title: Raltitrexed induces mitochondrial‑mediated apoptosis in SGC7901 human gastric cancer cells.
abstract: Raltitrexed is a specific inhibitor of thymidylate synthase (TS), which has been considered as a potential chemotherapeutic agent for the treatment of advanced gastric cancer. In the present study, the apoptosis mechanisms of raltitrexed in SGC7901 human gastric cancer cells were investigated. The cytotoxic activity of raltitrexed on SGC7901 cells was determined by cell counting kit-8 (CCK-8) assay. The CCK‑8 assay indicated that raltitrexed inhibits SGC7901 cell growth in a dose- and time-dependent manner. The morphological changes were observed by fluorescent microscopy, and characteristic morphological changes, including nuclear shrinkage and apoptotic bodies, were observed following Hoechst 33258 staining. The effects on apoptosis, cell cycle, mitochondrial transmembrane potential and reactive oxygen species (ROS) were measured by flow cytometry. The analysis revealed that raltitrexed exerted a growth inhibitory effect by inducing time-dependent apoptosis and cell-cycle arrest at the G0/G1 phase. In addition, a compromised mitochondrial membrane potential and overproduction of ROS demonstrated the involvement of the mitochondrial signaling pathway. Raltitrexed‑induced caspase‑3‑dependent apoptosis was identified using a caspase-3 activity assay and pretreatment with the caspase-3 inhibitor, Ac‑DEVD‑CHO (sequence, Ac-Asp-Glu-Val-Asp-CHO). The activity of caspase-3 was analyzed with a spectrometer. The protein expression levels of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and TS were examined by western blot and the mRNA expression level of TS was detected by quantitative polymerase chain reaction. The analysis revealed that the protein levels of Bax, cytochrome c and cleaved caspase‑3 were significantly increased by raltitrexed, while Bcl-2 expression levels were reduced. Furthermore, raltitrexed increased the expression of the TS protein and mRNA in a time‑dependent manner. These results indicate that raltitrexed induces the apoptosis of SGC7901 cells through the caspase‑3‑dependent mitochondrial signaling pathway and upregulates the expression of the TS protein and mRNA.
authors.affiliation: Department of Medical Oncology, 81 Hospital of PLA, 81 Clinical College, Anhui Medical University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Song
authors.initials: S
authors.lastname: Xue
authors.affiliation: Department of Medical Oncology, 81 Hospital of PLA, 81 Clinical College, Anhui Medical University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Ying-Xia
authors.initials: YX
authors.lastname: Chen
authors.affiliation: Department of Medical Oncology, 81 Hospital of PLA, 81 Clinical College, Anhui Medical University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Shu-Kui
authors.initials: SK
authors.lastname: Qin
authors.affiliation: Central Laboratory, 81 Hospital of PLA, 81 Clinical College, Anhui Medical University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Ai-Zhen
authors.initials: AZ
authors.lastname: Yang
authors.affiliation: Department of Medical Oncology, 81 Hospital of PLA, 81 Clinical College, Anhui Medical University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.affiliation: Central Laboratory, 81 Hospital of PLA, 81 Clinical College, Anhui Medical University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Hai-Jun
authors.initials: HJ
authors.lastname: Xu
authors.affiliation: Department of Medical Oncology, 81 Hospital of PLA, 81 Clinical College, Anhui Medical University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Hai-Yun
authors.initials: HY
authors.lastname: Geng
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011799:Quinazolines / Q000633:toxicity*; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013274:Stomach Neoplasms / Q000378:metabolism / Q000473:pathology; D013876:Thiophenes / Q000633:toxicity*; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011799:Quinazolines; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013876:Thiophenes; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D013940:Thymidylate Synthase; D053148:Caspase 3; C068874:raltitrexed