title: Cisplatin (CDDP) specifically induces apoptosis via sequential activation of caspase-8, -3 and -6 in osteosarcoma.
abstract: OBJECTIVE
  Osteosarcoma is a common malignant tumor. The first choice of treatment plan for osteosarcoma is chemotherapy. In particular, preoperative chemotherapy is most important in clinical treatment in orthopedics. In these chemotherapies, multiple anticancer drugs such as Adriamycin (ADM), CDDP, cyclophosphamide (CPM), methotrexate (MTX) and vincristine (VCR) are commonly used in combination. Recently, anticancer drugs have been shown to trigger apoptosis in various cancer cells. However, many studies on this topic have been examined using leukemia cell lines, and many kinds of cancer cells established from solid tumor are resistant to the induction of apoptosis by anticancer drugs. So in this study, we examined the effects of the anticancer drugs ADM, CDDP, CPM, MTX and VCR on osteosarcoma cells in vitro. We also examined the signaling pathways of each anticancer drug by studying the induction of apoptosis and activation of caspases in the osteosarcoma cells.
  
  
  METHODS
  We examined the effects of the anticancer drugs ADM, CDDP, CPM, MTX and VCR, which are used clinically for the treatment of osteosarcoma, on cells of the human osteosarcoma (HOS) cell line. The cytotoxic effects of the anticancer drugs were evaluated using the MTT assay. We used both flow cytometry and activation of caspases to confirm the induction of apoptosis in the HOS cells. To dissect the pathway of the caspase cascade in apoptosis in HOS cells, we used the tetrapeptides YVAD-CHO, DMQD-CHO, VEID-CHO and IETD-CHO, which selectively inhibit caspase-1, -3, -6 and -8, respectively.
  
  
  RESULTS
  ADM, CDDP, CPM and VCR, but not MTX, induced death of HOS cells in a dose-dependent manner. CDDP at 10 microM, CPM at 7.5 microM, ADM at 20 microM and VCR at 150 microM caused 80% cell death of HOS cells after 12 h. However, the percentages of apoptotic cells were 5.6% (medium alone), 75.9% (CDDP), 20.0% (CPM), 22.2% (ADM), 20.5% (VCR) and 13.1% (MTX). In addition, direct measurement of caspase-3 activity revealed that CDDP but not the other drugs activated caspase-3 in HOS cells. These analyses revealed that only CDDP induced apoptosis of HOS cells via activation of caspases. Furthermore, DMQD-CHO, VEID-CHO and IETD-CHO inhibited CDDP-induced apoptosis of HOS cells, suggesting that caspase-3, -6 and -8 are involved in the signaling pathway of CDDP-induced apoptosis. In contrast, none of the caspase inhibitors inhibited cell death induced by the other anticancer drugs.
  
  
  CONCLUSIONS
  This study demonstrates that CDDP specifically induces apoptosis via activation of caspases and the other anticancer drugs induce death of HOS cells via different signaling pathways. It also demonstrates that caspase-8 is a key molecule in the earliest stage of the signaling pathway of CDDP-induced apoptosis of HOS cells, and caspase-3 works downstream of caspase-8.
authors.affiliation: Department of Orthopedics, Yamanashi Medical University, Tamaho-cho, Yamanashi 409-3898, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Seki
authors.forename: H
authors.initials: H
authors.lastname: Yoshikawa
authors.forename: K
authors.initials: K
authors.lastname: Shiiki
authors.forename: Y
authors.initials: Y
authors.lastname: Hamada
authors.forename: N
authors.initials: N
authors.lastname: Akamatsu
authors.forename: K
authors.initials: K
authors.lastname: Tasaka
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D053178:Caspase 6; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D003520:Cyclophosphamide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D012516:Osteosarcoma / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D014750:Vincristine / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; C110922:acetyl-aspartyl-methionyl-glutaminyl-aspartyl-aldehyde; C401206:acetyl-isoleucyl-glutamyl-threonyl-aspartyl-aldehyde; C110923:acetyl-valyl-isoleucyl-aspartyl-aldehyde; D014750:Vincristine; D004317:Doxorubicin; D003520:Cyclophosphamide; C505407:CASP3 protein, human; C505514:CASP6 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053178:Caspase 6; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D002945:Cisplatin; D008727:Methotrexate