GRP78 obviously increased, suggesting that ER stress wasinduced after paclitaxel treatment. The cell proliferation assay indicated that a knockdownof Beclin 1 sensitized HeLa cells to paclitaxel. Furthermore, paclitaxel-mediated apoptoticcell death was further potentiated by the pretreatment with autophagy inhibitor chloroquineor small interfering RNA against Beclin 1. These results suggest that an induction ofautophagy 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 ofdownstream apoptosis. ConclusionOur results reveal autophagy induced by paclitaxel conferred protection of tumor cellsagainst apoptosis, and blockade of autophagy subsequently aggravated ER stress, enhancingthe apoptosis associated with paclitaxel treatment in HeLa cells.
Abstract
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 autophagyleads to aggravated endoplasmic reticulum stress, which then induces cells apoptosis inHeLa cells treated with paclitaxel. Materials and MethodsAutophagy activation and the proapoptotic effects were characterized using monodansylcadaverinelabeling and Hoechest staining, respectively. A Western blot analysis was usedto detect the expression of apoptotic and autophagy-related genes. A flow cytometry wasused to assess the cell apoptosis ratio. ResultsPaclitaxel exposure induced the aggregation of autophagosomes in the cytoplasms ofcervical cancer HeLa cells. The expression of Beclin 1 and LC3 II were upregulated, but p62was downregulated, which suggests that autophagy was promoted by paclitaxel. On theother hand, the expression of GRP78 obviously increased, suggesting that ER stress wasinduced after paclitaxel treatment. The cell proliferation assay indicated that a knockdownof Beclin 1 sensitized HeLa cells to paclitaxel. Furthermore, paclitaxel-mediated apoptoticcell death was further potentiated by the pretreatment with autophagy inhibitor chloroquineor small interfering RNA against Beclin 1. These results suggest that an induction ofautophagy 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 ofdownstream apoptosis. ConclusionOur results reveal autophagy induced by paclitaxel conferred protection of tumor cellsagainst apoptosis, and blockade of autophagy subsequently aggravated ER stress, enhancingthe apoptosis associated with paclitaxel treatment in HeLa cells.
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