title: Synergistic antitumor activity of pro-apoptotic agent PAC-1 with cisplatinum by the activation of CASP3 in pulmonary adenocarcinoma cell line H1299.
abstract: OBJECTIVE
  Evasion of apoptosis is a hallmark of human cancer cells. We sought to explore the potential synergistic antitumor activity and underlying mechanisms of the pro-apoptotic agent PAC-1 plus cisplatinum (Cis) in non-small cell lung cancer (NSCLC) cell lines.
  
  
  METHODS
  The adenocarcinoma cell lines H1299, A549, PC9, H1650 and H1975 were used as in vitro models. Colorimetric MTT assays, Western blotting and flow cytometry were used to evaluate the anti-growth effects of PAC-1 and/or Cis and apoptosis status. The activated form of CASP3 (C-CASP3) was assessed by immunofluorescent staining.
  
  
  RESULTS
  Single-agent Cis and PAC-1 were able to inhibit the cancer cell growth in certain dose ranges, with IC50 values of 1.9-11.7 and 5.6-14.8 μM, respectively. Sequential Cis→PAC-1 or concurrent Cis + PAC-1, but not PAC-1→Cis combinations showed synergistic effects on cell growth inhibition in H1299 cells (combination index, CI ≤ 0.6). In contrast, other combination modes mostly showed seemingly antagonistic effects (CI > 1.0). Flow cytometric analysis showed that Cis→PAC-1 sequential combination showed strong pro-apoptotic effects in H1299 cells. Western blots showed that in H1299, PC9 and H1975 cells, PAC-1 promoted the C-CASP3, but only in H1299 cells was there a synergistic effect with Cis on the CASP3 activation.
  
  
  CONCLUSIONS
  PAC-1 showed anti-tumor activity in NSCLCs in vitro and a synergistic effect with cisplatin in EGFR(wt)KRAS(wt) H1299 cells. Our data suggest a potential treatment approach using cisplatin plus a pro-apoptotic agent acting via CASP3 activation for this subgroup of pulmonary adenocarcinomas.
authors.affiliation: Affiliated Cancer Hospital, Guangzhou Medical University, Guangzhou, China.
authors.forename: Jian-qing
authors.initials: JQ
authors.lastname: Huang
authors.affiliation: Affiliated Cancer Hospital, Guangzhou Medical University, Guangzhou, China.
authors.forename: Hong-ling
authors.initials: HL
authors.lastname: Liang
authors.affiliation: Guangdong Lung Cancer Institute, Guangdong General Hospital, Guangzhou, China.
authors.forename: Xu-chao
authors.initials: XC
authors.lastname: Zhang
authors.affiliation: Guangdong Lung Cancer Institute, Guangdong General Hospital, Guangzhou, China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Affiliated Cancer Hospital, Guangzhou Medical University, Guangzhou, China.
authors.forename: Tian-en
authors.initials: TE
authors.lastname: Jin
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism*; D000077192:Adenocarcinoma of Lung; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology*; D008175:Lung Neoplasms / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology*
chemical_list: C514579:(4-benzylpiperazin-1-yl)acetic acid (3-allyl-2-hydroxybenzylidene)hydrazine; D006835:Hydrazones; D010879:Piperazines; C505407:CASP3 protein, human; D053148:Caspase 3; D002945:Cisplatin
keywords: CASP3; PAC-1; apoptosis; non-small cell lung cancer