Endoplasmic Reticulum Stress Signaling Is Involved In Mitomycin C (MMC)-induced Apoptosis In Human

Mitomycin C (MMC), is a potent reactive oxygen species (ROS)-generatingantitumor drug used to treat cancer. MMC has been reported to prevent scarringduring lumbar laminectomy and tendon surgery in a rat modelObjectivePart IHuman fibroblasts were cultured from epidural scar. We investigated whether5minutes of MMC exposure induced the apoptosis of human fibroblasts.Part IITo explore whether endoplasmic reticulum stress (ERS) is involved inMMC-induced apoptosis in human fibroblastsPart IIITo study the effect of ROS-generating in ERS involved MMC-induced apoptosisin human fibroblasts MethodsPart I1. Primary cultured human fibroblasts were obtained from epidural scar. Thehuman fibroblasts were identified by immunocytochemistry for subsequentexperiments.10mg MMC was dissolved in fresh medium. The mediumcontaining various concentrations of MMC was prepared before everyexperiment.2. CCK-8(Cell Counting Kit-8) assay was used to evaluate the viability ofhuman fibroblasts after MMC treatment.3. Flow cytometry was used to detect the apoptosis percentage and cell cycle ofhuman fibroblasts after MMC treatment4. TUNEL assay was used to observed the late stage apoptotic humanfibroblasts after MMC treatment5. Western blotting was used to detect the expression levels of apoptosisassociated proteins.Part II1. Primary cultured human fibroblasts were obtained from epidural scar. Thehuman fibroblasts were identified by immunocytochemistry for subsequentexperiments.10mg MMC was dissolved in fresh medium. The mediumcontaining various concentrations of MMC was prepared before everyexperiment.2. Real-time PCR and reverse transcription PCR were used to detect the transcription levels of XBP-1mRNA, CHOP mRNA and GRP78mRNA.3. CHOP siRNA, PERK, ATF6and IRE1shRNA knockdown was used todemonstrate that the ERS associated proteins involved in the apoptosis ofhuman fibroblasts.4. Western blotting was used to detect the expression levels of ERS andapoptosis associated proteins.Part II1. Primary cultured human fibroblasts were obtained from epidural scar. Thehuman fibroblasts were identified by immunocytochemistry for subsequentexperiments.10mg MMC was dissolved in fresh medium. The mediumcontaining various concentrations of MMC was prepared before everyexperiment.2. Lipid peroxidation assay was used to detect the MDA levels in humanfibroblasts.3. DCFH-DA was used to observe the ROS levels in human fibroblasts.4. CCK-8(Cell Counting Kit-8) assay was used to evaluate the viability ofhuman fibroblasts.5. Flow cytometry was used to detect the apoptosis percentage of humanfibroblasts.6. Western blotting was used to detect the expression levels of ERS andapoptosis associated proteins. ResultsPart I1. MMC significantly reduced human epidural scar fibroblasts viability in adose and time-dependent manner.2. MMC significantly increased the apoptosis rates of human epidural scarfibroblasts in a time-dependent manner, and the cell cycle was significantlyarrested at G1phase.3. MMC notably increased the expression levels of Cleaved Caspase-3andCleaved PARP in a time-dependent manner. MMC also remarkably reducedthe expression level of total PARP in a time-dependent manner.Part II1. MMC significantly increased the transcription levels of CHOP and GRP78mRNA and remarkably increased the splicing level of XBP1.2. MMC notably increased the activation or expression levels of PERK, eIF2α,ATF6, IRE1and CHOP in a dose and time-dependent manner.3. The expression level of CHOP was silenced by CHOP siRNA and theapoptosis rate of human epidural scar fibroblasts was significantly reduced aswell as the expression level of Bim.4. The expression level of PERK was silenced by PERK shRNA and theapoptosis rate of human epidural scar fibroblasts was significantly reduced aswell as the expression levels of CHOP, Bim and Cleaved Caspase-3. Theexpression level of IRE1and ATF6were silenced by IRE1and ATF6shRNA but the apoptosis rate of human epidural scar fibroblasts was not reduced aswell as the expression levels of CHOP, Bim and Cleaved Caspase-3.Part III1. MMC significantly increased the ROS and MDA levels in human epiduralscar fibroblasts.2. NAC, GSH and Edaravone pre-treatment significantly reduced the ROS andMDA levels in human epidural scar fibroblasts and significantly increased thecell viability.3. NAC, GSH and Edaravone pre-treatment significantly reduced the apoptosisrates of human epidural scar fibroblasts which increased by MMC treatment.4. NAC, GSH and Edaravone pre-treatment notably reduced the activation orexpression levels of GRP78、PERK、CHOP、Bim、Cleaved Caspase-3andPARP.ConclusionsPart I1. MMC significantly reduced the viability of human epidural scar fibroblasts.2. MMC significantly increased the apoptosis rate of human epidural scarfibroblasts and the cell cycle was significantly arrested at G1phase.3. MMC notably increased the expression levels of Cleaved Caspase-3andCleaved PARP, leading to the apoptosis of human epidural scar fibroblasts. Part II1. ERS associated proteins involved in MMC-induced apoptosis of humanepidural scar fibroblasts.2. The PERK pathway of ERS played a critical role in MMC-induced apoptosisof human epidural scar fibroblasts.Part III1. MMC-induced apoptosis of human epidural scar fibroblasts is triggered byROS generation.