5-aza-2’-deoxycytidine Induced Autophagy Of Breast Cancer Cells And Its Role In Cytotoxicity Of The Drug

BackgroundBreast cancer is one of the most commonly seen malignance in women. As people’s life style, including diet, have changed in recent years, the incidence of breast cancer increases obviously in China. Being ranked the second in female malignant tumor, breast cancer has not only severely affected women’s health, but has aslo caused extremely high mortality. Although there are many selections of anti-cancer drugs for clinical use, most anti-cancer drugs have the limitation of drug resistance in use. Thus, it is still an important subject to investigate the sensitive tumor type of new anti-cancer drugs and the underlying mechanism.5-aza-2’-deoxycytidine is an epigenetic anticancer drug, which was proved by Food and Drug Administration (FDA) of USA to be used for treatment of myelodysplastic syndrome and leukemia.5-aza-2’-deoxycytidine also has anticancer effects on solid tumors. Clinical trials of5-aza-2’-deoxycytidine have shown antineoplastic activity in patients with breast cancer and non-small cell lung cancer. Neverthless, compared to its effects on leukemia,5-aza-2’-deoxycytidine showed limited effects on soild tumor. However, in vitro,5-aza-2’-deoxycytidine showed cytotoxic effect on solid tumor cells, and that some kind of tumor cells are sensitive to it. Thus,5-aza-2’-deoxycytidine might be used to treat some specific types of tumors. The anti-cancer effect of5-aza-2’-deoxycytidine is either dependent on its demethylation effect or not. The former is associated with direct inhibition of DNA methyltransferase to activate tumor suppressor genes, and the later is probably through induction of DNA damage of cancer cells.Autophagy is a general phenomenon found in eukaryotes. It is a process, under the stimulation of some factors, e.g. DNA damage, nutrition deficiency and hypoxia, in which celluar membrane structures surround damaged proteins or organelles and fused with lysosomes to degrade damaged proteins or organelles. The degradation products can be recycled to use again. Autophagy is a stress response under the circumstance of disadvantage growth conditions. In response to the differences in stimulation, autophagy can either protect cells or lead cells to death. Autophagy play an important role in cancer development and treatment. It is reported that tumor cells actived autophagy to overcome hypoxia and nutrition deficiency so that it could delay apoptosis and increase survival ability in early non-invading breast cancers. In the response to anticancer drugs, autophagy can be either pretctive to cancer cells or promoting cell death.Anti-cancer drug Etopside can induce autophagy and its use in combination with autophagy showed better therapeutic effects.. It reported that DNA damage may induce autophagy but but it is still little reported that whether DNA damage caused by anti-cancer drugs is correlated to autophagy. In this study, we first observed the effects of5-aza-2’-deoxycytidine on autophagy, and then further studed the relationship between autophagy and its cytotoxity. This study deepened the understanding of anticancer mechanism of5-aza-2’-deoxycytidine and offered further theroy basement for its clinical use. ObjectivesTo investigate the effect of5-aza-2’-deoxycytidine on autophagy in different human breast cancer cell lines. To investigate whether5-aza-2’-deoxycytidine can induce autophagy of breast cancer cells and its association with DNA damage. To investigate the role of5-aza-2’-deoxycytidine induced autophagy on its cytotoxicty to human breast cancer cells, in another words. To investigate whether autophagy inhibits cell proliferation or promotes cell proliferation. To investigate whether5-aza-2’-deoxycytidine induced autophagy affects cell proliferation through modulation of cell cycle or apoptosis..MethodsHuman breast cancer cells MDA-MB-468, MCF-7and T-47D were treated with5-aza-2’-deoxycytidine and DNA damage, autophagy, cell proliferation, cell cycle and apoptosis of the treated cells were assayed.To investigate the causal relationship of DNA damage and autophagy induced by5-aza-2’-deoxycytidine, the effects of Etoposide and Cisplatin on DNA damage and autophagy in breast cancer cells MDA-MB-468, MCF-7and T-47D were first observed. Because it is generally accepted that the anti-cancer effects of Etoposide and Cisplatin was correlated to DNA damage caused and, meanwile, DNA damage could induce autophagy, so, if autophagy induced by Etoposide and Cisplatin is associated with DNA damage it caused, the causal relationship between DNA damage and autophagy induced by anti-cancer drug exists. In agrrement with the same principle, if DAC induced DNA damage and autophagy of breast cancer cells the same time, the causal relationship of the two exists.DNA damage of cell was evaluated by Comet Assay and changes of protein p53and p21expression were detected by Westen Blotting to validate the effects of DNA damage on p53pathway.Autophagy of breast cancer cells was monitored by three methods, including detecting the change of LC3II expression by Westen Blotting, monodansylcadaverin staining of breast cancer cells and transfectinon of pEGFP-LC3vector to breast cancer cells.Cell viability was assayed by MTT and cell proliferation rate was calculated according to change of cell viability.Four different autophagy inhibitors Bafilomycin A1,3-Methyladenine, Wortmannin or NH4Cl were combined with DAC respectively. The cell proliferation rate were compared between the groups of combination treatments and DAC treatment alone.Cell cycle was examined by flow cytometer.Apoptosis was examined by Annexin V kit.ResultsCompared with the untreated controls, Etoposide and Cisplatin induced DNA damage in breast cancer cell MDA-MB-468, MCF-7and T-47D. The difference between the groups is statistically significant. For MDA-MB-468cells, the comet length for control, Etoposide and Cisplatin treatment groups were65.939±11.753μm,142.788±16.575μm and86.121±37.612μm respectively (F=336.220, P=0.000). For MCF-7cells, the comet length for control, Etoposide and Cisplatin treatment groups were43.879±5.237μm,77.818±12.963μm,69.576±17.825μm, respectively (F=146.013, P=0.000). For T-47D cells, the comet length for control, Etoposide and Cisplatin treatment groups were55.152±7.755μm,154.333±24.762μm,153.182±24.979μm, respectively(F=284.347, P=0.000). Etoposide and Cisplatin increased p53and p21protein expression level in human breast cancer cells. It demonstratrated that Etoposide and Cisplatin induced DNA damage in human breast cancer cells. Etoposide and Cisplatin also increasd LC3-II expression level in MDA-MB-468, MCF-7and T-47D. It demonstratrated that Etoposide and Cisplatin induce autophagy in human breast cancer cells.Compared with the untreated controls, DAC induced DNA damage in breast cancer cell MDA-MB-468, MCF-7and T-47D. The difference between the groups is statistically significant. For MDA-MB-468cells, the comet length for control, DAC treatment groups were65.939±11.723μm,194.667±27.882μm, respectively (F=336.220, P=0.000). For MCF-7cells, the comet length for control, DAC treatment groups were43.879±5.237μm,86.818±15.288μm, respectively(t=-15.264, P=0.000). For T-47D cells, the comet length for control, DAC treatment groups were55.152±7.755μm,76.000±16.668μm, respectively (t=-6.515, P=0.000). DAC increased p53and p21protein expression level in human breast cancer cells. It showed that DAC induce DNA damage in human breast cancer cells. DAC also increasd LC3-II expression level in MDA-MB-468, MCF-7and T-47D. For MCF-7cells. With breast cancer cell stained with MDC and counted positive staining cells, DAC was found to increas percentage of MDC positive staining cells. Compared with the untreated controls, the difference between the groups is statistically significant, the percentage for control and DAC treatment groups were26.24±11.46%,58.30±6.72%respectively (t=-6.057, P=0.000). For MCF-7cells, after DAC treating cells transfected with pEGFP-LC3vector, percentage of pEGFP-LC3positive cells increasd. Compared with the untreated controls, the difference between the groups is statistically significant. The percentage for control and DAC treatment groups were35.096±2.898%,55.109±3.896%, respectively (t=-8.234, P=0.000). It showed that DAC induce autophagy in human breast cancer cells.Cell viability of control and DAC treatment groups in MDA-MB-468, MCF-7and T-47D was evaluated by MTT. DAC at100nmol/L could significantly inhibit cell proliferation in MDA-MB-468. The proliferation rate of control, DAC treatment groups were101.222±3.735%,78.917±3.093%, respectively (t=19.516, P=0.000); but, same concentration of DAC failed to inhibit growth of MCF-7and T-47D. For MCF-7, the proliferation rate for control, DAC treatment groups were 100.833±4.288%,92.811±13.099%(t=2.469, P=0.119). For T-47D, the proliferation rate for control, DAC treatment groups were100.167±6.270%,98.468±11.916%(t=0.535,P=0.596)Breast cancer cells MDA-MB-468, MCF-7and T-47D were first pre-treated with four differernt autophay inhibitors Bafilomycin Al,3-Methyladenine, Wortmannin and NH4Cl, and then treated with DAC. The results showed that inhibition of autophagy by BA1could enhance the cytotoxic effect of DAC in three breast cancer cells. After treating cells with BA1in combination with DAC, the cell proliferation rate of MDA-MB-468, MCF-7, T-47D were67.733±4.048%,65.254±7.433%,74.421±6.042%, respectively (P=0.000). Pre-treated by3MA could enhance the cytotoxity of DAC in MDA-MB-468and MCF-7, but not in T-47D. Pre-treated by WM and NH4Cl could enhance the cytotoxity of DAC in MDA-MB-468, but not in MCF-7and T-47D. The results showed that autophagy inhibitors BA1,3MA and WM inconbination with DAC could increase growth inhibition of breast cancer cells by DAC, but different autophagy inhibitors showed different effects of growth inhibition.DAC induced G2/M arrest in MDA-MB-468. The percentage of G2/M phase for control and DAC treatment groups were11.887±2.650%,28.020±1.495%respectively (P<0.05). Autophagy inhibitor BA1could increase G2/M arrest induced by DAC in MDA-MB-468. The percentage of G2/M phase for DAC and BA1in combination with DAC treatment groups were28.020±1.495%,36.907±3.003%, respectively (P<0.05).DAC could slightly induce apotosis in breast cancer cell MDA-MB-468. The apoptosis percentage for control and DAC treatment groups were11.255±2.943%,15.254±2.578%, respectively (P<0.05). Pre-treatment with autophagy inhibitor BA1had no effect on apoptosis induced by DAC. The apoptosis percentage for BA1 in combination of DAC treatment group was15.312±1.503%.ConclusionsDAC induced DNA damage, which could cause autophagy of breast cancer cells.DAC showed different cytotoxicity in different breast cancer cells, indicating that5-aza-2’-deoxycytidine may be just effective in some types of breast cancers.DAC in combination with autophagy inhibitors can enhance the cytotoxity of DAC in breast cancer cells, indicating that autophagy induced by DAC protects breast cancer cells.DAC induced G2/M cell cycle arrest in breast cancer cell MDA-MB-468, indicating that the cell proliferation inhibition by DAC is associated with G2/M cell cycle arrest caused.In combination with autophagy inhibitor BA1, DAC could increase G2/M arrest, indicating that autophagy may protect cells via promoting G2/M phase of cell cycle.DAC slightly induced apoptosis in breast cancer cell MDA-MB-468, indicating that DAC inhibit cell proliferation mainly via G2/M cell cycle arrest, but not apoptosis.Compared with DAC treatment group, apoptosis percentage was not increased in the group of BA1in combination with DAC treatment of MDA-MB-468, indicating that autophagy contributes little to apoptosis.To sum up, DAC induces DNA damage and autophagy in breast cancer cells. DNA damgae may be one of the factors inducing autophagy. Autophagy protects breast cancer cells from the cytotoxity of DAC. Autophagy inhibitors in combination with DAC may increase therapeutic effects of DAC in some types of breast cancers.