c-Jun-NH2-kinase-1 inhibition leads to antitumor activity in ovarian cancer

Abstract

Purpose: To show the functional, clinical, and biological significance of c-Jun-NH(2)-kinase (JNK)-1 in ovarian carcinoma.

Experimental design: Analysis of the impact of JNK on 116 epithelial ovarian cancers was conducted. The role of JNK in vitro and in experimental models of ovarian cancer was assessed. We studied the role of N-5-[4-(4-methyl piperazine methyl)-benzoylamido]-2-methylphenyl-4-[3-(4-methyl)-pyridyl]-2-pyrimidine amine (WBZ_4), a novel JNK inhibitor redesigned from imatinib based on targeting wrapping defects, in cell lines and in experimental models of ovarian cancer.

Results: We found a significant association of pJNK with progression-free survival in the 116 epithelial ovarian cancers obtained at primary debulking therapy. WBZ_4 led to cell growth inhibition and increased apoptosis in a dose-dependent fashion in four ovarian cancer cell lines. In vivo, whereas imatinib had no effect on tumor growth, WBZ_4 inhibited tumor growth in orthotopic murine models of ovarian cancer. The antitumor effect was further increased in combination with docetaxel. Silencing of JNK-1 with systemically administered siRNA led to significantly reduced tumor weights compared with nonsilencing siRNA controls, indicating that indeed the antitumor effects observed were due to JNK-1 inhibition.

Conclusions: These studies identify JNK-1 as an attractive therapeutic target in ovarian carcinoma and that the redesigned WBZ_4 compound should be considered for further clinical development.

Figure 1

WBZ_4 inhibits JNK activity in ovarian cancer cells. A. Basal p-JNK levels in cells treated with WBZ_4. Protein extracts from A2780CP20 cells treated with 10 μM WBZ_4 and collected at different time points were probed with pJNK, total JNK and β-actin. U, untreated sample; D, vehicle treated sample; S, serum shocked positive control. B. UV-dependent JNK activation and c-Jun phosphorylation. Protein extracts from cells treated with 10 μM of WBZ_4 or SP600125 and exposed to UV lights were probed with antibodies against the phosphorylated and total form of JNK and c-Jun. To show equal loading, blots were also probed with β-actin. UV, level of energy in J/m²; WBZ, WBZ_4; SP, SP600125. C-D. Densitometric analysis of blots shown in Figure 1B.

Figure 2

WBZ_4 and the JNK inhibitor SP600125 inhibit growth of ovarian cancer cells. A. Percentage growth inhibition of ovarian cancer cells vs. different concentrations of WBZ_4. Cells were treated with the drug for 72 hr followed by MTS assay. B. Percentage of FITC-Annexin V positive cells. A2780CP20 cells were treated with WBZ_4 and 72hr later, Annexin V labeling was assayed by FACS. 5 μM (*p<0.05), 10 μM (***p<0.001) and 20 μM (***p<0.001) C. Percentage growth inhibition vs. WBZ_4 and SP600125. Cell viability was assessed by MTS assay 72 hr after treatment. A2780CP20: 5 μM WBZ_4 (*p<0.05), 10 μM WBZ_4 (***p<0.001) and 20 μM WBZ_4 (***p<0.001); 5 μM SP600125 (*p<0.05), 10 μM SP600125 (**p<0.01) and 20 μM SP600125 (**p<0.01). HEYA8: 5 μM WBZ_4 (p>0.05), 10 μM WBZ_4 (*p<0.05) and 20 μM WBZ_4 (***p<0.001); 5 μM SP600125 (*p<0.05), 10 μM SP600125 (**p<0.01) and 20 μM SP600125 (***p<0.001). D. Cell cycle distribution of HeyA8 and A2780CP20 cells treated with WBZ_4 and SP600125. Cell cycle was evaluated by FACS based on the PI staining of cells treated with WBZ_4 and SP600125 for 72 hr. Error bars, SEM.

Figure 3

Therapeutic efficacy of WBZ_4 in combination with docetaxel. HeyA8 (A) or A2780CP20 (B) cells were implanted intraperitoneally as described in Materials and Methods. Mice were randomly allocated to one of the following groups, with therapy beginning 1week after tumor cell inoculation: PBS, PBS+ docetaxel, WBZ_4 and WBZ_4 + docetaxel. Left columns, number of nodules; center columns, mean tumor weights (bars, SD). Right, individual weights. C. WBZ_4 therapeutic activity was compared to that of imatinib in HeyA8 tumor bearing mice. Nude mice were injected i.p. with HeyA8 cells and randomly allocated to one of the following groups, with therapy beginning 1week after tumor cell inoculation: Vehicle, imatinib, WBZ_4, PBS+ docetaxel, WBZ_4 + docetaxel and WBZ_4 + imatinib. D. pJNK levels in tumor samples treated with vehicle control, WBZ_4 or imatinib (3 mice per group are shown). * p<0.05; ** p<0.01; *** p<0.001.

Figure 4

Effect of WBZ_4 with or without docetaxel on cell proliferation, apoptosis and microvessel density. A. MVD was determined after immunohistochemical peroxidase staining for CD31. B. Ki67 immunohistochemistry (IHC). Original magnification, 100x. Columns, mean percentage of Ki67-positive cells; bars, SD. Five animals per group and at least five fields per animal were counted. C. TUNEL IHC. Tumor sections from each group were stained for TUNEL. Representative slides from each group are shown. The number of apoptotic cells was counted. Columns, mean number of TUNEL-positive cells; bars, SD. Ten fields per slide and at least five slides per group (all from different animals) were counted.

Figure 5

JNK1 siRNA inhibits growth of ovarian cancer cells. A. Effect of JNK1 siRNA on JNK1 expression in different ovarian cancer cells. Western blot of extracts from cells transfected with control and JNK1 siRNA and probed with JNK and β-actin antibodies. B. Effect of JNK1siRNA on cell growth. Seventy two hr after transfection, total cell number was counted on the different groups and results are expressed as % growth inhibition relative to the cells transfected with the control siRNA. Error bars, SD. C. Cell death after JNK1 silencing. Cells transfected with JNK1 siRNA were stained with FITC Annexin V and PI and cell death was assessed by FACS.

Figure 6

In vivo therapeutic efficacy of JNK1 siRNA in combination with docetaxel. Nude mice were injected i.p. with HeyA8 (A-C) or SKOV3ip1 (D-F) cells and randomly allocated to one of the following groups, with therapy beginning 1week after tumor cell inoculation: control siRNA DOPC, control siRNA DOPC + Docetaxel, JNK1 siRNA DOPC and JNK1 siRNA DOPC + Docetaxel. Number of nodules (A and D); mean tumor weight (B and E); bars, SD. Individual tumor weights (C and F). G: Kaplan-Meier survival curves for progression free survival (PFS) for all stage tumors based on pJNK (JNKp183_5) levels.