Oncolytic measles virus strains have activity against multiple
tumor types and are currently in phase I clinical testing. Induction of the
heat shock protein 70 (HSP70) constitutes one of the earliest changes in cellular gene expression following
infection with RNA viruses including measles virus, and HSP70 upregulation induced by heat shock has been shown to result in increased measles virus cytotoxicity. HSP90 inhibitors such as
geldanamycin (GA) or
17-allylaminogeldanamycin result in pharmacologic upregulation of HSP70 and they are currently in clinical testing as
cancer therapeutics. We therefore investigated the hypothesis that
heat shock protein inhibitors could augment the measles virus-induced cytopathic effect. We tested the combination of a measles virus derivative expressing soluble human
carcinoembryonic antigen (MV-CEA) and GA in MDA-MB-231 (breast), SKOV3.IP (ovarian) and TE671 (
rhabdomyosarcoma)
cancer cell lines. Optimal synergy was accomplished when GA treatment was initiated 6-24 h following MV
infection. Western immunoblotting confirmed HSP70 upregulation in combination-treated cells. Combination treatment resulted in statistically significant increase in syncytia formation as compared to MV-CEA
infection alone. Clonogenic assays demonstrated significant decrease in
tumor colony formation in MV-CEA/GA combination-treated cells. In addition there was increase in apoptosis by
4,6-diamidino-2-phenylindole staining. Western immunoblotting for
caspase-9,
caspase-8,
caspase-3 and
poly(ADP-ribose) polymerase (PARP) demonstrated increase in cleaved
caspase-8 and PARP. The pan-
caspase inhibitor
Z-VAD-FMK and
caspase-8 inhibitor
Z-IETD-FMK, but not the
caspase-9 inhibitor Z-IEHD-FMK, protected
tumor cells from MV-CEA/GA-induced PARP activation, indicating that apoptosis in combination-treated cells occurs mainly via the extrinsic
caspase pathway. Treatment of normal cells, such as normal human fibroblasts, however, with the MV-CEA/GA combination, did not result in cytopathic effect, indicating that GA did not alter the MV-CEA specificity for
tumor cells. One-step viral growth curves, western immunoblotting for MV-N
protein expression, QRT-PCR quantitation of MV-genome copy number and CEA levels showed comparable proliferation of MV-CEA in GA-treated vs -untreated
tumor cells. Rho activation assays and western blot for total RhoA, a
GTPase associated with the actin cytoskeleton, demonstrated decrease in RhoA activation in combination-treated cells, a change previously shown to be associated with increase in paramyxovirus-induced cell-cell fusion. The enhanced cytopathic effect resulting from measles virus/GA combination supports the translational potential of this approach in the treatment of
cancer.