Glycolysis Inhibitor3-bromopyruvate Induces Necroptosis In Breast Cancer Cells Response To Autocrine Secretion Of TNF-α

Necrosis as a merely accidental cell death modality has been conclusivelyabandoned in the past. As research continues, a number of experts demonstrated thatnecrosis, similar to apoptosis, can also be a highly regulated process.3-bromopyruvate (3-BrPA) is a halogenated analog of pyruvic acid which inhibitsglycolysis and has antineoplastic activity on malignant cells. In this study, we foundthat caspase inhibitor (z-VAD-fmk) enchanced3-BrPA induced cell death in breastcancer MDA-MB-231cells which has necrotic morphological characteristics.Objectives:1. To investigate the effect of hexokinase Ⅱ inhibitor3-BrPA (3-bromopyruvate) onthe proliferation and cell death of breast cancer cells.2. To investigate whether the3-BrPA induced cell death depend on caspase.3. To explore whether the3-BrPA plus z-VAD-fmk induced necroptosis require RIP1and RIP3.4. To explore whether the3-BrPA plus z-VAD-fmk induced necroptosis requiresMLKL.5. To explore the function of TNF-α in3-BrPA plus z-VAD-fmk induced necroptosis.Methods:1. MTT assay were used to detect the growth inhibition induced by3-BrPA in breastcancer cells.2. The intracellular ATP level was measured by the ATP Assay Kit afterMDA-MB-231cells and MDA-MB-435cells treated with3-BrPA（80,160,320 μmol·L-1).3. Colony-forming assay were used to detect the growth inhibition induced by3-BrPA in breast cancer cells.4. Cell death was assessed by flow cytometry with propidium iodide (PI) staining orPI/Annexin V staining.5. The ultrastructural details of two breast cancer cells treated with vehicle DMSO,3-BrPA or3-BrPA plus z-VAD-fmk were analyzed by electron microscopy (EM).6. Western blot was used to explore the expressions of necrotic key proteins RIP1,RIP3, MLKL, TNF-α and TNFR1.7. Real-time PCR was used to explore the mRNA expressions of necrotic keyproteins RIP3and TNF-α.8. Small interfering RNA silenced RIP1, RIP3, MLKL and TNFR1gene expression,and the expression of related proteins were analyzed by Western blot.Results:1. Different concentrations of3-BrPA regulates the cell proliferation and death inMDA-MB-231and MDA-MB-435cells1.1In order to examine the effect of3-BrPA-induced inhibition of cellularproliferation in breast cancer cells MDA-MB-231and MDA-MB-435, cells weretreated with3-BrPA (20，40，80，160，320μmol·L-1).3-BrPA at concentrationsgreater than80μmol·L-1inhibited MDA-MB-231cell viability in a dose-andtime-dependent manner. In contrast, MDA-MB-435cells almost did not respondto3-BrPA.1.2In an assay to estimate malignant cell colony formation, MDA-MB-231cellsdecreased after3-BrPA (30，40，50μmol·L-1) treatment for7days. No obviousanti-proliferative effects with low-dose3-BrPA occurred in MDA-MB-435cells.1.3To confirm that the reduction in the cell numbers was reflective of cell death, flowcytometric detection was performed using PI staining and PI/Annexin V staining.PI staining was revealed that MDA-MB-231cells had greater sub-G1DNAcontent (an indicator of cellular damage) after treatment with increasing3-BrPA concentrations, but changes were not observed obviously in MDA-MB-435cells.PI/Annexin V staining confirmed these findings. Spontaneous cell death wasminimal in untreated (Control) cultures. MDA-MB-231had increased Annexin Vand PI staining (upper right quadrant) or Annexin V staining alone (lower rightquadrant) under the same treatment conditions mentioned above. The changeswere also not obvious in MDA-MB-435cells.1.4In addition, it is well documented that cancer cell death induced by3-BrPAdepends on the depletion of the cellular ATP pool. Our studies in two breast cancercell lines confirm this mechanism. When MDA-MB-231and MDA-MB-435cellswere treated with3-BrPA (80，160，320μmol·L-1) for6h, ATP levels rapidlydecreased in both cell lines in a dose-dependent manner.2.3-BrPA plus z-VAD-fmk induced necroptosis in MDA-MB-231cells2.1When MDA-MB-435cells treated with500μmol·L-13-BrPA, cells appearedobviously injury. In order to determine whether the cell death induced by3-BrPAin two breast cancer cell lines were caspase-dependent, a combination treatment of3-BrPA and z-VAD-fmk was applied. Surprisingly, z-VAD-fmk was notcytoprotective to MDA-MB-231cells injured by3-BrPA, but damage increased.Meanwhile, cell death was completely blocked by z-VAD-fmk in MDA-MB-435cells.2.2We took advantage of this cell death model, cells treated with either vehicleDMSO or3-BrPA plus z-VAD-fmk were analyzed by electron microscopy. MostDMSO-treated cells had normal cellular morphology with intact cytoplasmicmembranes, a significant percentage of cells treated with3-BrPA plus z-VAD-fmkhad swelled mitochondria and discontinuous cytoplasmic membranes. No obviousapoptotic morphological changes were observed. Electron micrographs ofMDA-MB-435cells treated with either vehicle DMSO or3-BrPA (320μmol·L-1)revealed ultrastructural details and a characteristic autophagosome inMDA-MB-435cells which may be one of the reasons that MDA-MB-435cellswere insensitive to relatively low concentrations of3-BrPA. 3.3-BrPA plus z-VAD-fmk induced necroptosis requires RIP1and RIP33.1Next, cells were pre-treated with necrostatin-1(Nec-1), a necroptoticpathway-related RIP1kinase inhibitor. Data shown that the combination3-BrPAwith z-VAD-fmk treatment induced loss of cell viability was modestly prevented.3.2Since smac mimetic plus z-VAD-fmk-induced necroptosis of HT29cells iscorrelated with the levels of RIP1and RIP3expression. We therefore focused oninvestigation of the mechanism whether RIP1and RIP3play important roles in3-BrPA plus z-VAD-fmk-induced cell death. To this end, we examined if3-BrPAplus z-VAD-fmk regulates RIP1total protein by Western blot and RIP3mRNAlevels by Real-time PCR, respectively.3-BrPA plus z-VAD-fmk increased thelevels of the RIP1total protein that could be detected by6hours after treatment.3.3Treatment with3-BrPA plus z-VAD-fmk up-regulated the levels of RIP3mRNAsignificantly.3.4Next, we silenced RIP1using RNAi. Knockdown of RIP1virtually abolished thisform of cell death.3.5Then we knocked down RIP3with two individual siRNA oligoes. Cell deathinduced by3-BrPA plus z-VAD-fmk was attenuated when RIP3was knockeddown.4.3-BrPA plus z-VAD-fmk induced necroptosis requires MLKL4.1In the issue of Cell, Wang and colleagues provided convincing evidence thatmixed lineage kinase domain-like (MLKL) is a functional substrate for RIP3kinase that serves as an adaptor protein for necrosis signal transduction. To testwhether it works in3-BrPA-induced necroptosis, we identify MLKL expression inMDA-MB-231cells treated with necroptosis inducers.3-BrPA plus z-VAD-fmkup regulated the expression of MLKL with a significant increase at6hours.4.2Consistent with Wang’s report, necrosulfonamide (NSA, an inhibitor of MLKL)repressed the expression of MLKL and cell death was modestly prevented byNSA.4.3Then we knocked down MLKL with three different oligos in MDA-MB-231cells.Cell death induced by3-BrPA plus z-VAD-fmk was attenuated when MLKL was knocked down in MDA-MB-231cells.5.3-BrPA–induced necroptosis in MDA-MB-231cells response to TNF-α5.1Since Smac mimetic induces necroptosis in HT-29cells as a result of autocrineTNF-α. We examined TNF-α mRNA levels and total protein of3-BrPA plusz-VAD-fmk treated cells by Real-time PCR and Western blot, respectively. Indeed,necroptosis inducer up-regulated the levels of TNF-α mRNA in MDA-MB-231cells obviously.5.2The expression of TNF-α total protein was also upregulated apparently afterexposure to necroptosis stimuli for6hours.5.3Moreover, addition of an increasing amount of anti-TNF-α to culture mediumpartially blocked this form of cell death and it did so in a dose-dependent manner.5.4We used siRNAs to deplete TNFR1in MDA-MB-231cells. Consistently,knockdown of TNFR1also prevented these cells from dying. Knockdownefficiencies of the siRNAs were determined by Western blot utilizing threeindividual siRNA oligos. Taken together, these results validate that TNF-α iscritical for the3-BrPA plus z-VAD-fmk-induced necroptosis.Conclusion:1. z-VAD-fmk enhanced3-BrPA induced cell death in MDA-MB-231cells.2. Autophagy may be one of the reasons that MDA-MB-435cells were insensitive torelatively low concentrations of3-BrPA.2.3-BrPA plus z-VAD-fmk induced necroptosis in MDA-MB-231cells requires RIP1,RIP3, MLKL and TNFR1.