Sildenafil increases chemotherapeutic efficacy of doxorubicin in prostate cancer and ameliorates cardiac dysfunction.

We have shown that the potent phosphodiesterase-5 (PDE-5) inhibitor sildenafil (Viagra) induces a powerful effect on reduction of infarct size following ischemia/reperfusion injury and improvement of left ventricular dysfunction in the failing heart after myocardial infarction or doxorubicin (DOX) treatment. In the present study, we further investigated the potential effects of sildenafil on improving antitumor efficacy of DOX in prostate cancer. Cotreatment with sildenafil enhanced DOX-induced apoptosis in PC-3 and DU145 prostate cancer cells, which was mediated by enhanced generation of reactive oxygen species, up-regulation of caspase-3 and caspase-9 activities, reduced expression of Bcl-xL, and phosphorylation of Bad. Overexpression of Bcl-xL or dominant negative caspase 9 attenuated the synergistic effect of sildenafil and DOX on prostate cancer cell killing. Furthermore, treatment with sildenafil and DOX in mice bearing prostate tumor xenografts resulted in significant inhibition of tumor growth. The reduced tumor size was associated with amplified apoptotic cell death and increased expression of activated caspase 3. Doppler echocardiography showed that sildenafil treatment ameliorated DOX-induced left ventricular dysfunction. In conclusion, these results provide provocative evidence that sildenafil is both a powerful sensitizer of DOX-induced killing of prostate cancer while providing concurrent cardioprotective benefit.

A serotype 5/3 adenovirus expressing MDA-7/IL-24 infects renal carcinoma cells and promotes toxicity of agents that increase ROS and ceramide levels.

Agents that generate reactive oxygen species (ROS) are recognized to enhance MDA-7/IL-24 lethality. The present studies focused on clarifying how such agents enhanced MDA-7/IL-24 toxicity in renal cell carcinoma cells (RCCs). Infection of RCCs with a tropism-modified serotype 5/3 adenovirus expressing MDA-7/IL-24 (Ad.5/3-mda-7) caused plasma membrane clustering of CD95 and CD95 association with pro-caspase 8, effects that were enhanced by combined exposure to 17-N-allylamino-17-demethoxygeldanamycin (17AAG), As(2)O(3), or fenretinide and that correlated with enhanced cell killing. Knockdown of CD95 or expression of cellular FADD (Fas-associated protein with death domain)-like interleukin-1ß-converting enzyme inhibitory protein, short form (c-FLIP-s) blocked enhanced killing. Inhibition of ROS generation, elevated cytosolic Ca(2+), or de novo ceramide synthesis blocked Ad.5/3-mda-7 ± agent-induced CD95 activation and the enhancement of apoptosis. Ad.5/3-mda-7 increased ceramide levels in a PERK-dependent fashion that were responsible for elevated cytosolic Ca(2+) levels that promoted ROS generation; 17AAG did not further enhance cytokine-induced ceramide generation. In vivo, infection of RCC tumors with Ad.5/3-mda-7 suppressed the growth of infected tumors that was enhanced by exposure to 17AAG. Our data indicate that in RCCs, Ad.5/3-mda-7-induced ceramide generation plays a central role in tumor cell killing and inhibition of multiple signaling pathways may have utility in promoting MDA-7/IL-24 lethality in renal cancer.

Poly(ADP-ribose) polymerase 1 modulates the lethality of CHK1 inhibitors in carcinoma cells.

Prior studies have demonstrated that inhibition of CHK1 can promote the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and phosphorylation of histone H2AX and that inhibition of poly(ADP-ribose) polymerase 1 (PARP1) can affect growth factor-induced ERK1/2 activation. The present studies were initiated to determine whether CHK1 inhibitors interacted with PARP1 inhibition to facilitate apoptosis. Transient expression of dominant-negative CHK1 raised basal ERK1/2 activity and prevented CHK1 inhibitors from activating ERK1/2. CHK1 inhibitors modestly increased the levels of PARP1 ADP ribosylation and molecular or small-molecule inhibition of PARP1 blocked CHK1 inhibitor-stimulated histone H2AX phosphorylation and activation of ERK1/2. Stimulated histone H2AX phosphorylation was ataxia telangiectasia-mutated protein-dependent. Multiple CHK1 inhibitors interacted in a greater than additive fashion with multiple PARP1 inhibitors to cause transformed cell-killing in short-term viability assays and synergistically killed tumor cells in colony-formation assays. Overexpression of BCL-xL or loss of BAX/BAK function, but not the function of BID, suppressed CHK1 inhibitor + PARP1 inhibitor lethality. Inhibition of BCL-2 family protein function enhanced CHK1 inhibitor + PARP1 inhibitor lethality and restored drug-induced cell-killing in cells overexpressing BCL-xL. Thus, PARP1 plays an important role in regulating the ability of CHK1 inhibitors to activate ERK1/2 and the DNA damage response. An inability of PARP1 to modulate this response results in transformed cell death mediated through the intrinsic apoptosis pathway.

BCL-2 family inhibitors enhance histone deacetylase inhibitor and sorafenib lethality via autophagy and overcome blockade of the extrinsic pathway to facilitate killing.

We examined whether the multikinase inhibitor sorafenib and histone deacetylase inhibitors (HDACI) interact to kill pancreatic carcinoma cells and determined the impact of inhibiting BCL-2 family function on sorafenib and HDACI lethality. The lethality of sorafenib was enhanced in pancreatic tumor cells in a synergistic fashion by pharmacologically achievable concentrations of the HDACIs vorinostat or sodium valproate. Overexpression of cellular FLICE-like inhibitory protein (c-FLIP-s) or knockdown of CD95 suppressed the lethality of the sorafenib/HDACI combination (sorafenib + HDACI). In immunohistochemical analyses or using expression of fluorescence-tagged proteins, treatment with sorafenib and vorinostat together (sorafenib + vorinostat) promoted colocalization of CD95 with caspase 8 and CD95 association with the endoplasmic reticulum markers calnexin, ATG5, and Grp78/BiP. In cells lacking CD95 expression or in cells expressing c-FLIP-s, the lethality of sorafenib + HDACI exposure was abolished and was restored when cells were coexposed to BCL-2 family inhibitors [ethyl [2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA14-1), obatoclax (GX15-070)]. Knockdown of BCL-2, BCL-XL, and MCL-1 recapitulated the effects of GX15-070 treatment. Knockdown of BAX and BAK modestly reduced sorafenib + HDACI lethality but abolished the effects of GX15-070 treatment. Sorafenib + HDACI exposure generated a CD95- and Beclin1-dependent protective form of autophagy, whereas GX15-070 treatment generated a Beclin1-dependent toxic form of autophagy. The potentiation of sorafenib + HDACI killing by GX15-070 was suppressed by knockdown of Beclin1 or of BAX + BAK. Our data demonstrate that pancreatic tumor cells are susceptible to sorafenib + HDACI lethality and that in tumor cells unable to signal death from CD95, use of a BCL-2 family antagonist facilitates sorafenib + HDACI killing via autophagy and the intrinsic pathway.

Sorafenib and vorinostat kill colon cancer cells by CD95-dependent and -independent mechanisms.

We examined the interaction between the multikinase inhibitor sorafenib and histone deacetylase inhibitors. Sorafenib and vorinostat synergized (sorafenib + vorinostat) to kill HCT116 and SW480 cells. In SW480 cells, sorafenib + vorinostat increased CD95 plasma membrane levels and promoted death-inducing signal complex (DISC) formation, and drug toxicity was blocked by knockdown of CD95 or overexpression of cellular FLICE-like inhibitory protein (c-FLIP-s). In SW620 cells that are patient-matched to SW480 cells, sorafenib + vorinostat toxicity was significantly lower, which correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing, whereas knockdown of LASS6 in SW480 cells suppressed CD95 activation. Knocking down LASS6 expression also suppressed CD95 activation in hepatoma, pancreatic, and ovarian cancer cells. In HCT116 cells, sorafenib + vorinostat treatment caused DISC formation without reducing c-FLIP-s expression and did not increase CD95 plasma membrane levels; sorafenib + vorinostat exposure killed HCT116 cells via an intrinsic pathway/caspase 9-dependent mechanism. In HCT116 cells, knockdown of CD95 enhanced sorafenib + vorinostat lethality, which correlated with less drug-induced CD95-dependent autophagy. Sorafenib + vorinostat treatment activated the c-Jun NH(2)-terminal kinase pathway, which was causal in promoting dissociation of Beclin1 from BCL-2, and in promoting autophagy. Knockdown of Beclin1 expression blocked autophagy and enhanced drug toxicity. Our data demonstrate that treatment of colon cancer cells with sorafenib + vorinostat activates CD95 via de novo ceramide synthesis that promotes viability via autophagy or degrades survival via either the extrinsic or intrinsic pathways.

Vorinostat and sorafenib synergistically kill tumor cells via FLIP suppression and CD95 activation.

PURPOSE AND DESIGN: Mechanism(s) by which the multikinase inhibitor sorafenib and the histone deacetylase inhibitor vorinostat interact to kill hepatic, renal, and pancreatic adenocarcinoma cells has been defined. RESULTS: Low doses of sorafenib and vorinostat interacted in vitro in a synergistic fashion to kill hepatic, renal, and pancreatic adenocarcinoma cells in multiple short-term viability (24-96 h) and in long-term colony formation assays. Cell killing was suppressed by inhibition of cathepsin proteases and caspase-8 and, to a lesser extent, by inhibition of caspase-9. Twenty-four hours after exposure, the activities of extracellular signal-regulated kinase 1/2, AKT, and nuclear factor-kappaB were only modestly modulated by sorafenib and vorinostat treatment. However, 24 h after exposure, sorafenib- and vorinostat-treated cells exhibited markedly diminished expression of c-FLIP-s, full-length BID, BCL-2, BCL-XL, MCL-1, XIAP, increased expression of BIM, and increased activation of BAX, BAK, and BAD. Expression of eIF2alpha S51A blocked sorafenib- and vorinostat-induced suppression of c-FLIP-s levels and overexpression of c-FLIP-s abolished lethality. Sorafenib and vorinostat treatment increased surface levels of CD95 and CD95 association with caspase-8. Knockdown of CD95 or FADD expression significantly reduced sorafenib/vorinostat-mediated lethality. CONCLUSIONS: These data show that combined exposure of epithelial tumor cell types to sorafenib and vorinostat diminishes expression of multiple antiapoptotic proteins and promotes activation of the CD95 extrinsic apoptotic and the lysosomal protease pathways, and that suppression of c-FLIP-s expression represents a critical event in transduction of the proapoptotic signals from CD95 to promote mitochondrial dysfunction and death.

Transient exposure of carcinoma cells to RAS/MEK inhibitors and UCN-01 causes cell death in vitro and in vivo.

The present studies were initiated to determine in greater molecular detail how MEK1/2 inhibitors [PD184352 and AZD6244 (ARRY-142886)] interact with UCN-01 (7-hydroxystaurosporine) to kill mammary carcinoma cells in vitro and radiosensitize mammary tumors in vitro and in vivo and whether farnesyl transferase inhibitors interact with UCN-01 to kill mammary carcinoma cells in vitro and in vivo. Expression of constitutively activated MEK1 EE or molecular suppression of JNK and p38 pathway signaling blocked MEK1/2 inhibitor and UCN-01 lethality, effects dependent on the expression of BAX, BAK, and, to a lesser extent, BIM and BID. In vitro colony formation studies showed that UCN-01 interacted synergistically with the MEK1/2 inhibitors PD184352 or AZD6244 and the farnesyl transferase inhibitors FTI277 and R115,777 to kill human mammary carcinoma cells. Athymic mice carrying approximately 100 mm(3) MDA-MB-231 cell tumors were subjected to a 2-day exposure of either vehicle, R115,777 (100 mg/kg), the MEK1/2 inhibitor PD184352 (25 mg/kg), UCN-01 (0.2 mg/kg), or either of the drugs in combination with UCN-01. Transient exposure of tumors to R115,777, PD184352, or UCN-01 did not significantly alter tumor growth rate or the mean tumor volume in vivo approximately 15 to 30 days after drug administration. In contrast, combined treatment with R115,777 and UCN-01 or with PD184352 and UCN-01 significantly reduced tumor growth. Tumor cells isolated after combined drug exposure exhibited a significantly greater reduction in plating efficiency using ex vivo colony formation assays than tumor cells that were exposed to either drug individually. Irradiation of mammary tumors after drug treatment, but not before or during treatment, significantly enhanced the lethal effects of UCN-01 and MEK1/2 inhibitor treatment. These findings argue that UCN-01 and multiple inhibitors of the RAS-MEK pathway have the potential to suppress mammary tumor growth, and to interact with radiation, in vitro and in vivo.

Mitogen-activated protein kinase kinase 1/2 inhibitors and 17-allylamino-17-demethoxygeldanamycin synergize to kill human gastrointestinal tumor cells in vitro via suppression of c-FLIP-s levels and activation of CD95.

Prior studies have noted that inhibitors of mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) enhanced geldanamycin lethality in malignant hematopoietic cells by promoting mitochondrial dysfunction. The present studies focused on defining the mechanism(s) by which these agents altered survival in carcinoma cells. MEK1/2 inhibitors [PD184352; AZD6244 (ARRY-142886)] interacted in a synergistic manner with geldanamycins [17-allylamino-17-demethoxygeldanamycin (17AAG) and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin] to kill hepatoma and pancreatic carcinoma cells that correlated with inactivation of extracellular signal-regulated kinase 1/2 and AKT and with activation of p38 MAPK; p38 MAPK activation was reactive oxygen species dependent. Treatment of cells with MEK1/2 inhibitors and 17AAG reduced expression of c-FLIP-s that was mechanistically connected to loss of MEK1/2 and AKT function; inhibition of caspase-8 or overexpression of c-FLIP-s abolished cell killing by MEK1/2 inhibitors and 17AAG. Treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent plasma membrane clustering of CD95 without altering the levels or cleavage of FAS ligand. In parallel, treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent association of caspase-8 with CD95. Inhibition of p38 MAPK or knockdown of BID, FAS-associated death domain, or CD95 expression suppressed MEK1/2 inhibitor and 17AAG lethality. Similar correlative data were obtained using a xenograft flank tumor model system. Our data show that treatment of tumor cells with MEK1/2 inhibitors and 17AAG induces activation of the extrinsic pathway and that suppression of c-FLIP-s expression is [Mol Cancer Ther 2008;7(9):2633-48].

Lapatinib resistance in HCT116 cells is mediated by elevated MCL-1 expression and decreased BAK activation and not by ERBB receptor kinase mutation.

We have defined some of the mechanisms by which the kinase inhibitor lapatinib kills HCT116 cells. Lapatinib inhibited radiation-induced activation of ERBB1/2, extracellular signal-regulated kinases 1/2, and AKT, and radiosensitized HCT116 cells. Prolonged incubation of HCT116 cells with lapatinib caused cell killing followed by outgrowth of lapatinib-adapted cells. Adapted cells were resistant to serum starvation-induced cell killing and were cross-resistant to multiple therapeutic drugs. Lapatinib was competent to inhibit basal and epidermal growth factor (EGF)-stimulated ERBB1 phosphorylation in adapted cells. Coexpression of dominant-negative ERBB1 and dominant-negative ERBB2 inhibited basal and EGF-stimulated ERBB1 and ERBB2 phosphorylation in parental and adapted cells. However, in neither parental nor adapted cells did expression of dominant-negative ERBB1 and dominant-negative ERBB2 recapitulate the cell death-promoting effects of lapatinib. Adapted cells had increased expression of MCL-1, decreased expression of BAX, and decreased activation of BAX and BAK. Overexpression of BCL-XL protected parental cells from lapatinib toxicity. Knockdown of MCL-1 expression enhanced lapatinib toxicity in adapted cells that was reverted by knockdown of BAK expression. Inhibition of caspase function modestly reduced lapatinib toxicity in parental cells, whereas knockdown of apoptosis-inducing factor expression suppressed lapatinib toxicity. Thus, in HCT116 cells, lapatinib adaptation can be mediated by altered expression of pro- and antiapoptotic proteins that maintain mitochondrial function.

Extrinsic pathway- and cathepsin-dependent induction of mitochondrial dysfunction are essential for synergistic flavopiridol and vorinostat lethality in breast cancer cells.

The present studies have determined whether interactions between the cyclin-dependent kinase inhibitor flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat; Zolinza) occur in breast cancer cells. MDA-MB-231 and MCF7 cells were treated with flavopiridol (25-100 nmol/L) and vorinostat (125-500 nmol/L) in vitro, and mechanisms of cell killing were determined. Concurrent treatment of cells with flavopiridol and vorinostat or treatment of cells with flavopiridol followed by vorinostat promoted cell killing in a greater than additive fashion. Similar data were obtained with the CDK inhibitor roscovitine. Flavopiridol suppressed c-FLIP-l/s and BCL-xL expression, whereas vorinostat reduced expression of BCL-xL, and combined exposure to flavopiridol and vorinostat reduced MCL-1 and X-chromosome-linked inhibitor of apoptosis protein (XIAP) levels. Pharmacologic or genetic inhibition of caspase-8 reduced flavopiridol toxicity, but abolished killing by vorinostat and cell death caused by the vorinostat/flavopiridol regimen. Loss of BAX/BAK function or loss of BID function modestly reduced flavopiridol toxicity, but abolished vorinostat-mediated potentiation of flavopiridol toxicity, as did inhibition of caspase-9. Inhibition and/or deletion of cathepsin B function significantly attenuated vorinostat/flavopiridol lethality. Flavopiridol suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT activity and expression of activated forms of AKT and mitogen-activated protein/ERK kinase 1 maintained c-FLIP-l/s, BCL-xL, and XIAP expression and protected cells against flavopiridol/vorinostat lethality. Overexpression of c-FLIP-s and BCL-xL abolished the lethality of flavopiridol/vorinostat. Collectively, these data argue that flavopiridol enhances the lethality of vorinostat in breast cancer cells in part through the inhibition of AKT and ERK1/2 function, leading to reduced expression of multiple inhibitors of the extrinsic and intrinsic apoptosis pathways, as well as activation of cathepsin protease-dependent pathways.

17-Allylamino-17-demethoxygeldanamycin enhances the lethality of deoxycholic acid in primary rodent hepatocytes and established cell lines.

Ansamycin antibiotics that target heat shock protein 90 function are being developed as anticancer agents but are also known to be dose limiting in patients due to hepatotoxicity. Herein, to better understand how the normal tissue toxicity of geldanamycins could be ameliorated to improve the therapeutic index of these agents, we examined the interactions of 17-allylamino-17-demethoxygeldanamycin (17AAG) and the secondary bile acid deoxycholic acid (DCA) in hepatocytes and fibroblasts. DCA and 17AAG interacted in a greater than additive fashion to cause hepatocyte cell death within 2 to 6 h of coadministration. As single agents DCA, but not 17AAG, enhanced the activity of extracellular signal-regulated kinase 1/2, AKT, c-Jun NH(2)-terminal kinase 1/2 (JNK1/2), and p38 mitogen-activated protein kinase (MAPK). Combined exposure of cells to DCA and 17AAG further enhanced JNK1/2 and p38 MAPK activity. Inhibition of JNK1/2 or p38 MAPK, but not activator protein-1, suppressed the lethality of 17AAG and of 17AAG and DCA. Constitutive activation of AKT, but not MAPK/extracellular signal-regulated kinase kinase 1/2, suppressed 17AAG- and DCA-induced cell killing and reduced activation of JNK1/2. DCA and 17AAG exposure promoted association of BAX with mitochondria, and functional inhibition of BAX or caspase-9, but not of BID and caspase-8, suppressed 17AAG and DCA lethality. DCA and 17AAG interacted in a greater than additive fashion to promote and prolong the generation of reactive oxygen species (ROS). ROS-quenching agents, inhibition of mitochondrial function, expression of dominant-negative thioredoxin reductase, or expression of dominant-negative apoptosis signaling kinase 1 suppressed JNK1/2 and p38 MAPK activation and reduced cell killing after 17AAG and DCA exposure. The potentiation of DCA-induced ROS production by 17AAG was abolished by Ca(2+) chelation and ROS generation, and cell killing following 17AAG and DCA treatment was abolished in cells lacking expression of PKR-like endoplasmic reticulum kinase. Thus, DCA and 17AAG interact to stimulate Ca(2+)-dependent and PKR-like endoplasmic reticulum kinase-dependent ROS production; high levels of ROS promote intense activation of the p38 MAPK and JNK1/2 pathways that signal to activate the intrinsic apoptosis pathway.

H-RAS V12-induced radioresistance in HCT116 colon carcinoma cells is heregulin dependent.

The abilities of mutated active K-RAS and H-RAS proteins, in an isogenic human carcinoma cell system, to modulate the activity of signaling pathways following exposure to ionizing radiation is unknown. Loss of K-RAS D13 expression in HCT116 colorectal carcinoma cells blunted basal extracellular signal-regulated kinase 1/2 (ERK1/2), AKT, and c-Jun NH2-terminal kinase 1/2 activity. Deletion of the allele to express K-RAS D13 also enhanced expression of ERBB1, ERBB3, and heregulin but nearly abolished radiation-induced activation of all signaling pathways. Expression of H-RAS V12 in HCT116 cells lacking an activated RAS molecule (H-RAS V12 cells) restored basal ERK1/2 and AKT activity to that observed in parental cells but did not restore or alter basal c-jun NH2-terminal kinase 1/2 activity. In parental cells, radiation caused stronger ERK1/2 pathway activation compared with that of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which correlated with constitutive translocation of Raf-1 into the plasma membrane of parental cells. Inhibition of mitogen-activated protein kinase/ERK1/2, but not PI3K, radiosensitized parental cells. In H-RAS V12 cells, radiation caused stronger PI3K/AKT pathway activation compared with that of the ERK1/2 pathway, which correlated with H-RAS V12-dependent translocation of PI3K into the plasma membrane. Inhibition of PI3K, but not mitogen-activated protein kinase/ERK1/2, radiosensitized H-RAS V12 cells. Radiation-induced activation of the PI3K/AKT pathway in H-RAS V12 cells 2 to 24 hours after exposure was dependent on heregulin-stimulated ERBB3 association with membrane-localized PI3K. Neutralization of heregulin function abolished radiation-induced AKT activation and reverted the radiosensitivity of H-RAS V12 cells to those levels found in cells lacking expression of any active RAS protein. These findings show that H-RAS V12 and K-RAS D13 differentially regulate radiation-induced signaling pathway function. In HCT116 cells expressing H-RAS V12, PI3K-dependent radioresistance is mediated by both H-RAS-dependent translocation of PI3K into the plasma membrane and heregulin-induced activation of membrane-localized PI3K via ERBB3.

Activated forms of H-RAS and K-RAS differentially regulate membrane association of PI3K, PDK-1, and AKT and the effect of therapeutic kinase inhibitors on cell survival.

The abilities of mutated active RAS proteins to modulate cell survival following exposure to ionizing radiation and small molecule kinase inhibitors were examined. Homologous recombination in HCT116 cells to delete the single allele of K-RAS D13 resulted in a cell line that exhibited an approximately 75% reduction in basal extracellular signal-regulated kinase 1/2, AKT, and c-jun-NH2-kinase 1/2 activity. Transfection of cells lacking K-RAS D13 with H-RAS V12 restored extracellular signal-regulated kinase 1/2 and AKT activity to basal levels but did not restore c-jun-NH2-kinase 1/2 phosphorylation. In cells expressing H-RAS V12, radiation caused prolonged intense activation of AKT. Inhibition of H-RAS V12 function, blockade of phosphatidylinositol 3-kinase (PI3K) function using small interfering RNA/small-molecule inhibitors, or expression of dominant-negative AKT abolished radiation-induced AKT activation, and radiosensitized these cells. Inhibition of PI3K function did not significantly radiosensitize parental HCT116 cells. Inhibitors of the AKT PH domain including perifosine, SH-(5, 23-25) and ml-(14-16) reduced the plating efficiency of H-RAS V12 cells in a dose-dependent fashion. Inhibition of AKT function using perifosine enhanced radiosensitivity in H-RAS V12 cells, whereas the SH and ml series of AKT PH domain inhibitors failed to promote radiation toxicity. In HCT116 H-RAS V12 cells, PI3K, PDK-1, and AKT were membrane associated, whereas in parental cells expressing K-RAS D13, only PDK-1 was membrane bound. In H-RAS V12 cells, membrane associated PDK-1 was phosphorylated at Y373/376, which was abolished by the Src family kinase inhibitor PP2. Inhibition of PDK-1 function using the PH domain inhibitor OSU-03012 or using PP2 reduced the plating efficiency of H-RAS V12 cells and profoundly increased radiosensitivity. OSU-03012 and PP2 did not radiosensitize and had modest inhibitory effects on plating efficiency in parental cells. A small interfering RNA generated against PDK1 also radiosensitized HCT116 cells expressing H-RAS V12. Collectively, our data argue that molecular inhibition of AKT and PDK-1 signaling enhances the radiosensitivity of HCT116 cells expressing H-RAS V12 but not K-RAS D13. Small-molecule inhibitory agents that blocked stimulated and/or basal PDK-1 and AKT function profoundly reduced HCT116 cell survival but had variable effects at enhancing tumor cell radiosensitivity.

Sildenafil (Viagra) sensitizes prostate cancer cells to doxorubicin-mediated apoptosis through CD95.

We previously reported that Sildenafil enhances apoptosis and antitumor efficacy of doxorubicin (DOX) while attenuating its cardiotoxic effect in prostate cancer. In the present study, we investigated the mechanism by which sildenafil sensitizes DOX in killing of prostate cancer (PCa) cells, DU145. The death receptor Fas (APO-1 or CD95) induces apoptosis in many carcinoma cells, which is negatively regulated by anti-apoptotic molecules such as FLIP (Fas-associated death domain (FADD) interleukin-1-converting enzyme (FLICE)-like inhibitory protein). Co-treatment of PCa cells with sildenafil and DOX for 48 hours showed reduced expression of both long and short forms of FLIP (FLIP-L and -S) as compared to individual drug treatment. Over-expression of FLIP-s with an adenoviral vector attentuated the enhanced cell-killing effect of DOX and sildenafil. Colony formation assays also confirmed that FLIP-S over-expression inhibited the DOX and sildenafil-induced synergistic killing effect as compared to the cells infected with an empty vector. Moreover, siRNA knock-down of CD95 abolished the effect of sildenafil in enhancing DOX lethality in cells, but had no effect on cell killing after treatment with a single agent. Sildenafil co-treatment with DOX inhibited DOX-induced NF-κB activity by reducing phosphorylation of IκB and nuclear translocation of the p65 subunit, in addition to down regulation of FAP-1 (Fas associated phosphatase-1, a known inhibitor of CD95-mediated apoptosis) expression. This data provides evidence that the CD95 is a key regulator of sildenafil and DOX mediated enhanced cell death in prostate cancer.

Transient exposure of mammary tumors to PD184352 and UCN-01 causes tumor cell death in vivo and prolonged suppression of tumor regrowth.

Previous studies from our group have demonstrated in vitro that UCN-01 (7-hydroxystaurosporine) and inhibitors of MEK1/2 interact to cause tumor cell death in a wide variety of malignant, but not in nontransformed, cell types. The present studies determined whether UCN-01 and MEK1/2 inhibitors interacted to cause tumor cell death in vivo. In vitro colony formation studies demonstrated that UCN-01 and the MEK1/2 inhibitor PD184352 interacted to synergistically kill human mammary carcinoma cells (MDA-MB-231, MCF7) with similar combination index values. Athymic mice were implanted in the rear flank with either MDA-MB-231 or MCF7 cells and tumors permitted to form to a volume of approximately 100 mm3 prior to a two day exposure of either Vehicle, PD184352 (25 mg/kg), UCN-01 (0.1-0.2 mg/kg) or the drug combination. Tumor volume was measured every other day and tumor growth determined over the following approximately 30 days. Transient exposure of MDA-MB-231 tumors or MCF7 tumors to either PD184352 or UCN-01 did not significantly alter tumor growth rate or the mean tumor volume in vivo approximately 15-30 days after drug administration. In contrast, combined treatment with PD184352 and UCN-01 significantly reduced MDA-MB-231, and largely abolished MCF7 tumor growth. Tumor control values for both cell lines were 0.36. Tumor cells isolated approximately 30 days after combined drug exposure exhibited a significantly greater reduction in plating efficiency using ex vivo colony formation assays than tumor cells that were exposed to either drug individually. Reduced tumor growth correlated with profound tumor cell death within five days of combined drug exposure, which was also evident approximately 30 days after exposure. In addition, tumor cell death correlated with a reduction in the phosphorylation of ERK1/2 and the immuno-reactivity of Ki67 and of CD31. Collectively, these findings argue that UCN-01 and MEK1/2 inhibitors have the potential to suppress mammary tumor growth in vivo which is independent of p53 status, estrogen dependency, caspase 3 levels or oncogenic K-RAS expression.

Inhibition of insulin/IGF-1 receptor signaling enhances bile acid toxicity in primary hepatocytes.

Modulation of ERBB and insulin-like growth factor 1 (IGF-1) receptor function is recognized as a potential mechanism to inhibit tumor growth. We and others have shown that inhibition of ERBB1 can enhance bile acid toxicity. Herein, we extend our analyses to examine the impact of insulin/IGF-1 receptor inhibition on primary hepatocyte survival when exposed to the secondary bile acid deoxycholic acid (DCA) and compare the impact inhibition of this receptor has on bile acid toxicity effects to that of ERBB1/MEK1/2 inhibition. The insulin/IGF-1 receptor inhibitor NVP-ADW742 at concentrations which inhibit both the insulin and IGF-1 receptors had a modest negative impact on hepatocyte viability, and strongly potentiated DCA-induced apoptotic cell death. Identical data were obtained expressing a dominant negative IGF-1 receptor in hepatocytes; a receptor which acts to inhibit both the IGF-1 receptor and the insulin receptor in trans. Inhibition of ERBB1 function using Iressa (gefitinib) or the tyrphostin AG1478 had more modest effects at enhancing DCA lethality than inhibition of the insulin/IGF-1 receptor function. In contrast, over-expression of a dominant negative ERBB1 protein had pleiotropic effects on multiple signaling pathways in an apparently non-specific manner. These findings suggest that novel therapeutic kinase inhibitors, targeted against growth factor receptors, have the potential to promote bile acid toxicity in hepatocyte when bile flow may be impaired.

Melanoma differentiation-associated 7 (interleukin 24) inhibits growth and enhances radiosensitivity of glioma cells in vitro and in vivo.

PURPOSE: Despite therapeutic interventions including surgery, chemotherapy, and radiotherapy, glioblastoma multiforme (GBM) has a very poor prognosis and novel therapies are required. EXPERIMENTAL DESIGN: Melanoma differentiation-associated 7 (mda-7) (interleukin 24), when expressed via a recombinant replication-defective adenovirus, adenovirus (Ad).mda-7, has profound antiproliferative and cytotoxic effects in a variety of tumor cells but not in nontransformed cells. The present studies examined the combined impact of Ad.mda-7 and ionizing radiation on the proliferation and survival of GBM cell lines. RESULTS: Ad.mda-7 caused a dose-dependent reduction in the proliferation of glioma cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The antiproliferative effects of Ad.mda-7 were enhanced by radiation in a greater than additive fashion. These effects were not observed in cultures of nontransformed primary astrocytes. Purified MDA-7 protein caused a similar dose-dependent reduction in GBM cell growth that was enhanced after radiation exposure. The enhanced reduction in growth correlated with increased necrosis and DNA degradation. These modifications in cell phenotype correlated with reduced expression of Bcl-(XL) and enhanced expression of BAX. Overexpression of Bcl-(XL) protected cells from the antiproliferative and cytotoxic effects of Ad.mda-7 + radiation. Incubation of cells with N-acetyl cysteine abolished the enhancing effects of radiation. In vitro, Ad.mda-7 and radiation reduced colony formation ability, which was significantly increased when the two treatments were combined. In vivo, Ad.mda-7 enhanced the survival of Fischer 344 rats implanted intracranially with glioma cells. Radiation did not alter survival in control infected animals, whereas it prolonged survival in those infected with Ad.mda-7. CONCLUSIONS: These findings demonstrate that mda-7 reduces the proliferation and enhances the radiosensitivity of GBM cells in vitro and in vivo.

MDA-7 (interleukin-24) inhibits the proliferation of renal carcinoma cells and interacts with free radicals to promote cell death and loss of reproductive capacity.

The median survival of metastatic renal cell carcinoma (RCC) is 12 months, and the majority of treatment options are palliative. MDA-7 (interleukin-24), when expressed via a recombinant replication defective adenovirus, Ad.mda-7, has profound antiproliferative and cytotoxic effects in a wide variety of tumor cells but not in nontransformed cells. The studies in this study examined the impact of MDA-7 on RCC proliferation and survival. RCC lines (A498 and UOK121N), but not primary renal epithelial cells, were resistant to adenoviral infection that correlated with a lack of coxsackievirus and adenovirus receptor expression. Additional studies were performed using purified preparations of bacterially synthesized glutathione S-transferase (GST)-MDA-7 protein. GST-MDA-7, but not GST, caused a dose-dependent inhibition of RCC proliferation but not of primary renal epithelial cells. Clinically achievable concentrations of the novel therapeutic agent arsenic trioxide (0.5-1 micro M) were found to have little effect on RCC growth. However, the combination of GST-MDA-7 and arsenic trioxide resulted in a greater than additive reduction in cell growth that correlated with a large increase in tumor cell death. The free radical scavenger N-acetyl cysteine abolished the potentiating effect of arsenic trioxide. Although pro-caspase 3, poly(ADP-ribose) polymerase, and Bcl-(XL) levels, as well as nucleosomal DNA integrity, were reduced by combined treatment, cell killing was predominantly nonapoptotic. Combined treatment of RCC lines with GST-MDA-7 and arsenic trioxide also resulted in a substantial reduction in clonogenic survival compared with either treatment individually. Collectively, these findings demonstrate that MDA-7 protein, in combination with agents that generate free radicals, may have potential in the treatment of RCC.

mda-7 (IL-24) Inhibits growth and enhances radiosensitivity of glioma cells in vitro via JNK signaling.

Despite therapeutic interventions including surgery, chemotherapy and radiotherapy, glioblastoma multiforme (GBM) has a very poor prognosis and novel therapies are required. MDA-7 (IL-24), when expressed via a recombinant replication defective adenovirus, Ad.mda-7, has profound anti-proliferative and cytotoxic effects in a variety of tumor cells, but not in non-transformed cells. The present studies examined the combined impact of Ad.mda-7 and ionizing radiation on the proliferation and survival of GBM cells. Ad.mda-7 reduced the proliferation of rodent and human glioma cells in MTT assays and in colony formation assays. The anti-proliferative effects of Admda-7 were enhanced by radiation in a greater than additive fashion. In vitro, this cellular change correlated with enhanced cell numbers in G1/G0 and G2/M phases of the cell cycle, implying Ad.mda-7 radiosensitizes tumor cells in a cell cycle-independent manner. The radiosensitizing effects were not observed in cultures of non-transformed primary astrocytes. The enhanced reduction in growth correlated with increased necrosis and DNA degradation. Ad.mda-7 enhanced p38 and ERK1/2 activity but did not alter JNK or Akt activity. Irradiation of cells expressing MDA-7 suppressed ERK1/2 activity and dramatically enhanced JNK1/2 activity without altering either Akt or p38 activity. Inhibition of JNK1/2, but not p38, signaling abolished the radiosensitizing properties of MDA-7. Inhibition of neither ERK1/2 nor PI3K signaling enhanced the anti-proliferative effects of Ad.mda-7, whereas combined inhibition of both pathways enhanced cell killing, suggesting that ERK and PI3K signaling can be protective against MDA-7 lethality.