title: Gold (I) N-heterocyclic carbene complex inhibits mouse melanoma growth by p53 upregulation.
abstract: BACKGROUND
  Cancer treatment using gold (I) complexes is becoming popular. In this study, a gold (I) N-heterocyclic complex designated as complex 3 was synthesized, its cytotoxicity was examined, and its anti-melanoma activity was evaluated in vitro and in vivo.
  
  
  METHODS
  Viability of cancer cells was determined by MTT assay upon treatment with various concentrations of a gold (I) N-heterocyclic carbene complex (complex 3) in a dose and time dependent manner. Mouse melanoma cells B16F10 were selected for further apoptotic studies, including flowcytometric analysis of annexin binding, cell cycle arrest, intracellular ROS generation and loss in the mitochondrial membrane potential. ELISA based assays were done for caspase activities and western blots for determining the expression of various survival and apoptotic proteins. Immunocytology was performed to visualize the translocation of p53 to the nucleus. B16F10 cells were inoculated into mice and post tumor formation, complex 3 was administered. Immunohistology was performed to determine the expressions of p53, p21, NF-κB (p65 and p50), MMP-9 and VEGF. Student's t test was used for determining statistical significance. The survival rate data were analyzed by Kaplan-Meier plots.
  
  
  RESULTS
  Complex 3 markedly inhibited the growth of HCT 116, HepG2, and A549, and induced apoptosis in B16F10 cells with nuclear condensation, DNA fragmentation, externalization of phosphatidylserine, activation of caspase 3 and caspase 9, PARP cleavage, downregulation of Bcl-2, upregulation of Bax, cytosolic cytochrome c elevation, ROS generation, and mitochondrial membrane potential loss indicating the involvement of an intrinsic mitochondrial death pathway. Further, upregulation of p53, p-p53 (ser 15) and p21 indicated the role of p53 in complex 3 mediated apoptosis. The complex reduced tumor size, and caused upregulation of p53 and p21 along with downregulation of NF-κB (p65 and p50), VEGF and MMP-9. These results suggest that it induced anti-melanoma effect in vitro and in vivo by modulating p53 and other apoptotic factors.
  
  
  CONCLUSIONS
  The gold (I) N-heterocyclic carbene complex (C22H26N6AuO2PF6) designated as complex 3 induced ROS and p53 dependent apoptosis in B16F10 cells involving the mitochondrial death pathway along with suppression of melanoma tumor growth by regulating the levels of pro and anti apoptotic factors (p53, p21, NF-κB, VEGF and MMP-9).
authors.forename: Abhishek
authors.initials: A
authors.lastname: Nandy
authors.forename: Sumit Kumar
authors.initials: SK
authors.lastname: Dey
authors.forename: Sujata
authors.initials: S
authors.lastname: Das
authors.forename: Rudra Narayan
authors.initials: RN
authors.lastname: Munda
authors.affiliation: Cancer Biology and Inflammatory Disorder division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal 700032, India. joydevdinda@gmail.com.
authors.forename: Joydev
authors.initials: J
authors.lastname: Dinda
authors.forename: Krishna Das
authors.initials: KD
authors.lastname: Saha
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D017612:Gold Compounds / Q000138:chemical synthesis / Q000494:pharmacology*; D045325:HCT116 Cells; D056945:Hep G2 Cells; D006571:Heterocyclic Compounds / Q000138:chemical synthesis / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D008545:Melanoma / Q000378:metabolism*; D008697:Methane / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology; D051379:Mice; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D000970:Antineoplastic Agents; D017612:Gold Compounds; D006571:Heterocyclic Compounds; D016159:Tumor Suppressor Protein p53; C030011:carbene; D008697:Methane