Abstract
We have examined the mechanisms by which the multinuclear platinum chemotherapeutic BBR3610 kills human colon cancer cells. BBR3610 more efficiently killed HCT116, DLD1, SW480, and HT29 cells than BBR3464, cisplatin, or oxaliplatin. The amount of platinum uptake per cell and its incorporation into DNA were identical for BBR3464 and BBR3610. BBR3610 lethality (IC75) was unaltered comparing HCT116 wild-type and p53–/– cells, was reduced in p21–/– cells, and was enhanced in K-RAS D13 null cells. Small molecule or molecular inhibition of epidermal growth factor receptor (ERBB1) or phosphatidyl inositol 3 kinase (PI3K) enhanced BBR3610 toxicity in HCT116, DLD1, and SW480 cells. Small molecule or molecular inhibition of caspase 8 function abolished the toxicity of BBR3610 and of BBR3610 + ERBB1 inhibitor treatments, whereas inhibition of caspase 9 suppressed the ability of ERBB1 inhibitors to enhance BBR3610 lethality. Treatment with BBR3610 reduced AKT activity; the expression of dominant-negative AKT enhanced and expression of constitutively active AKT suppressed, respectively, the toxicity of BBR3610 and of BBR3610 + ERBB1 inhibitor treatments. Treatment with BBR3610 reduced expression of c-FLIP-s and MCL-1, levels that were maintained in cells expressing constitutively active AKT. Overexpression of c-FLIP-s or loss of BID function suppressed BBR3610 toxicity, whereas overexpression of XIAP or Bcl-xL suppressed the potentiation of cell killing by ERBB1 inhibitors. Collectively, our data argue that BBR3610 promotes cell killing via a caspase 8-dependent mechanism, which can be enhanced by ERBB1/PI3K inhibitors that promote additional BBR3610-dependent cell killing via activation of BAX and caspase 9.
Footnotes
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P.D. is the holder of the Universal Inc. Professorship in Signal Transduction Research. These studies were funded by The Goodwin Foundation and P01 CA104177 Core B for virus production.
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N.P.F. and S.G. contributed equally to direction of the studies.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.038406.
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ABBREVIATIONS: ERK, extracellular signal-regulated kinase; MEK, mitogen-activated extracellular regulated kinase; PI3K, phosphatidyl inositol 3 kinase; –/–, null/gene deleted; MAPK, mitogen-activated protein kinase; PD184352, 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; JNK, c-Jun NH2-terminal kinase; FAS, fatty acid synthase; FADD, fatty acid synthase-associating death domain protein; WT, wild type; BBR3464, (SP-4 –1)-diamminebis((SP-4 –2)-diamminechloroplatinum(π)(μ-hexane-1,6-diamine))platinum tetranitrate; ECL, enhanced chemiluminescence; DMSO, dimethyl sulfoxide; JNK-IP, c-Jun NH2-terminal kinase inhibitor peptide; ERBB1, epidermal growth factor receptor; HRP, horseradish peroxidase; PAGE, polyacrylamide gel electrophoresis; siRNA, small interfering RNA; MEF, mouse embryonic fibroblast; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; AG1478, 4-(3′-chloroanilino)-6,7-dimethoxy-quinazoline; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; PD98059, 2′-amino-3′-methoxyflavone; ABT-737, (R)-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-N-{4-[4-(4′-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-3-nitro-benzenesulfonamide; BMS-354825, N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide.
- Received May 23, 2007.
- Accepted June 18, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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