Investigation of the role of Bax, p21/Waf1 and p53 as determinants of cellular responses in HCT116 colorectal cancer cells exposed to the novel cytotoxic ruthenium(II) organometallic agent, RM175.
Ruthenium(II) organometallic complexes form monofunctional adducts with guanine in DNA in vitro and have a cytotoxic anticancer activity spectrum in preclinical models suggesting lack of cross-resistance with cisplatin. The primary cytotoxic lesion remains to be identified but the downstream mechanism of action is nevertheless of interest. Using isogenic derivatives of the HCT116 colorectal cancer cell line, we investigated the role of p53, p21/WAF1 and Bax in the cellular response to the novel ruthenium(II) organometallic complex RM175, [(eta(6)-C(6)H(5)C(6)H(5))RuCl (H(2)NCH(2)CH(2)NH(2)-N,N')](+) PF(6)(-). Western blotting demonstrated dose-dependent accumulation of p53, Bax and p21/WAF1 within 48 h of the start of RM175 treatment in wild-type HCT116 cells. HCT116 wild-type and Bax-null cells arrested in the G(1) and G(2) phases of the cell cycle. This pattern of cell cycle arrest was not observed in p53-null or in p21/WAF1-null cells. Following RM175 treatment, HCT116 wild-type and p21/WAF1 null cells underwent a dose-dependent induction of apoptosis (Annexin-V and sub-G(1) apoptosis assays). This apoptotic response was not observed in p53-null or Bax-null cells. In short-term sulphorhodamine B assays, the IC(50) for RM175 was 16 microM for p53-null HCT116, and 8 microM for wild-type cells (P<0.05). However, the sensitivity to RM175 in clonogenic assays at 16 days was independent of p53 status. These results identify determinants of the short-term in vitro response to RM175 demonstrating a role for p53 and p21/WAF1 in the growth arrest and for p53 and Bax in the apoptotic response. The mechanism of p53-independent suppression of long-term clonogenicity remains to be determined.
Hayward
R L
RL
Cancer Research UK Edinburgh Oncology Unit, University of Edinburgh Cancer Research Centre, Edinburgh, EH4 2XR, UK.
Schornagel
Q C
QC
Tente
R
R
Macpherson
J S
JS
Aird
R E
RE
Guichard
S
S
Habtemariam
A
A
Sadler
P
P
Jodrell
D I
DI
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D045325:HCT116 Cells; D006801:Humans; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D009942:Organometallic Compounds / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012428:Ruthenium / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein
D000970:Antineoplastic Agents; C494826:BAX protein, human; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D009942:Organometallic Compounds; D019253:Proto-Oncogene Proteins c-bcl-2; C501327:RM175; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D012428:Ruthenium