Therapeutic efficacy of a novel βIII/βIV-tubulin inhibitor (VERU-111) in pancreatic cancer

Abstract

Background: The management of pancreatic cancer (PanCa) is exceptionally difficult due to poor response to available therapeutic modalities. Tubulins play a major role in cell dynamics, thus are important molecular targets for cancer therapy. Among various tubulins, βIII and βIV-tubulin isoforms have been primarily implicated in PanCa progression, metastasis and chemo-resistance. However, specific inhibitors of these isoforms that have potent anti-cancer activity with low toxicity are not readily available.

Methods: We determined anti-cancer molecular mechanisms and therapeutic efficacy of a novel small molecule inhibitor (VERU-111) using in vitro (MTS, wound healing, Boyden chamber and real-time xCELLigence assays) and in vivo (xenograft studies) models of PanCa. The effects of VERU-111 treatment on the expression of β-tubulin isoforms, apoptosis, cancer markers and microRNAs were determined by Western blot, immunohistochemistry (IHC), confocal microscopy, qRT-PCR and in situ hybridization (ISH) analyses.

Results: We have identified a novel small molecule inhibitor (VERU-111), which preferentially represses clinically important, βIII and βIV tubulin isoforms via restoring the expression of miR-200c. As a result, VERU-111 efficiently inhibited tumorigenic and metastatic characteristics of PanCa cells. VERU-111 arrested the cell cycle in the G2/M phase and induced apoptosis in PanCa cell lines via modulation of cell cycle regulatory (Cdc2, Cdc25c, and Cyclin B1) and apoptosis - associated (Bax, Bad, Bcl-2, and Bcl-xl) proteins. VERU-111 treatment also inhibited tumor growth (P < 0.01) in a PanCa xenograft mouse model.

Conclusions: This study has identified an inhibitor of βIII/βIV tubulins, which appears to have excellent potential as monotherapy or in combination with conventional therapeutic regimens for PanCa treatment.

Keywords: Pancreatic cancer; VERU-111; miR-200c; β –tubulins; βIII/βIV-tubulin inhibitor.

Fig. 1

VERU-111 inhibits growth characteristics of PanCa cells. (A) Effect of VERU-111 on viability of Panc-1, AsPC-1, and HPAF-II cells. Structure of VERU-111 (2-(1H-indol-3-yl)-1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl)) – methanone) is shown as insert. Cells were treated with indicated concentrations of VERU-111 for 24 (i) and 48 (ii) hrs and cell viability was determined by MTT assay. (B) Effect of VERU-111 on PanCa cells in a real time cell proliferation assay. Cells (5000 cells/well) were seeded in E-plate (xCELLigence) and placed into the xCELLigence Real Time Cell Analyzer (RTCA) DP. After 8–10 h, VERU-111 or the vehicle control was added and the experiment was allowed to run for 80 h. Line graphs show the average baseline cell index of control and VERU-111-treated cells. (C-E) Effect of VERU-111 on clonogenic potential of PanCa cells as determined by anchorage dependent colony formation assay. Representative images of colony formation assay and bar graphs indicating quantification of cells are shown at indicated concentrations of VERU-111. (Values means ± SEM; n = 3). Asterisk (*) denotes the significant value p < 0.05

Fig. 2

Effect of VERU-111 on the expression of β-tubulin isotypes in PanCa cells. (A) Effect of VERU-111 on mRNA expression of βI, βIIa, βIIb, βIII, βIVa, βIVb, βV and βVI-tubulins in Panc-1 (i) and AsPC-1 (ii) cells. Briefly, cells were treated with the indicated concentrations of VERU-111 for 24 h. RNAs were isolated and transcribed for cDNA preparation. qPCR was performed to determine the mRNA expression of indicated tubulin isotypes. GAPDH was used as an internal control. Bar graphs represent relative fold expression of various tubulins mRNA. (Values mean ± SEM; n = 3). Asterisk (*) denotes the significant value p < 0.05. (B) Effect of VERU-111 on protein levels of various β-tubulin isotypes in Panc-1 (i) and AsPC-1(ii) cells. Cells were treated with vehicle or indicated concentrations of VERU-111 for 24 h and cell lysates were subjected for Western blot analysis. Equal loading of protein in each well was confirmed by stripping and re-probing of the blots with GAPD for all isoform were provided in Additional file 7: Figure S2

Fig. 3

VERU-111 repressed the expression of βIII-tubulin and restored miR-200c expression in PanCa cells. (A) Effect of VERU-111 (i), colchicine (ii), vinorelbine (iii) and paclitaxel (iv) treatment on the mRNA expression of βIII-tubulin in PanCa cells as determined by qPCR analysis. Bar graphs represent fold change mRNA expression of βIII-tubulin compared to control group. (Values means ±SEM; n = 3). p < 0.05. (B) Western blot analysis results indicating the effect of VERU-111, colchicine and vinorelbine on β-tubulin III in Panc-1 cells at 24 h post-treatment. (C) PanCa cells (Panc-1) were treated with control (vehicle) or VERU-111, colchicine, vinorelbine and paclitaxel at 5–10 nM for 18 h. These cells were processed for immunofluorescence analysis using anti- βIII-tubulin antibody (green) and DAPI (blue). The images were captured with a Zeiss 710 Confocal microscope and Zen imaging software (Zeiss) at × 63 magnifications. (D) Effect of VERU-111 on the expression of miR-200c in Panc-1 (i), AsPC-1 (ii) and HPAF-II (iii) cells as determined by qPCR analysis. RNU6B was used as an internal control. (E) Effect of VERU-111 on the expression of βIII-tubulin in miR-200c mimic or inhibitor transfected Panc-1 cells as determined by qPCR (i) and WB analysis (ii). Cells were transfected with 100 nM of miR-200c mimic (pre-200c) or miR-200c inhibitor or scrambled miRNA (negative control) for 48 h followed by VERU-111 (20 nM) treatment for 24 h. RNA was isolated and transcribed for cDNA and mRNA expression of βIII-tubulin was determined by qPCR (i). Data in bar graph indicate fold change mRNA expression of βIII-tubulin. (Values mean ± SEM; n = 3). Asterisk (*) denote the significant value p < 0.05. In a same parallel experiment, protein lysates were prepared and subjected for Western blot analysis to determine the protein levels of βIII-tubulin. Equal loading of protein was determined by stripping and probing the blot with GAPDH antibody. (F) Comparative effect of VERU-111, colchicine and vinorelbine, on cell viability of Panc-1 (i), AsPC-1 (ii), and HPAF-II (iii) cells as determined by MTT assay. Line bar graphs indicate percent cell viability compared to control group in response to VERU-111, colchicine, vinorelbine, and paclitaxel treatment after 48 h treatment. Values in graph represent mean ± SEM of three independent experiments. Asterisk (*) denotes the significant value p < 0.05

Fig. 4

Effect of VERU-111 on invasion and migration of PanCa cells. (A) Effect of VERU-111 on migration of Panc-1 and AsPC-1 cells determine by wound healing assays. The wounded monolayer was incubated in different concentrations of VERU-111 for 24 h. Images of wound healing assays (magnification, × 10) of Panc-1 (i) and AsPC-1 (ii) cells of control and VERU-111 treatment groups after 24 h. (B) Effect of VERU-111 on migration of AsPC-1 and Panc-1 cells using 96-transwell chamber plate. Representative images of migratory Panc-1 and AsPC-1 cells (i) of control and VERU-111 treatment groups after 24 h. Bar graphs (ii) indicating number of migratory Panc-1 and AsPC-1 cells in control and VERU-111 treatment groups. (C) Effect of VERU-111 on invasion of Panc-1 and AsPC-1 cells (i) as determined by Boyden’s Chamber assay. Representative images of control and VERU-111 treatment groups were captured at 20x magnification after 24 h. Bar graphs (ii) indicate number of invaded Panc-1 and AsPC-1 cells. (D) Effect of VERU-111 on real time migration (i) and invasion (ii) of Panc-1 cells using xCELLigence assay. Results are presented as means ± SEM (n = 3). Asterisk (*) denotes the significant value p < 0.05

Fig. 5

Effect of VERU-111 on cell cycle distribution and apoptosis in PanCa cells. (A) Effect of VERU-111 on cell cycle distribution of Panc-1 and AsPC-1 cells. Briefly, Cells were treated with VERU-111 for 24 h and analyzed by flow cytometric analysis using Propidium Iodide. Bar graph showing cell cycle distribution at different phases in G0-G1, S, and G2M as analyzed by MOTFIT Analysis Software (i-ii). (B) Effect of VERU-111 on protein levels of cell cycle regulatory proteins (Cyclin B1, Cdc25c, Cdc2, and pCdc2Tyr15) in Panc-1(i) and AsPC-1(ii) cells as determined by Western blot analysis. (C) Effect of VERU-111 on apoptosis induction. Briefly, cells were treated with indicated concentrations of VERU-111 for 24 h and apoptosis induction was analyzed by flow cytometry using Annexin V-7AAD Apoptosis kit. Bar graphs showing dose-dependent increase of apoptotic cells in VERU-111 treatment. (D) Effect of Protein levels of Bax, Bcl-2, Bad and Bcl-xl in Panc-1(i) and AsPC-1(ii) cells in response to VERU-111 treatment for 24 h as determined by Western blot analysis. (E) Effect of VERU-111 on protein levels of pro-caspase-3 and 9, cleaved caspase-3 and 9 and PARP cleavage in Panc-1(i) and AsPC-1(ii) cells as determined by Western blot analysis. (F) Effect of VERU-111 alone or in combination with caspase inhibitor Z-VAD-FMK on protein level of pro-caspase-3 and 9, cleaved caspase-3 and 9 and PARP cleavage in Panc-1(i) and AsPC-1(ii) cells as determined by Western blot analysis. (G) Effect of VERU-111 alone or in combination with caspase inhibitor Z-VAD-FMK on apoptosis induction analyzed by flow cytometry using Annexin V-7AAD Apoptosis kit. Data was acquired by using the Bio-RAD ZE5/Evererst Software v2.1 and analyzed using FlowJo v.10.3. Asterisk (*) denotes the significant value p < 0.05

Fig. 6

VERU-111 inhibits pancreatic tumor growth. (A) Effect of VERU-111 on AsPC-1 cells derived xenograft tumors in athymic nude mice. Representative images of AsPC-1 cells derived xenograft tumor bearing mice of control and VERU-111 treated group. (B) Line graph showing average tumor volume of control and VERU-111 treatment group different time points. Data shown in the graph represent mean ± SEM of six tumors of each group. (C) Average tumor weight of control and VERU-111 treatment group. (D) Net tumor growth of control and VERU-111 treatment group. Data in bar graph represent mean ± SEM of six tumors in each group. Asterisk (*) denotes the significant value p < 0.05. (E) Representative images of H&E staining of excised xenograft tumors of control (i) and VERU-111 treatment (ii) group. Effect of VERU-111 on the expression of PCNA, βI, βIII, βIVb and βIVb in excised tumor tissues of control (i) and VERU-111 treated (ii) mice as determined by Immunohistochemistry. (F) Effect of VERU-111 on mRNA expression of βI, βIII, βIVb and βIVb in excised xenograft tumors of control and VERU-111 treated mice as determined by qPCR. Bar graph represents fold mRNA expression of βI, βIII, βIVb and βIVb (mean ± SEM; n = 4). Asterisk (*) denotes the significant value p < 0.05. (G) Effect of VERU-111 on the expression of miR-200c in excised xenograft tumors of control and VERU-111 treated mice as determined by qPCR (H) and representative images of in situ hybridization. (I) Proposed model illustrating possible molecular mechanisms of VERU-111 for the inhibition of pancreatic tumor growth. VERU-111 destabilizes microtubule fiber integrality (de-polymerization) via inhibitions of βIII/βIV isotypes, cell cycle arrest and induction of apoptosis. Moreover, VERU-111 also induces miR-200c expression, which negatively regulates β-tubulin III, leading to apoptosis induction and inhibition of invasion/migration of PanCa cells