2-Phenyl-5-(pyrrolidin-1-yl)-1-(3,4,5-trimethoxybenzyl)-1H-benzimidazole, a benzimidazole derivative, inhibits growth of human prostate cancer cells by affecting tubulin and c-Jun N-terminal kinase

Abstract

Background and purpose: The c-Jun N-terminal kinase (JNK) and tubulin are, frequently, targets for developing anti-cancer drugs. A major obstacle to successful development is P-glycoprotein (P-gp)-mediated resistance. Here, we have assessed a compound that inhibited growth of cancer cells, for effects on JNK and tubulin and as a substrate for P-gp.

Experimental approach: Several pharmacological and biochemical assays were used to characterize signalling pathways of 2-phenyl-5-(pyrrolidin-1-yl)-1-(3,4,5-trimethoxybenzyl)-1H-benzimidazole (PPTMB), a benzimidazole analogue, in prostate cancer cells.

Key results: PPTMB inhibited proliferation of several human prostate cancer cell lines. It displayed similar activity against a P-gp-rich cell line, indicating that PPTMB was not a substrate for P-gp. PPTMB induced G2/M arrest of the cell cycle and subsequent apoptosis, using flow cytometry. Tubulin polymerization assays and Western blot analysis showed that PPTMB directly acted on tubulin and caused disruption of microtubule dynamics, inducing mitotic arrest and sustained high levels of cyclin B1 expression and Cdk1 activation. Subsequently, mitochondria-related apoptotic cascades were induced, including Bcl-2 and Bcl-xL phosphorylation, Mcl-1 down-regulation, truncated Bad formation and activation of caspase-9 and -3. PPTMB stimulated JNK phosphorylation at Thr(183)/Tyr(185). SP600125, a specific JNK inhibitor, significantly inhibited apoptotic signalling, indicating that JNK plays a key role in PPTMB action. PPTMB showed a 10-fold higher potency against prostate cancer cells than normal prostate cells.

Conclusions and implications: PPTMB is an effective anti-cancer agent. It disrupted microtubule dynamics, leading to mitotic arrest of the cell cycle and JNK activation, which in turn stimulated the mitochondria-related apoptotic cascades in prostate cancer cells.

Figure 1

Effects of PPTMB on cell proliferation and cell cycle progression. (A) The chemical structure of PPTMB. (B) Various human prostate cancer cell lines were treated with PPTMB for 48 h. Cell proliferation was examined by SRB assay. Data are expressed as mean ± SEM of four determinations. (C) The cells were incubated in the absence or presence of PPTMB (10 µM) for the indicated times. Then, the cells were fixed and stained with PI to analyse DNA content by FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). Data are representatives of three independent experiments. Vincristine (positive control), 0.1 µM.

Figure 2

Effect of PPTMB on tubulin polymerization assays. (A) Purified tubulins were incubated at 37°C with GTP in the absence (control) or presence of PPTMB (30 µM) or taxol (0.1 µM). Tubulin polymerization was examined turbidimetrically. Data are expressed as mean ± SEM of three determinations. (B) PC-3 cells were incubated with vehicle, PPTMB, taxol or vincristine for 24 h. Then, the cells were harvested and separated into soluble (S, tubulin monomer) and particulate form (P, tubulin polymers), and the proteins were separated by Western blot analysis, and α-tubulin was detected. The expression of particulate fraction was quantified using the computerized image analysis system ImageQuant (Amersham Biosciences). Data are expressed as mean ± SEM of three determinations. **P < 0.01 and ***P < 0.001 compared with the control. (C) PC-3 cells were incubated with vehicle, PPTMB (10 µM), taxol (0.1 µM) or vincristine (0.1 µM) for the indicated times. Then, the cells were fixed and stained with primary antibody to α-tubulin. Then, FITC-labelled secondary antibodies were used (green fluorescence), and the protein was detected by a confocal laser microscopic system. The nuclei were detected by DAPI staining (blue fluorescence). Data are representatives of two independent experiments. Scale bar: 20 µM.

Figure 3

Effect of PPTMB on MEK1/2 and phospho-MEK1/2 expression. PC-3 cells were incubated in the absence or presence of PPTMB (10 µM) for the indicated times. The cells were harvested and lysed for the detection of protein expression by Western blot analysis. The expression was quantified using the computerized image analysis system ImageQuant (Amersham Biosciences). Data are expressed as mean ± SEM of three determinations. **P < 0.01 and ***P < 0.001 compared with the control.

Figure 4

Effect of PPTMB on cell cycle regulators and apoptosis-related proteins. PC-3 cells were incubated in the absence or presence of PPTMB for the time-dependent effect (10 µM, A and B) or concentration-dependent effect (12 h, C). The cells were harvested and lysed for the detection of protein expression by Western blot analysis. Data are representatives of three independent experiments. The expression was quantified using the computerized image analysis system ImageQuant (Amersham Biosciences).

Figure 5

Determination of the functional role of JNK on PPTMB-mediated effects. (A) PC-3 cells were incubated in the absence or presence of PPTMB (10 µM) for the indicated times. Then, the cells were harvested and lysed for the detection of protein expression by Western blot analysis. Data are representatives of three independent experiments. (B) PC-3 cells were incubated in the indicated agent (PPTMB, 10 µM; SP600125, 20 µM) for 24 h. The cells were analysed for DNA content by FACScan flow cytometer. Data are expressed as mean ± SEM of four determinations. (C) The cells were harvested and lysed for the detection of protein expression by Western blot analysis. Data are representatives of three independent experiments. (D) PC-3 cells were incubated with the indicated agent (PPTMB, 10 µM; SP600125, 20 µM) for 12 h. The cells were fixed and stained with primary antibody to α-tubulin. FITC-labelled secondary antibodies were used (green fluorescence), and the protein was detected by a confocal laser microscopic system. The nuclei were detected by DAPI staining (blue fluorescence). Data are representatives of two independent experiments. Scale bar: 20 µM.

Figure 6

Selectivity of PPTMB for cancer cells. (A) Prostate cancer cells or primary prostate cells were treated with PPTMB at the indicated concentrations for 48 h. Cell proliferation was examined by SRB assay. Data are expressed as mean ± SEM of four determinations. (B) The cells were treated with PPTMB, and the DNA content was analysed by FACScan flow cytometer. Data are representatives of two independent experiments.