Autophagy inhibition contributes to radiation sensitization of esophageal squamous carcinoma cells

Radiotherapy is a useful component of treatment strategies for esophageal cancer. The role of autophagy in response to ionizing radiation was investigated in human esophageal squamous carcinoma cells. Cell viability and clonogenic survival assay were used to evaluate the radiosensitivity of autophagy inhibitor (3‐MA) on esophageal squamous carcinoma cells. The percentage of apoptotic cells and cell cycle analysis were assessed by flow cytometry; DAPI staining was used to detect apoptotic cells. The expression of beclin‐1 and LC3 was measured using a Western blot. The ultrastructural analysis was under the electron microscope. 6 Gy irradiation induced a massive accumulation of autophagosomes accompanied by strong upregulation of beclin‐1 and LC3‐II expression in TE‐1 cells. Compared with radiation alone, 3‐MA combined with radiation significantly decreased cell viability, as well as autophagic ratio, beclin‐1, and LC3‐II protein level. Inhibition of autophagy increased radiation‐induced apoptosis and the percentage of G2/M‐phase cells. Blockade of autophagy with 3‐MA enhanced cytotoxicity of radiotherapy in human esophageal squamous carcinoma cells. It suggests that inhibition of autophagy could be used as adjuvant therapy to treat esophageal squamous cell carcinoma.     

DNA—PKCS subunits in radiosensitization by hyperthermia on hepatocellular carcinoma hepG2 cell line

AIM：To investigate the role ofDNA-PKcs subunits in radiosensitization by hyperthermia on hepatocellular carcinoma HepG2 cell lines.METHODS:Hep G2 cells were exposed to hyperthermia and irradiation.Hyperthermia was given at 45.5℃.Cell survival was determined by an in vitro clonogenic assay for the cells treated with or without hyperthermia at various time points,DNA DSB rejoining WAS measured using asymmitirc ficld inversion gel electrophoresis(AFIGE).The DNA-PKcs activities were measured usingDNA-PKcs enzyme assay system.RESULTS:Hyperthermia can significantly enhance irradiation-Killing cells.Thermal enhancement ratio as calculated at 10%survival was2.02 The difference in radiosensitivity between two treatment modes manifested as a difference in the αcomponents and the almost same βcomponents,whichαvalue was considerably higher in the cells of combined radiation and hyperthermia as compared with irradiating cells(1.07Gy^-1versus0.44Gy^-1).Survival fraction showed 1logarithm increase after an 8-hour interval between heat and irradiation.whereasDNA-PKcs activity did not show any recovery.The cells were exposed to heat 5minutes only,DNA-PKcs activity was inhibited at the nadir,even though the exposure time was lengthened.Whereas the ability of DNADSBrejoining was inhibited with the increase of the length of hyperthermic time,The repair kinetics of DNA DSB rejoining after treatment with Wortmannin is different from the hyperthermic group due to the striking high slow rejoining component.CONCLUSION:Determination with the cell extracts and the peptied phosphorylation assay,DNA-PKcs activity was inactivated by heat treatment at 45.5℃,and could not restore,Cell survival is not associated with the DNA-PKcs inactivity after heat,DNA-PKcs in not a unique factor affecting the DNA DSBrepair.This suggests that DNA-PKcs do not play a crucial role in the enhancement of cellular radiosensitivity by hyperthermia.     

棕榈酸对肝癌HepG2细胞自噬和凋亡的影响

目的探讨棕榈酸对肝癌HepG2细胞自噬和凋亡的影响。方法采用含浓度为800txmol／L棕榈酸的DMEM高糖培养基培养肝癌HepG2细胞24h,向HepG2细胞转染绿色荧光蛋白（GFP）-LC3质粒,通过观察LC3II绿色荧光斑点确定自噬发生情况。利用BafAl抑制自噬后,采用M30免疫荧光检测细胞凋亡情况。结果棕榈酸刺激24h明显增加自噬及凋亡细胞数量。阻断自噬,棕榈酸刺激引起的HepG2细胞凋亡水平明显增加。结论棕榈酸可刺激自噬发生,该自噬对肝癌HepG2细胞有一定的保护作用。     

Endoplasmic reticulum stress sensitizes human esophageal cancer cell to radiation

AIM:To investigate the role of endoplasmic reticulum(ER) stress in cancer radiotherapy and its molecular mechanism.METHODS:Tunicamycin(TM) was applied to induce ER stress in human esophageal cancer cell line EC109,and the radiosensitization effects were detected by acute cell death and clonogenic survival assay.Cell cycle arrest induced by TM was determined by flow cytometric analysis after the cellular DNA content was labeled with propidium iodide.Apoptosis of EC109 cells induced by TM was detected by annexin V staining and Western blotting of caspase-3 and its substrate poly ADP-ribose polymerase.Autophagic response was determined by acridine orange(AO) staining and Western blotting of microtubule-associated protein-1 light chain-3(LC3) and autophagy related gene 5(ATG5).In order to test the biological function of autophagy,specific inhibitor or Beclin-1 knockdown was used to inhibit autophagy,and its effect on cell apoptosis was thus detected.Additionally,involvement of the phosphatidylinositol-3 kinase(PI3K)/Akt/mammalian target of the rapamycin(mTOR) pathway was also detected by Western blotting.Finally,male nude mice inoculated subcutaneously with EC109 cells were used to confirm cell model observations.RESULTS:Our results showed that TM treatment enhanced cell death and reduced the colony survival fraction induced by ionizing radiation(IR),which suggested an obvious radiosensitization effect of TM.Moreover,TM and IR combination treatment led to a significant increase of G2/M phase and apoptotic cells,compared with IR alone.We also observed an increase of AO positive cells,and the protein level of LC3-II and ATG5 was induced by TM treatment,which suggested an autophagic response in EC109 cells.However,inhibition of autophagy by using a chemical inhibitor or Beclin-1 silencing led to increased cell apoptosis and decreased cell viability,which suggested a cytoprotective role of autophagy in stressed EC109 cells.Furthermore,TM treatment also activated mTORC1,and in turn reduced Akt phosphorylation,which sugge     

Role of autophagy in differential sensitivity of hepatocarcinoma cells to sorafenib

AIM: To investigate the role of sorafenib(SFN) in autophagy of hepatocellular carcinoma(HCC). We evaluated how SFN affects autophagy signaling pathway in human HCC cell lines. METHODS: Two different human HCC cell lines, Hep3 B and Huh7, were subjected to different concentrations of SFN. Cell viability and onset of apoptosis were determined with colorimetric assay and immunoblotting analysis, respectively. The changes in autophagy-related proteins, including LC3, ULK1, AMPK, and LKB, were determined with immunoblotting analysis in the presence or absence of SFN. To assess autophagic dynamics, autophagic flux was measured with chloroquine, a lysosomal inhibitor. The autophagic responsiveness between different HCC cell lines was compared under the autophagy enhancing conditions.RESULTS: Hep3 B cells were significantly more resistant to SFN than Huh7 cells. Immunoblotting analysis revealed a marked increase in SFN-mediated autophagy flux in Huh7 cells, which was, however, absent in Hep3 B cells. While both starvation and rapamycin enhanced autophagy in Huh7 cells, only rapamycin increased autophagy in Hep3 B cells. Immunoblotting analysis of autophagy initiation proteins showed that SFN substantially increased phosphorylation of AMPK and consequently autophagy in Huh7, but not in Hep3 B cells.CONCLUSION: The autophagic responsiveness to SFN is distinct between Hep3 B and Huh7 cells. Resistance of Hep3 B cells to SFN may be associated with altered autophagy signaling pathways.     

Ginseng polysaccharide serves as a potential radiosensitizer through inducing apoptosis and autophagy in the treatment of osteosarcoma

Recent studies have confirmed that the combined use of anti-cancer drugs with ionizing radiation (IR) could improve the sensitivity of osteosarcoma (OS) cells. Therefore, it is necessary to identify potential effective drugs for the enhancement of IR-radiosensitivity. In the current study, we found that 20, 10, 5, and 1 μM of ginseng polysaccharide (GPS) significantly suppressed MG-63 cell viability with or without γ-ray radiation in a dose- and time-dependent manner. Strikingly, 20 μM of GPS combined with 5 Gy treatment suppressed colony formation capacity by nearly 13.75～fold compared with IR treatment alone. Our results showed that GPS could markedly induce early apoptosis and autophagy in MG-63 cells. A higher drug concentration and a greater exposure dose were directly associated with more apoptosis and autophagy in cells. Western blot analysis showed that GPS decreased the phosphorylation of p38 and AKT as well as the protein expression of Bax and cleaved-caspase3. In summary, GPS inhibited proliferation and increased apoptosis and autophagic death in OS cells, indicating that GPS may be a potential effective auxiliary drug for improving the IR sensitivity of OS patients.     

Novel poly (ADP‐ribose) polymerase inhibitor,AZD2281, enhances radiosensitivity of both normoxic and hypoxic esophageal squamous cancer cells

Radiotherapy plays an important role in the treatment of esophageal squamous cell carcinoma (ESCC). However, the outcome of radiotherapy in ESCC remains unsatisfactory because esophageal squamous cancer cells, particularly those under hypoxic condition, exhibit radioresistance. The aim of this study was to determine whether or not AZD2281, a potent poly (ADP‐ribose) polymerase (PARP) inhibitor, could enhance the radiation sensitivity of two ESCC cell lines, namely ECA109 and TE13. The radiosensitizing effect of AZD2281 was evaluated on the basis of cell death, clonogenic survival and tumor xenograft progression. AZD2281 alone was slightly toxic to ESCC cell lines. Apoptosis was increased and clonogenic survival was decreased in both cell lines when AZD2281 was combined with ionizing radiation (IR) under normoxic condition. AZD2281 enhanced IR‐induced apoptosis to a more significant level under chronic hypoxic condition (0.2% O₂, 48 hour) than under normoxic condition. AZD2281 also slightly enhanced clonogenic cell death under chronic hypoxic condition compared with that under normoxic condition. This result could be associated with increased radiation‐induced DNA double‐strand breaks (DSB), decreased DSB repair and increased apoptosis of ESCC cells. Furthermore, homologous recombination (HR) protein Rad51 expression and focus formation were decreased in ESCC cells exposed to moderate chronic hypoxic condition (0.2% O₂, 48 hour); this result indicated that chronic hypoxic ESCC cells were HR deficient, possibly causing contextual synthetic lethality with PARP inhibitor in radiation sensitization. AZD2281 was also a radiation sensitizer in ESCC tumor xenograft models. Hence, in vitro and in vivo findings provide evidence that AZD2281 potently sensitizes ESCC cells to X‐ray irradiation. The selective cell killing of HR‐defective hypoxic cells contributes to radiosensitization by PARP inhibitor in ESCC cells under hypoxic condition.     

Blocking NF-κB nuclear translocation leads to p53-related autophagy activation and cell apoptosis

AIM: To investigate the anti-tumor effects of nuclear factor-κB (NF-κB) inhibitor SN50 and related mechanisms of SGC7901 human gastric carcinoma cells.METHODS: MTT assay was used to determine the cytotoxic effects of SN50 in gastric cancer cell line SGC7901. Hoechst 33258 staining was used to detect apoptosis morphological changes after SN50 treatment. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after SN50 treatment.Immunofluorescence staining was used to detect the expression of light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. Western blotting analysis were used to determine the expression of proteins involved in apoptosis and autophagy including p53, p53 upregulated modulator of apoptosis (PUMA), damage-regulated autophagy modulator (DRAM), LC3 and Beclin 1. We detected the effects of p53-mediated autophagy activation on the apoptosis of SGC7901 cells with the p53 inhibitor pifithrin-α.RESULTS: The viability of SGC7901 cells was inhibited after SN50 treatment. Inductions in the expression of apoptotic protein p53 and PUMA as well as autophagic protein DRAM, LC3 and Beclin 1 were detected with Western blotting analysis. SN50-treated cells exhibited punctuate microtubule-associated protein 1 LC3 in immunoreactivity and MDC-labeled vesicles increased after treatment of SN50 by MDC staining. Collapse of mitochondrial membrane potential Δψ were detected for 6 to 24 h after SN50 treatment. SN50-induced increases in PUMA, DRAM, LC3 and Beclin 1 and cell death were blocked by the p53 specific inhibitor pifithrin-α.CONCLUSION: The anti-tumor activity of NF-κB inhibitors is associated with p53-mediated activation of autophagy.     

Sunitinib modulates the radiosensitivity of esophageal squamous cell carcinoma cells in vitro

This study aims to explore the radiosensitivity of sunitinib on esophageal cancer cell lines. For in vitro studies, human esophageal squamous cell carcinoma (ESCC) cell lines were treated with sunitinib 24 hours before irradiation. ESCC cell lines were treated with sunitinib with or without radiation. Cell proliferation was detected by Cell Counting Kit 8 assay. Radiosensitization was evaluated by clonogenic survival assay. Cell apoptosis and cell cycle analysis were detected by flow cytometry. Deoxyribonucleic acid (DNA) double‐strand breaks were performed by immunocytofluorescence analysis. Western blot analysis was used to determine the effect of sunitinib on radiation induced signal transduction. Sunitinib potently sensitized ESCC cells to radiation with a sensitization enhancement ratio of 1.13–1.72. Furthermore, sunitinib increased radiation induced DNA double‐strand breaks, promoted the apoptosis of ESCC cells and induced the G2/M arrest. Radiosensitization was accompanied with enhanced apoptosis and regulated by the intrinsic pathway of apoptosis. Sunitinib sensitized ESCC cells to the cytotoxic effects of radiation. This compound is promising for future clinical trials with chemoradiation in esophageal cancer.     

Effect of ionizing radiation on acinar morphogenesis of human prostatic epithelial cells under three-dimensional culture conditions

Homeostasis is maintained by the interplay of multiple factors that directly or indirectly regulate cell proliferation and cell death. Complex multiple interactions between cells and the extracellular matrix occur during acinar morphogenesis and changes in these might indicate carcinogenesis of cells from a normal to a malignant, invasive phenotype. In this study, the human prostatic epithelial cell line RWPE-1 was cultured under three-dimensional (3-D) culture conditions, and the effect of ionizing radiation on acinar morphogenesis and its association with autophagy were discussed. The results illustrated that formation of specific spheroid (acinar) structures was detectable under 3-D culture conditions. Radiation induced the disruption of acini in different cell models using either gene overexpression (Akt) or gene knock-down (Beclin 1 and ATG7). Introduction of Akt not only accelerated the growth of cells (i.e., caused the cells to manifest elongating and microspike-like structures that are obviously different from structures seen in wild-type RWPE-1 cells under two-dimensional conditions), but also changed their morphological characteristics under 3-D culture conditions. Knock-down of autophagy-related genes (Beclin 1 and ATG7) increased the radiosensitivity of cells under 3-D culture conditions, and cells died of non-apoptotic death after radiation. The results suggested that ionizing radiation may change the cell phenotype and the formation of acini. Additionally even the autophagy mechanism may play a role in these processes.     

Docetaxel shows radiosensitization in human hepatocellular carcinoma cells

AIM: To determine the radiosensitizing potential of docetaxel in human hepatocellular carcinoma SMMC-7721 cells and its mechanisms. METHODS: SMMC-7721 cells were incubated with docetaxel at 0.125, 0.25, and 0.5 nmoL/L for 24 h and at 0.125 and 0.25 nmol/L for 48 h before irradiation. Radiation doses were given from 0 to 10 Gy. Cell survival was measured by a standard clonogenic assay after a 9-d incubation. The reactive oxygen species (ROS) and glutathione (GSH) are detected after being given the same dose of docetaxel for the same time. RESULTS: The sensitization enhancement ratios (SER) for SMMC-7721 cells determined at the 50% survival level were 1.15, 1.21 and 1.49 at 0.125, 0.25, and 0.5 nmol/L for pre-incubation of 24 h, respectively; the SER were 1.42, 1.67 at 0.125 and 0.25 nmol/L, for pre-incubation of 48 h, respectively. The ROS of SMMC-7721 cells increased and GSH decreased after pretreatment with the same doses of docetaxel for 24 or 48 h. CONCLUSION: A radiosensitizing effect of docetaxel could be demonstrated unambiguously in this cell line used. In addition, our data showed that the mechanism of radiopotentiation by docetaxel probably does not involve a G2/M block in SMMC-7721 cells, and ROS generation and GSH deletion may play a key role in the radiosensitizing effect of docetaxel.     

Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent endogenous activator of the cell death pathway and functions by activating the cell surface death receptors 4 and 5 (DR4 and DR5). TRAIL is nontoxic in vivo and preferentially kills neoplastically transformed cells over normal cells by an undefined mechanism. Radiotherapy is a common treatment for breast cancer as well as many other cancers. Here we demonstrate that ionizing radiation can sensitize breast carcinoma cells to TRAIL-induced apoptosis. This synergistic effect is p53-dependent and may be the result of radiation-induced up-regulation of the TRAIL-receptor DR5. Importantly, TRAIL and ionizing radiation have a synergistic effect in the regression of established breast cancer xenografts. Changes in tumor cellularity and extracellular space were monitored in vivo by diffusion-weighted magnetic resonance imaging (diffusion MRI), a noninvasive technique to produce quantitative images of the apparent mobility of water within a tissue. Increased water mobility was observed in combined TRAIL- and radiation-treated tumors but not in tumors treated with TRAIL or radiation alone. Histological analysis confirmed the loss of cellularity and increased numbers of apoptotic cells in TRAIL- and radiation-treated tumors. Taken together, our results provide support for combining radiation with TRAIL to improve tumor eradication and suggest that efficacy of apoptosis-inducing cancer therapies may be monitored noninvasively, using diffusion MRI.     

IGF-1 contributes to liver cancer development in diabetes patients by promoting autophagy

Introduction and objectivesType 2 diabetes mellitus (T2DM) increases the occurrence and mortality of liver cancer. Insulin growth factor (IGF) plays a crucial role in the development of diabetes and liver cancer, and specifically, IGF-1 may be involved in the development of liver cancer with preexisting T2DM. Autophagy contributes to cancer cell survival and apoptosis. However, the relationship between IGF-1 and autophagy has rarely been evaluated. The purpose of this study was to investigate whether IGF-1 promotes the development of liver cancer in T2DM patients by promoting autophagy.Materials and methodsThirty-three hepatocellular carcinoma (HCC) patients with T2DM and 33 age-matched patients with HCC without T2DM were included in this study. We analyzed the expression of IGF-1 and autophagy-related LC3 and p62 mRNA and the prognosis of two groups. In vitro, we stimulated HepG2 cells with IGF-1 and then detected changes in autophagy and cell proliferation, apoptosis, and migration in the presence/absence of wortmannin, an autophagy inhibitor.ResultsIGF-1 promoted autophagy, resulting in inhibition of apoptosis and induction of growth and migration of HepG2 cells. Inhibition of autophagy by wortmannin impaired IGF-1 function. Higher expression of IGF-1 was detected in HCC patients with T2DM. IGF-1 expression was higher in liver cancer tissue compared to paracancerous tissue. Elevated IGF-1 was associated with a poor prognosis in patients with HCC.ConclusionsIGF-1 participates in the development of liver cancer by inducing autophagy. Elevated IGF-1 was a prognostic factor for patients with HCC, especially when accompanied by T2DM.     

Apoptosis occurs in lymphoma cells but not in hepatoma cells following ionizing radiation and photodynamic therapy

The aim of this study was to determine the relative role of apoptosis in photodynamically-induced cytotoxicity or radiation-induced cytotoxicity for hepatoma and lymphoma cells. Human hepatoma cells and mouse lymphoma cells were treated with either photodynamic therapy (PDT) or ionizing radiation. Dosimetry studies of immediate cell death following photodynamic therapy in the hepatoma cell line demonstrated second-order kinetics, similar to that seen in the lymphoma cells. No immediate cell death was noted in the hepatoma or lymphoma cells following ionizing radiation, but experiments measuring delayed cell death demonstrated a dose-dependent response. Maximum DNA ladder formation occurred 2 hr after PDT and 24 hr after ionizing radiation in the lymphoma cells, which was consistent with the time courses of cell death for these treatments. The hepatoma cell line did not demonstrate DNA ladder formation despite dosages of PDT or ionizing radiation sufficient to cause high levels of cytotoxicity.     

Inhibiting MT2‐TFE3‐dependent autophagy enhances melatonin‐induced apoptosis in tongue squamous cell carcinoma

Autophagy modulation is a potential therapeutic strategy for tongue squamous cell carcinoma (TSCC). Melatonin possesses significant anticarcinogenic activity. However, whether melatonin induces autophagy and its roles in cell death in TSCC are unclear. Herein, we show that melatonin induced significant apoptosis in the TSCC cell line Cal27. Apart from the induction of apoptosis, we demonstrated that melatonin‐induced autophagic flux in Cal27 cells as evidenced by the formation of GFP‐LC3 puncta, and the upregulation of LC3‐II and downregulation of SQSTM1/P62. Moreover, pharmacological or genetic blockage of autophagy enhanced melatonin‐induced apoptosis, indicating a cytoprotective role of autophagy in melatonin‐treated Cal27 cells. Mechanistically, melatonin induced TFE3^(Ser321) dephosphorylation, subsequently activated TFE3 nuclear translocation, and increased TFE3 reporter activity, which contributed to the expression of autophagy‐related genes and lysosomal biogenesis. Luzindole, a melatonin membrane receptor blocker, or MT2‐siRNA partially blocked the ability of melatonin to promote mTORC1/TFE3 signaling. Furthermore, we verified in a xenograft mouse model that melatonin with hydroxychloroquine or TFE3‐siRNA exerted a synergistic antitumor effect by inhibiting autophagy. Importantly, TFE3 expression positively correlated with TSCC development and poor prognosis in patients. Collectively, we demonstrated that the melatonin‐induced increase in TFE3‐dependent autophagy is mediated through the melatonin membrane receptor in TSCC. These data also suggest that blocking melatonin membrane receptor‐TFE3‐dependent autophagy to enhance the activity of melatonin warrants further attention as a treatment strategy for TSCC.     

ATF3转录调控HSP110对低氧诱导的肺动脉平滑肌细胞自噬和增殖的影响

目的 探讨ATF3调控HSP110表达对低氧刺激下人肺动脉平滑肌细胞增殖及自噬的影响。 方法 体外培养人肺动脉平滑肌细胞，单独感染ATF3沉默腺病毒或共感染HSP110过表达腺病毒，48 h后加入自噬激活剂雷帕霉素预处理3 h，建立缺氧细胞模型，24 h后，CCK-8法检测细胞增殖能力，流式细胞术分析细胞凋亡，Real-time PCR检测ATF3、HSP110 mRNA的表达，Western blot检测ATF3、HSP110、LC3Ⅱ/Ⅰ、Beclin-1、p62蛋白的表达，免疫荧光观察LC3斑点形成，荧光素酶报告系统鉴定ATF3对HSP110的调控作用。 结果 缺氧组ATF3和HSP110的表达显著增加（P<0.01），ATF3沉默后，细胞增殖能力下降，凋亡率增加（P<0.01），同时自噬蛋白LC3 Ⅱ/Ⅰ、Beclin-1的表达及LC3荧光斑点显著减少(P<0.01)，自噬降解底物p62的表达明显增加(P<0.01)，而加入雷帕霉素或共感染HSP110过表达腺病毒后，ATF3沉默对细胞增殖和自噬的抑制作用显著减弱(P<0.01)。双荧光素酶报告基因检测结果显示，ATF3可上调HSP110启动子活性。 结论 ATF3调控HSP110表达增加而促进人肺动脉平滑肌细胞增殖，其机制可能与介导细胞自噬有关。     

Ethanol enhances susceptibility to apoptotic cell death via down-regulation of autophagy-related proteins

Background: Alcohol induces cellular stress and promotes cell death in immune cells. Molecular mechanisms by which ethanol impairs the function of immune cells are largely unknown. Autophagy is a degradation pathway, acting either as a pro‐survival or pro‐death mechanism activated during stress conditions. We examined whether ethanol influences autophagy in monocytic human U937, CD4 Jurkat, and MCF‐7 cells. Methods: Effects of ethanol during starvation‐induced autophagy were investigated, treating cells with ethanol alone and in combination with activation of autophagy by rapamycin or inhibition by wortmannin. Apoptotic and necrotic cell death features such as the breakdown of the mitochondrial membrane potential, DNA fragmentation, and cell permeability were assessed using FACS analyses. Expression level of Beclin‐1, LC3‐II, Bcl‐2, and the activation of caspase‐3, and PARP‐1 were determined using Western blot analyses. Influence of ethanol on formation of LC3‐II complexes was assessed using fluorescence microscopy in MCF‐7 cells stable transfected with a GFP‐LC3‐II‐expression vector. Results: Ethanol down regulated autophagy proteins such as Beclin‐1 and LC3‐II. Apoptosis was enhanced as shown by breakdown of mitochondrial potential, up‐regulation of cleaved caspase‐3 and PARP‐1 and down‐regulation of anti‐apoptotic protein Bcl‐2. Formation of LC3‐II complexes was inhibited by ethanol in caspase‐3 deficient MCF‐7 cells. Stimulation of autophagy by rapamycin prevented ethanol‐induced apoptotic cell death. Inhibition of autophagy by wortmannin aggravated ethanol‐mediated necrotic cell death. Conclusion: Inhibition of autophagy via ethanol enhances susceptibility to cell death.     

大黄素诱导肝癌细胞氧化损伤的实验研究

目的：研究大黄素抑制肝癌细胞增殖的作用机理。方法：采用流式细胞仪法观察了大黄素对肝癌细胞内活性氧的影响；采用MTT比色法观察了大黄素对细胞增殖的影响。结果：大黄素作用于肝癌细胞后，在2小时内引起活性氧的产生；大黄素抑制了肝癌细胞的增殖。结论：大黄素能够通过诱导细胞内活性氧的产生，引起细胞脂质过氧化，抑制了肝癌细胞的增殖。