Jianrui Song

Hypoxia-induced autophagy contributes to the chemoresistance of hepatocellular carcinoma cells.

Hypoxia commonly exists in solid tumors. In this adverse condition, adaptive responses including autophagy are usually provoked to promote cell survival. In our study, autophagy, a lysosomal-mediated degradation pathway, is demonstrated a protective way to make hepatocellular carcinoma cells be resistant to chemotherapy under hypoxia. Compared with normoxia, chemotherapeutic agents-induced cell death under hypoxia was significantly decreased, as a result of the reduced apoptosis. However, when autophagy was inhibited by 3-MA or siRNA targeted Beclin 1, this reduction was reversed i.e. chemoresistance was attenuated, which means autophagy mediates the chemoresistance under hypoxia. In conclusion, autophagy decreases hepatoma cells sensitization to chemotherapeutic agents by affecting their apoptotic potential.

Targeting autophagy potentiates chemotherapy-induced apoptosis and proliferation inhibition in hepatocarcinoma cells

Induction of cell death and inhibition of cell growth are the main targets of cancer therapy. Here we evaluated the role of autophagy on chemoresistance of human hepatocarcinoma (HCC) cell lines, focusing on its crosstalk with cell apoptosis and proliferation. In this study, a chemotherapeutic agent (cisplatin or 5FU) induced the formation of autophagosomes in three human HCC cell lines and upregulated the expression of autophagy protein LC3-II. Inhibition of autophagy by 3-methyladenine or si-beclin 1 increased chemotherapy-induced apoptosis in HCC cells. Meanwhile, increased damage of the mitochondrial membrane potential was also observed in HCC cells when autophagy was inhibited. Furthermore, inhibition of autophagy reduced clone formation and impaired cell growth of HCC cells when treated with chemotherapy. Co-administration of an autophagy inhibitor (chloroquine) and chemotherapy significantly inhibited tumor growth in a mouse xenograft tumor model, with greater extent of apoptosis and impaired proliferation of tumor cells. This study suggests that autophagy is a potential novel target to improve therapy efficiency of conventional chemotherapeutics towards HCC.

Increased p38-MAPK is responsible for chemotherapy resistance in human gastric cancer cells.

BackgroundChemoresistance is one of the main obstacles to successful cancer therapy and is frequently associated with Multidrug resistance (MDR). Many different mechanisms have been suggested to explain the development of an MDR phenotype in cancer cells. One of the most studied mechanisms is the overexpression of P-glycoprotein (P-gp), which is a product of the MDR1 gene. Tumor cells often acquire the drug-resistance phenotype due to upregulation of the MDR1 gene. Overexpression of MDR1 gene has often been reported in primary gastric adenocarcinoma.MethodsThis study investigated the role of p38-MAPK signal pathway in vincristine-resistant SGC7901/VCR cells. P-gp and MDR1 RNA were detected by Western blot analysis and RT-PCR amplification. Mitgen-activated protein kinases and function of P-gp were demonstrated by Western blot and FACS Aria cytometer analysis. Ap-1 activity and cell apoptosis were detected by Dual-Luciferase Reporter Assay and annexin V-PI dual staining.ResultsThe vincristine-resistant SGC7901/VCR cells with increased expression of the multidrug-resistance 1 (MDR1) gene were resistant to P-gp-related drug and P-gp-unrelated drugs. Constitutive increases of phosphorylated p38-MAPK and AP-1 activities were also found in the drug-resistant cells. Inhibition of p38-MAPK by SB202190 reduced activator protein-1 (AP-1) activity and MDR1 expression levels and increased the sensitivity of SGC7901/VCR cells to chemotherapy.ConclusionActivation of the p38-MAPK pathway might be responsible for the modulation of P-glycoprotein-mediated and P-glycoprotein-unmediated multidrug resistance in the SGC7901/VCR cell line.

Inhibition of autophagy enhances anticancer effects of bevacizumab in hepatocarcinoma

Angiogenesis inhibitors have long been considered desirable anticancer agents. However, it was found that many tumors could develop resistance to antiangiogenesis inhibitors. Antiangiogenic therapy results in metabolic stress. Autophagy is an important survival mechanism in cancer cells under metabolic stress; however, it remains unknown if autophagy contributes to antiangiogenesis resistance. In this study, we reported that bevacizumab treatment reduced the development of new blood vessels and inhibited cell growth in xenografts of hepatocellular carcinoma (HCC) tumors. Bevacizumab treatment also upregulated expression of the autophagy-related genes (Beclin1 and LC3) and increased autophagosome formation. Our in vitro studies demonstrated that autophagy inhibition significantly increased apoptosis of HCC cells during nutrient starvation or hypoxia. In addition, the combined treatment of an autophagy inhibitor and bevacizumab markedly inhibited the tumor growth of HCC xenografts, led to enhanced apoptosis, and impaired the proliferation of tumor cells compared with treatment with either drug alone. Furthermore, autophagy inhibition led to enhanced reactive oxygen species (ROS) generation in HCC cells exposed to nutrient starvation or hypoxia in vitro and increased DNA oxidative damage in vivo. Antioxidants reduced nutrient starvation or the hypoxia-induced cell death of HCC cells after autophagy inhibition. Our results suggest that autophagy modulates ROS generation and contributes to cell survival under metabolic stress. Therefore, autophagy inhibition may be a novel way of increasing the efficicacy of antiangiogenic agents in the treatment of HCC.

Autophagic cell death induced by 5-FU in Bax or PUMA deficient human colon cancer cell

Autophagy is a membrane process that results in the transporting of cellular contents to lysosomes for degradation. Autophagic cell death is another way of programed cell death called type II PCD, which has complicated connection with apoptosis, both of these two types of cell death play an important role in tumor development. In this study, we investigated chemotherapeutic agent induced cell death pathway in wild type (WT), Bax(-/-) and PUMA(-/-) HCT116 cells. Bax or PUMA deficient cells had similar chemosensitivity to WT cells but were defective in undergoing apoptosis. The results of electron microscopy and GFP-LC3 localization assay showed that autophagy was induced in Bax or PUMA deficient cells but not in WT cells. mTOR activity was decreased in Bax or PUMA deficient cells which further indicated the up-regulation of autophagy. Inhibition of autophagy by 3-Methyladenine (3-MA) decreased the cell death in Bax or PUMA deficient cells. Taken together, these results suggest that autophagic cell death can be used as an alternative cell death pathway in apoptosis defective cells and may bring a new target for cancer therapy.

Autophagy in hypoxia protects cancer cells against apoptosis induced by nutrient deprivation through a beclin1‐dependent way in hepatocellular carcinoma

Oxygen deficiency and nutrient deprivation widely exists in solid tumors because of the poor blood supply. However, cancer cells can survive this adverse condition and proliferate continuously to develop. To figure out the way to survive, we investigated the role of autophagy in the microenvironment in hepatocellular carcinoma. In order to simulate the tumor microenvironment more veritably, cells were cultured in oxygen‐nutrient‐deprived condition following a hypoxia preconditioning. As a result, cell death under hypoxia plus nutrient deprivation was much less than that under nutrient deprivation only. And the decreased cell death mainly attributed to the decreased apoptosis. GFP‐LC3 and electron microscopy analysis showed that autophagy was significantly activated in the period of hypoxia preconditioning. However, autophagic inhibitor—3‐MA significantly abrogated the apoptosis reduction in hypoxia, which implied the involvement of autophagy in protection of hepatocellular carcinoma cells against apoptosis induced by starvation. Furthermore, Beclin 1 was proved to play an important role in this process. siRNA targeting Beclin 1 was transfected into hepatocellular carcinoma cells. And both data from western blot detecting the expression of LC3‐II and transmission microscopy observing the accumulation of autophagosomes showed that autophagy was inhibited obviously as a result of Beclin 1 knockdown. Besides, the decreased apoptosis of starved cells under hypoxia was reversed. Taken together, these results suggest that autophagy activated by hypoxia mediates the tolerance of hepatocellular carcinoma cells to nutrient deprivation, and this tolerance is dependent on the activity of Beclin 1. J. Cell. Biochem. 112: 3406–3420, 2011.

Paradoxical roles of autophagy in different stages of tumorigenesis: protector for normal or cancer cells

Autophagy serves as a dynamic degradation and recycling system that provides biological materials and energy in response to stress. The role of autophagy in tumor development is complex. Various studies suggest that autophagy mainly contributes to tumor suppression during the early stage of tumorigenesis and tumor promotion during the late stage of tumorigenesis. During the tumorization of normal cells, autophagy protects genomic stability by retarding stem cells-involved damage/repair cycle, and inhibits the formation of chronic inflammatory microenvironment, thus protecting normal cell homeostasis and preventing tumor generation. On the other hand, autophagy also protects tumor cells survival during malignant progression by supporting cellular metabolic demands, decreasing metabolic damage and supporting anoikis resistance and dormancy. Taken together, autophagy appears to play a role as a protector for either normal or tumor cells during the early or late stage of tumorigenesis, respectively. The process of tumorigenesis perhaps needs to undergo twice autophagy-associated screening. The normal cells that have lower autophagy capacity are prone to tumorization, and the incipient tumor cells that have higher autophagy capacity possibly are easier to survival in the hash microenvironment and accumulate more mutations to promote malignant progression.

Autophagy lessens ischemic liver injury by reducing oxidative damage

BackgroundHepatic ischemia/reperfusion is a multi-factorial process which causes liver injury. It is reported that ischemia alone is sufficient to induce liver injury. Nutrient deprivation is a crucial factor impacting ischemic injury of the liver. Therefore, we explored the role of autophagy in ischemia through using hepatic ischemia rat model in vivo and nutrient-free model in vitro.ResultsWe found that both ischemia in vivo and nutrient deprivation in vitro activated autophagy, inhibition of which aggravated ischemia- or nutrient deficiency-induced injury. In the nutrient-free condition, autophagy inhibition enhanced liver cell necrosis but not apoptosis by promoting reactive oxygen species (ROS) accumulation, and antioxidant NAC could reverse this trend. Inhibition of autophagy also resulted in the increase of the percentage of necrotic cell but not apoptotic cell in the ischemia-treated rat livers. Further studies showed that under nutrient deprivation, autophagy inhibition promoted mitochondrial ROS generation, which further aggravated mitochondria damage. These changes formed a “vicious cycle” that accelerated the process of cell necrosis. Autophagy inhibition also increased mitochondrial oxidative stress during hepatic ischemia, and antioxidant could suppress the aggravation of ischemia-induced liver damage in the co-treatment of autophagy inhibitor.ConclusionsTaken together, our results suggested that autophagy suppressed ischemic liver injury by reducing ROS-induced necrosis. This finding will contribute to the development of the therapeutic strategy about the pre-treatment of liver surgery.

Chloroquine Promotes the Anticancer Effect of TACE in a Rabbit VX2 Liver Tumor Model

Background: To investigate the efficacy of TACE combined with CQ, an autophagic inhibitor, in a rabbit VX2 liver tumor model. Methods: Tumor size was measured. And tumor growth rate was calculated to examine the effect of the combined treatment. Apoptosis was detected by TUNEL assay. Meanwhile, autophagic activity was detected by immunohistochemistry and Western blotting to investigate the mechanism underlying. Liver function was also examined to assess feasibility and safety of the combined therapy. Results: Tumors in the control grew more than 4 times bigger after 14 days, while that in the group of TACE alone just showed mild growth. But a slight shrinkage was shown after the treatment of CQ+TACE. Growth ratio of TACE alone was 96.45% ± 28.958% while that of CQ+TACE was -28.73% ± 12.265%. Compared with TACE alone, necrosis in CQ+TACE showed no significant difference, however, the apoptosis was much higher. There were only 14.8±3.11% apoptotic cells in TACE, but 33±4.18% in CQ+TACE, which suggests the increased apoptosis in CQ+TACE contributed to the decrease of tumor volume. In terms of autophagic activity, the result is negative when we immunostained sections of the control with LC3 antibody, but positive in TACE alone and CQ+TACE. And the result of Western blot showed that there was just a low level of LC3Ⅱexpressed in the control and CQ alone, but higher in TACE, and much higher in CQ+TACE because CQ inhibited its degradation in autophagy. Compared with control, p62 decreased in TACE, but the decrease was partially reversed in CQ+TACE. In addition, toxicity of CQ+TACE was assessed not higher than TACE alone, which supports the safety of CQ+TACE. Conclusion: CQ+TACE works better than TACE alone in rabbit VX2 liver tumor model because CQ inhibits autophagy induced by TACE. The inhibited autophagy loses its resistance to apoptosis that apoptosis increased, which contributes to the inhibition of tumor growth. This study indicates CQ may be a promising adjuvant to promote the effect of TACE.

Involvement of proapoptotic genes in autophagic cell death induced by irradiation

Irradiation is widely used in anticancer therapy; however, the efficiency is limited. Most cancer cells have mutations in apoptosis that they can easily escape the apoptosis induced by irradiation. Autophagy has been known as type II programmed cell death that can be activated by irradiation, especially when apoptosis is blocked, but the underlying molecular mechanism is largely unknown. We demonstrated that proapoptotic genes PUMA and Bid are involved in the regulation of autophagic cell death. When wild-type (WT), Bax−/− and PUMA−/− HCT116 cells were exposed to irradiation, we found that, compared with WT, Bax−/− cells showed significantly decreased cell death because of Bax deficiency; however, surprisingly PUMA−/− cells showed significant increase in cell death although the proapoptotic gene PUMA was knocked out. By analyzing apoptosis via Annexin V-fluorescein isothiocyanate (FITC) assay with flow cytometry, both Bax−/− and PUMA−/− cells showed less apoptosis than WT, suggesting the existence of another type of cell death in PUMA−/− cells. Autophagy was then examined in three cell lines by counting the percentage of cells with punctate GFP-LC3. Although all three cell lines showed significantly increased autophagy activity after irradiation, that of PUMA−/− cells was much higher than the other two cell lines, which suggests that PUMA−/− cells may die through autophagy. This was then confirmed by the decreased cell death in PUMA−/− cells when autophagy was blocked by 3-MA. In addition, we also tested the responses of WT and Bid−/− MEFs to irradiation. Bid−/− MEFs but not WT died through autophagy after irradiation. These results imply the involvement of apoptosis-associated genes such as PUMA and Bid in autophagic cell death, which contributes to identifying the molecular mechanism by which autophagy drives cells to death.