[Show abstract][Hide abstract] ABSTRACT: The present study uses cell-based screening assays to assess the anticancer effects of targeting phosphatidylinositol 3-kinase-regulated integrin-linked kinase (ILK) in combination with small-molecule inhibitors of Raf-1 or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK). The objective was to determine if synergistic interactions are achievable through the use of agents targeting two key cell signaling pathways involved in regulating glioblastoma cancer. The phosphatidylinositol 3-kinase/protein kinase B (PKB)/Akt and the Ras/MAPK pathway were targeted for their involvement in cell survival and cell proliferation, respectively. The glioblastoma cell lines U87MG, SF-188, and U251MG were transiently transfected with an antisense oligonucleotide targeting ILK (ILKAS) alone or in combination with the Raf-1 inhibitor GW5074 or with the MEK inhibitor U0126. Dose and combination effects were analyzed by the Chou and Talalay median-effect method and indicated that combinations targeting ILK with either Raf-1 or MEK resulted in a synergistic interaction. Glioblastoma cells transfected with ILKAS exhibited reduced levels of ILK and phosphorylated PKB/Akt on Ser473 but not PKB/Akt on Thr308 as shown by immunoblot analysis. These results were confirmed using glioblastoma cells transfected with ILK small interfering RNA, which also suggested enhanced gene silencing when used in combination with U0126. U87MG glioblastoma cells showed a 90% (P < 0.05) reduction in colony formation in soft agar with exposure to ILKAS in combination with GW5074 compared with control colonies. A substantial increase in Annexin V-positive cells as determined by using fluorescence-activated cell sorting methods were seen in combinations that included ILKAS. Combinations targeting ILK and components of the Ras/MAPK pathway result in synergy and could potentially be more effective against glioblastoma cancer than monotherapy.
Molecular Cancer Therapeutics 03/2006; 5(3):645-54. DOI:10.1158/1535-7163.MCT-05-0099 · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Overexpression of Bcl-2 protein in cancer cells can inhibit programmed cell death and engender chemoresistance. Reducing Bcl-2 protein levels by using antisense oligonucleotides targeting the gene message can increase the sensitivity of cancer cells to cytotoxic agents. The objective of this work was to investigate the antitumor efficacy of the Bcl-2 antisense oligonucleotide oblimersen (Genasense; G3139), alone and in combination with vinorelbine (VNB), in an ectopic and orthotopic xenograft model of NCI-H460 human non-small-cell lung cancer. In addition to assessing therapeutic effect, Bcl-2 protein expression in tumor tissue isolated from lung and heart was measured. In the ectopic xenograft model, oblimersen at 5 and 10 mg/kg significantly inhibited tumor growth compared with saline-treated control groups, and furthermore, the antitumor effect of oblimersen was associated with down-regulation of Bcl-2 protein in isolated tumor tissue. Moreover, the combination of oblimersen with VNB was more active in inhibiting tumor growth than either drug used alone. In the orthotopic model, oblimersen treatment (5 mg/kg) increased the median survival time of mice to 33 days in comparison with a median survival time of 21 days in the control animals. With this model, the anticancer effect was demonstrated by assessing tumor growth in lung and heart tissues by hematoxylin and eosin staining and Bcl-2 expression by immunohistochemistry. When VNB at 5 mg/kg was combined with oblimersen administered at 5 mg/kg, 33% of mice survived more than 90 days. These data suggest that the combination of oblimersen and VNB may provide enhanced antitumor activities against non-small-cell lung cancer.
Clinical Cancer Research 12/2004; 10(22):7662-70. DOI:10.1158/1078-0432.CCR-04-1036 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of HER-2/neu overexpression on the tumor microenvironment in an aggressive breast cancer xenograft model were investigated. These studies focused on tumors derived following the subcutaneous injection of MDA-MB-435/LCC6 cells transfected with human c-erbB2 (LCC6HER-2) into SCID-Rag2M mice. LCC6HER-2 tumors were more viable (H&E-stained tumor sections) than isogenic vector control tumors (LCC6Vector). Correspondingly, a 2.7-fold increase in trypan blue-excluding cells (P = 0.00056) and a 4.8-fold increase in clonogenic cells (P = 0.00146) were noted in cell suspensions derived from disaggregated LCC6HER-2 versus LCC6Vector tumors. Tumor sections stained with the antibody detecting 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5), a marker of hypoxia, showed a greater fraction of hypoxic tissue in LCC6 HER-2 tumors compared with control tumors. Flow cytometric analyses based on viable tumor cells (DNA content ≥ 2N) in cell suspensions from disaggregated tumors confirmed that there were significantly more EF5-positive cells (i.e., hypoxic) in LCC6HER-2 than in LCC6Vector tumors (16.41 ± 8.1% and 5.96 ± 4.1%, respectively; P = 0.0015). Protein levels of phosphorylated (Ser536) nuclear factor-κB p65 were significantly elevated in LCC6HER-2 tumors (P = 0.00048), and a trend in increased hypoxia-inducible factor-1α protein levels was observed in LCC6HER-2 compared with LCC6Vector tumors. Despite the substantial viable hypoxic cell fraction and a 1.7-fold increase of vascular endothelial growth factor protein (P = 0.05) in LCC6HER-2 tumors, no significant differences were found (P > 0.05) between LCC6 HER-2 and LCC6Vector vasculature (CD31 staining and Hoechst 33342 perfusion). These results suggest that HER-2/neu overexpression may be linked with overall increased tumor viability and a significant increase in the population of viable hypoxic cells, which is not due to differences in tumor vascularization.
Molecular Cancer Research 11/2004; 2(11):606-19. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pyrazoloacridine (NSC 366140, PD115934, PZA) is a new class of acridine anticancer agents under investigation in Phase II clinical trials in patients with advanced cancers. Although poor responses in patients to the treatment with PZA alone have been observed, this class of agents remains of interest because of its distinct mechanism of action from other topoisomerase poisons. Therefore, the combination of PZA with conventional anticancer agents presents an attractive approach to treat drug-resistant human tumors. In the present study, the cytotoxic effects of PZA combined with doxorubicin, topotecan, and etoposide were determined using paired parental and doxorubicin-resistant human colon carcinoma (SW-620 and SW620/AD-300) and breast cancer cell lines (MCF-7 and MCF-7/TH). Cytotoxicity was measured by soft agar clonogenic assays. Dose effect and combination effects were analyzed by the method of Chou and Talalay. The combination of PZA with doxorubicin, topotecan, and etoposide in fixed ratios demonstrated synergistic cytotoxicity on both SW-620 and SW620/AD-300 cell lines. The combination of PZA with doxorubicin also exhibited synergistic cytotoxicity against both MCF-7 and MCF-7/TH cell lines. The mechanism of synergism appeared independent of topoisomerase I and II inhibition, and interference with protein-DNA complexes. Strategies to define optimal drug combinations are proving to be of significant value when considering potential clinical applications of new and established agents.
Clinical Cancer Research 03/2004; 10(3):1160-9. DOI:10.1158/1078-0432.CCR-1044-3 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of Dox (Dox), paclitaxel (Taxol), and serum starvation on the regulation of XIAP (X-linked inhibitor of apoptosis), Bcl-2 phosphorylation, and apoptosis were evaluated in human H460 non-small cell lung cancer cells. Protein kinases that responded to these treatments as prosurvival elements in signal transduction were identified by simultaneously screening phosphorylation of protein kinases in H460 cells cultured in serum-free medium or treated with Dox. We demonstrated that Dox and Taxol induced apoptosis through down-regulation of XIAP and phosphorylation of Bcl-2 in a concentration-dependent manner without changing expression of Bcl-xL in H460 cells. These effects were paralleled by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase protein. We identified that serum starvation and Dox reduced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK), protein kinase C (PKC) alpha/beta and c-Jun NH(2)-terminal kinase. The MEK-specific inhibitor U0126 or PKC inhibitor staurosporine (STP) also down-regulated XIAP expression and induced apoptosis. Thus, our data suggest that apoptosis and down-regulation of XIAP induced by Dox exposure or serum starvation may be mediated through inactivation of the MEK/ERK and PKCalpha/beta pathways. In support of this we demonstrated that the cytotoxic effects of Dox when combined with U0126 or STP were enhanced, i.e., synergistic cytotoxic activities were demonstrated. The synergistic interaction of U0126 or STP with Dox was sequence- and concentration-dependent.
Molecular Cancer Therapeutics 08/2003; 2(7):641-9. · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of programmed cell death in cancer cells offers novel and potentially useful approaches to improving patient responses to conventional chemotherapy. X-linked inhibitor of apoptosis (XIAP), is the most potent member of the IAP gene family in terms of its ability to inhibit caspases and suppress apoptosis. In this study, we investigated the effect of XIAP down-regulation by antisense oligonucleotides (AS ODNs) on human non-small cell lung cancer (NIH-H460) growth in vitro and in vivo. In cultured H460 cells, G4 AS ODN was identified as the most potent compound. It down-regulated XIAP mRNA by 55% and protein levels up to 60% as determined by real-time quantitative reverse transcription-PCR and Western blotting, respectively, and induced 60% cell death. In contrast, the scrambled control ODN caused minimal XIAP loss and less than 10% cell death. Treatment with G4 AS ODN induced apoptosis as revealed by degradation of procaspase-3 and poly(ADP-ribose) polymerase proteins with significant nuclear DNA condensation and fragmentation. In addition, G4 AS ODNs sensitized H460 cells to the cytotoxic effects of doxorubicin, Taxol, vinorelbine, and etoposide. In animal models, administration of G4 AS ODN had significant sequence-specific inhibitory effects on H460 solid tumor establishment in a xenograft model. This antitumor activity was associated with an 85% down-regulation of XIAP protein in the tumors. In addition, the combination of 15 mg/kg G4 AS ODN with 5 mg/kg vinorelbine significantly delayed tumor establishment, more than either agent alone. These studies support the contention that XIAP is a viable target for cancer therapy in human non-small cell lung cancer.
Clinical Cancer Research 08/2003; 9(7):2826-36. · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We studied the possibility of increasing sensitization of drug-resistant MDA435/LCC6 multidrug-resistant (MDR) human breast cancer cells to doxorubicin (DOX) by increasing cellular drug retention with P-glycoprotein (P-gp) inhibitor PSC833 in combination with induction of cell death through down-regulation of Bcl-2 protein using Bcl-2 antisense (G3139). In in vitro cytotoxicity assays, the combination of G3139 with DOX exhibited 40% increased cytotoxicity in both wild-type (WT) and MDR cells. PSC833 increased the cytotoxicity of DOX and Taxol with complete and partial reversal of the resistance of MDR cells to DOX and Taxol, respectively. The presence of G3139 did not increase the cytotoxicity of PSC833 combined with DOX or Taxol in both cell lines. In vivo studies with WT and MDR cell lines transplanted into severely combined immunodeficient mice demonstrated that G3139 (5 mg/kg) was able to suppress the growth of both WT and MDR tumors to an equivalent extent. PSC833 (100 mg/kg) partially restored the sensitivity of resistant tumors to DOX, and the combination of G3139 and PSC833 with liposomal DOX showed maximum growth suppression of MDR tumors compared with individual treatments. The improved efficacy of this treatment was attributed to Bcl-2 antisense-induced apoptosis, combined with cellular retention of DOX in tumor cells via P-gp blockade.
Journal of Experimental Therapeutics and Oncology 03/2003; 3(2):72-82. DOI:10.1046/j.1359-4117.2003.01075.x
[Show abstract][Hide abstract] ABSTRACT: We examined the role of Mcl-1 and Bcl-2 expression in the induction of apoptosis, through blocking protein tyrosine kinase (PTK), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK)/Erk kinase (MEK) signaling pathways by various kinase inhibitors in MCF-7 breast cancer cells. The PTK inhibitor genistein (GEN) and PKC inhibitor staurosporine (STP) down-regulated Mcl-1 and Bcl-2 expression, and induced growth inhibition by blocking at the G2/M phase of cell cycle, followed by apoptosis, leading to chromatin condensation and DNA fragmentation. LY294002 (LY)-mediated inhibition of PI3-K activity down-regulated Bcl-2 but not Mcl-1 expression, triggered growth arrest at the G1/G0 phase of cell cycle and also led to apoptosis marked with chromatin condensation and DNA fragmentation. The MEK inhibitor U0126 (U0) decreased Bcl-2 expression but not Mcl-1 expression, inhibited cells growth and induced G1/G0 arrest, but in this case cell death occurred without significant apoptotic features. The kinase inhibitor concentration dependence of cytotoxicity correlated well with down-regulation of Bcl-2 but not with changes in Mcl-1 levels. This suggests that Bcl-2 plays a predominant role in the regulation of cell death induced by cell signaling alterations whereas Mcl-1 does not appear to control cell survival under these conditions in MCF-7 cells. Further studies showed that the combination of GEN, STP and LY with U0 can produce synergetic cytotoxic effects on MCF-7 cells. Our results suggest that PTK, PKC, PI3-K and MEK signaling pathways can regulate Bcl-2 expression and form an integrated network that plays a critical role in cell survival.
Breast Cancer Research and Treatment 10/2001; 70(1):11-20. DOI:10.1023/A:1012564620853 · 3.94 Impact Factor