Autophagy Blocker Chloroquine Promoting Formation Of ROS And Enhancing The Sensitivity Of Human Breast Cancer Cells To3-bromine Pyruvic Acid

Hexokinase II (HKII), a key enzyme of glycolysis, is widelyover-expressed in cancer cells.3-bromopyruvate (3-BrPA), an inhibitor of HK II, hasbeen proposed as a specific antitumor agent. Autophagy is a process that regulates thebalance between protein synthesis and protein degradation. Autophagy in mammaliansystems occurs under basal conditions and can be stimulated by stresses, includingstarvation, oxidative stress. Therefore, we hypothesized that3-BrPA could induce autophagy. In the present study, we explored the mechanism of3-BrPA and itscombined action with chloroquine. Our results demonstrate that in MDA-MB-435andin MDA-MB-231cells,3-BrPA induces autophagy, which can be inhibited bychloroquine. Furthermore, the combined treatment synergistically decreased the numberof viable cells. Interestingly, the combined treatment triggered apoptosis inMDA-MB-435cells, while it induced necroptosis in MDA-MB-231cells. ROSmediated cell death when3-BrPA and CQ were co-administered. Finally, CQ enhancedthe anticancer efficacy of3-BrPA in vivo. Collectively, our results show that3-BrPAtriggers autophagy, increasing breast cancer cell resistance to3-BrPA treatment and thatCQ enhanced3-BrPA-induced cell death in breast cancer cells by stimulating ROSformation. Thus, inhibition of autophagy may be an innovative strategy for adjuvantchemotherapy of breast cancer.Aims:1. To explore the effects of3-BrPA on the proliferation of human breast cancer cells,and whether3-BrPA could induce autophagy.2. To explore the effects of autophagy inhibitor3-MA and CQ coordinated with3-BrPA on the prolifertion and cell death of human breast cancer cells, whetherautophagy blocker could enhance the sensitivity of human breast cancer cells to3-BrPA.3. To explore the cell death type induced by3-BrPA coordinated with CQ and itsmechanism.4. To explore whether CQ could enhance the antitumor ability of3-BrPA in vivo.Methods:1. MDA-MB-231, MDA-MB-435cells were treated with different concentrations of3-BrPA(20、40、80、160、320μmol·L-1) for24,36,48h, the viability rate was detectedin order to make clear the dose-effect relationship by MTT assay; Then cell morphologywas photographed by inverted microscope.2. Breast cancer cells MDA-MB-435and MDA-MB-231were treated with320μM3-BrPA and160μM3-BrPA respectively for12h as indicated. Then cells were collectedand prepared for electron microscopy (EM) analysis. Cells were treated as above for0, 1,2,4,8, and12h as indicated, and the extracted protein was immunoblotted againstLC3and Beclin-1antibody. β-actin was used to normalize the data for equal proteinloading. In the end, cells were transiently transfected with GFP-tagged LC3plasmidDNA (GFP-LC3), treated with3-BrPA (320μM) and3-BrPA (160μM) as above for12h,then subjected to confocal microscopy analysis (scale bar=10μm).3. After pre-incubation with40μM CQ/5mM3-MA for1h, MDA-MB-435cells andMDA-MB-231cells were treated with320μM3-BrPA and160μM3-BrPA respectivelyfor12h, and the relative cell viability was assessed with an MTT assay. Cells weretreated as above. Then cells were photographed using an inverted microscope. AnnexinV/PI double staining was used to detecte dead cell and Western blotting assay was usedto explore the expressions of apoptosis-related protein Bcl-2, Mcl-1, Bax and Bak andautophagy-related proteins Beclin1, LC3. The cells were knocked downautophagy-related proteins Atg7and then MDA-MB-435cells and MDA-MB-231cellswere treated with320μM and160μM3-BrPA respectively. The viability rate wasdetected by MTT.4. After pre-incubation with10μM z-VAD-fmk for1h, cells were treated with160μM3-BrPA and/or with20μM CQ for24h. Then, relative cell viability was assessed with anMTT assay. Then MDA-MB-231cells were treated with160μM3-BrPA and/or with20μM CQ for24h, and then cells were collected and prepared for EM analysis. Afterpre-incubation with10μM Nec-1for1h, MDA-MB-435and MDA-MB-231cells weretreated with160μM3-BrPA and/or with20μM CQ for24h. Then, relative cell viabilitywas assessed with an MTT assay. Finally, two cells were knocked down RIPK1andRIPK3, and the viability was detected after receiving treatment of3-BrPA(160μmol·L-1)and CQ(20μmol·L-1) for24h.5. MDA-MB-435cells and MDA-MB-231cells were treated with320μM3-BrPA and160μM3-BrPA respectively, and/or with40μM CQ for6h. ROS production wasanalyzed with DHE staining and flow cytometry. After pre-incubation with5mM NACfor1h, cells were treated as above. Relative cell viability was measured with an MTTassay, and morphology was detected by inverted microscope. In the end, cells weretreated as above. Loss of mitochondrial membrane potential (MMP) was quantified withJC-1staining and fluorescent microscopy.6. The nude mice (5-6weeks) used in these studies were obtained from Beijingvitalriver and weighed20–25g at the time of tumor implantation. The mice were keptunder a12:12h light–dark cycle, at24±2°C and fed with clean food and water. Human MDA-MB-231cells (107cells/ml) were inoculated subcutaneously to formtumors. Tumors (100-200mm3) bearing mice were assorted to four groups (5mice/group). Vehicle (0.9%NS) or CQ (40mg/kg/d,24days) or3-BrPA (5mg/kg/d,24days) alone or in combination was administrated intraperitoneally. Tumor growth wasmonitored every three days by two-dimensional measurements of individual tumors foreach mouse. Tumor volume was calculated using the formula: length×width2/2. Aftertreatment ended, mice were sacrificed. Tumors were excised, calculated and fixed in4%formalin solution, embedded in paraffin, and then stained with hematoxillin-eosin(H&E).Results:Ⅰ.3-BrPA has different effects on cell proliferation in breast cancer cell linesMDA-MB-435and MDA-MB-231.3-BrPA can induce cell death in certain tumor cell lines. May3-BrPA inhibit cellgrowth in breast cancer cells (MDA-MB-231, MDA-MB-435) too? The cell viabilitywas measured with an MTT assay after incubation of each cell line with3-BrPA forvarious periods. The result showed3-BrPA (0–320μM) reduced MDA-MB-231cellgrowth in a dose-and time-dependent manner, however,3-BrPA had little effect onMDA-MB-435cell growth. Most MDA-MB-231cells treated by3-BrPA shrank andbecame rounded before detaching from the culture plates under inverted microscope,whereas only modest decrease of viable MDA-MB-435cells has been found. Theseprompted us to explore further the unique effects of3-BrPA on the two cells.Ⅱ.3-BrPA induces autophagy in breast cancer cells.First electron microscopy (EM) was used to visualize cell morphology after the twotumour cell lines were treated with3-BrPA.3-BrPA treatment increased the presence ofautophagosomes filled with debris in both cell lines; only a few vacuoles were observedin control cells. Autophagy-specific markers such as microtubule-associated protein1LC3, Beclin-1were used to quantify autophagy with immunoblot analysis. Conversionof LC3I/II and up-regulation of Beclin-1suggested increased formation ofautophagosomes in a time-dependent manner in the breast cancer cells. Induction ofautophagy by3-BrPA was then examined by imaging the cellular distribution of GFP-LC3, a fusion construct of green fluorescent protein with LC3. In control cells,GFP-LC3puncta were mainly distributed to the cytosol, that indicats a low level ofautophagy under these conditions. In contrast, cells incubated with3-BrPA for12hshowed sharply increase of autophagosomes with accumulated GFP-LC3(presumablyas GFP-LC3-II), and that indicated the indeed occurrance of autophagy in the breastcancer cells.Ⅲ. Autophagy inhibition enhances3-BrPA-induced cell death.To investigate the role of autophagy in cell death, cell autophagy was inhibited withCQ or3-Methyladenine (3-MA) and cell death was measured. As expected, a significantincrease in3-BrPA-induced cell death was observed in the breast cancer cells afterautophagy was inhibited with CQ or3-MA. Under inverted microscope, viableMDA-MB-231and MDA-MB-435cells were significantly decreased after3-BrPA plusCQ treatment for12h. The majority of cells shrank and became rounded beforedetaching from the culture plates. Moreover, annexin V-propidium iodide stainingrevealed a significant increase in cell death (defined as annexin V+and PI V+cells)after3-BrPA plus CQ treatment.To ascertain what happened about the expression of autophagy-related proteinsafter the treatment of3-BrPA combined with CQ, Western blotting was used to measureLC3-II. The result showed the turnover of LC3Ⅰto LC3Ⅱdecreased andautophagy-related proteins Beclin1reduced too. These clearly exhibited the autophagywas suppressed. Meanwhile the reduction of expression of anti-apoptotic proteinsBcl-2and Mcl-1and the augment of expression of pro-apoptotic proteins Bax and Bak,These suggest that autophagy may be a mechanism of resistance to3-BrPA and thatcould be reversed by pharmacologic blocker.Treatment of3-BrPA after knockdown of autophagy-related proteins Atg7significantly increased the tuome viability, compared to control. These showed theinhibition of autophagy by genetic blockade of autophagy initiation can enhance thesensitivity of the breasr cancer cell to3-BrPA. Ⅳ.3-BrPA plus CQ treatment induces RIPK1-dependent apoptosis inMDA-MB-435cells and RIPK1/3-dependent necroptosis in MDA-MB-231cells.To confirm the cell death type induced by the combination of3-BrPA and CQ, weused a broad spectrum caspase inhibitor, z-VAD-fmk to rescue cells from death inducedby3-BrPA plus CQ treatment. An MTT assay revealed that z-VAD-fmk rescuedMDA-MB-435cells, but exacerbated cell death in MDA-MB-231cells. Using EM, weobserved that the plasma membranes of MDA-MB-231cells treated by3-BrPA plus CQhad ruptured, and cell necrosis has been indicated. Death may also occur by aprogrammed form of necrosis, necroptosis, which requires RIPK1and RIPK3activationby death receptors or other death receptor-independent mechanisms. To assess whether3-BrPA plus CQ treatment induced necroptosis, MDA-MB-231cells were treated with3-BrPA plus CQ with/without the specific RIPK1and necroptosis inhibitornecrostatin-1(Nec-1). Nec-1alone had no effect on cells, but dramatically restored cellsurvival by after treatment with3-BrPA plus CQ in MDA-MB-231cells andMDA-MB-435cells. Knockdown of RIPK1in both cell lines also suppressed cell deathand enhanced cell viability. Similarly, cells treated with a validated siRNA againstRIPK3were significantly more viable after treatment with3-BrPA plus CQ inMDA-MB-231cells，but not in MDA-MB-435cells. These data suggest that3-BrPAand CQ induce two different cell death types in the two tumor cells.Ⅴ. ROS production causes cell death with3-BrPA and autophagy inhibition.Next, to elucidate the mechanism which leads to the initiation of3-BrPA-relatedautophagy, we quantified intracellular ROS in breast cancer cells after a6h incubationwith3-BrPA, CQ, or a combination of both drugs. ROS generation was markedlyincreased in cells treated with3-BrPA plus CQ compared to cells treated with3-BrPAalone. Because elevated ROS occurred in cells with suppressed autophagy, weinvestigated whether ROS inhibition influenced3-BrPA-plus-CQ-mediated cell death.ROS formation was inhibited with the ROS scavenger N-acetyl cysteine (NAC) and cellviability was measured with an MTT assay. Cell morphology was observed andphotographed under an inverted microscope. The data indicate that cell sensitization to 3-BrPA-and CQ-induced completely blocked cell death when ROS formation wasinhibited. In addition, the mitochondrial membrane potential (MMP), an indicator ofROS generation, was detected in these cells using JC-1. Higher MMP was observed inthe control culture group compared to the dually treated groups. With co-treatment with3-BrPA and CQ, JC-1revealed higher green fluorescence, while adding NAC makesMMP turn to be higher again.Ⅵ. CQ enhanced anti-tumor efficacy of3-BrPA in nude mice.To ascertain whether a combination of3-BrPA and CQ could suppress tumorgrowth, MDA-MB-231xenografts were examined. Tumors continued to grow inxenografted mice treated with vehicle, CQ, or3-BrPA, or a combination of CQ and3-BrPA. The combination of CQ plus3-BrPA prevented tumor growth. After treatmentended, mice were sacrificed, and tumors were excised and evaluated. Tumor weights ofmice treated with vehicle, CQ, and3-BrPA alone were greater than those from micetreated with CQ plus3-BrPA. Hematoxylin and eosin (HE) staining indicated largernecrotic areas in CQ plus3-BrPA-treated mice. Thus, CQ enhanced the anti-tumorefficacy of3-BrPA in vivo.Conclusions:1.3-BrPA has different effects on cell proliferation in breast cancer cell linesMDA-MB-435and MDA-MB-231.2.3-BrPA induces autophagy in breast cancer cells.3. Autophagy inhibition enhances3-BrPA-induced cell death.3-BrPA plus CQtreatment induces RIPK1-dependent apoptosis in MDA-MB-435cells andRIPK1/3-dependent necroptosis in MDA-MB-231cells.4. ROS production causes cell death with3-BrPA and autophagy inhibition.5. CQ enhanced anti-tumor efficacy of3-BrPA in nude mice.