Microtubule inhibitor, SP-6-27 inhibits angiogenesis and induces apoptosis in ovarian cancer cells

Abstract

In ovarian cancer (OVCA), treatment failure due to chemo-resistance is a serious challenge. It is therefore critical to identify new therapies that are effective against resistant tumors and have reduced side effects. We recently identified 4-H-chromenes as tubulin depolymerizing agents that bind to colchicine site of beta-tubulin. Here, we screened a chemical library of substituted 4-H-chromenes and identified SP-6-27 to exhibit most potent anti-proliferative activity towards a panel of human cisplatin sensitive and resistant OVCA cell lines with 50% inhibitory concentration (IC₅₀; mean ± SD) ranging from 0.10 ± 0.01 to 0.84 ± 0.20 μM. SP-6-27 exhibited minimum cytotoxicity to normal ovarian epithelia. A pronounced decrease in microtubule density as well as G2/M cell cycle arrest was observed in SP-6-27 treated cisplatin sensitive/resistant OVCA cells. The molecular mechanism of SP-6-27 induced cell death revealed modulation in cell-cycle regulation by upregulation of growth arrest and DNA damage inducible alpha transcripts (GADD45). An enhanced intrinsic apoptosis was observed in OVCA cells through upregulation of Bax, Apaf-1, caspase-6, -9, and caspase-3. In vitro wound healing assay revealed reduced OVCA cell migration upon SP-6-27 treatment. Additionally, SP-6-27 and cisplatin combinatorial treatment showed enhanced cytotoxicity in chemo-sensitive/resistant OVCA cells. Besides effect on cancer cells, SP-6-27 further restrained angiogenesis by inhibiting capillary tube formation by human umbilical vein endothelial cells (HUVEC). Together, these findings show that the chromene analog SP-6-27 is a novel chemotherapeutic agent that offers important advantages for the treatment of ovarian cancer.

Keywords: chromene; cisplatin resistance; microtubule inhibitor; ovarian cancer.

Figure 1. Microtubule inhibitor SP-6-27 exhibits cytotoxicity towards human ovarian cancer cells

(A) Structure of SP-6-27 as derived from the parent compound GRI-394837. (B) Changes in the cell morphology of the ovarian cancer cells upon treatment with SP-6-27. Cisplatin sensitive (A2780) and resistant (cis-A2780) ovarian carcinoma cells were incubated with vehicle control (DMSO) or SP-6-27 (0.5 μM, 24 h). Cell morphology was assessed by phase-contrast microscopy and representative images (20X magnification; scale bar- 10 μm) are shown here. (C) Dose–response curves of SP-6-27 in a panel of (i) cisplatin sensitive (A2780, SKOV-3, TOV-112D) and (ii) cisplatin resistant (OVCAR-3, cis-A2780, cis-TOV-112D) ovarian cancer cell lines following 72h treatment. (D) IC₅₀ values (mean± SD) for SP-6-27 in different human ovarian cancer cell lines compared to normal ovarian epithelial cells (HOSEpiC). The cell viability was assessed by Alamar Blue assay. Data represents mean of 3 independent experiments performed at least in triplicate.

Figure 2. Chromene analog SP-6-27 disrupts the microtubular dynamics and inhibits migration in ovarian cancer cells

Ovarian cancer cells (A2780) were treated with control (DMSO, vehicle) or SP-6-27 (0.5μM) for 24 h and stained with DAPI (blue) and an (A) anti–α-tubulin or (B) anti-β-tubulin antibody (green). Images were captured using an Olympus FluoView confocal microscope. Representative confocal micrographs (original magnification: 80X) are shown. (C) Effect of SP-6-27 on OVCA cell migration. In vitro wound healing assay was performed using A2780 cells cultured in 6 well plates. Confluent cultures were scratched with a 1 mL pipette tip as described in the Methods section. Representative phase-contrast images of cells migrating into the wounded area in SP-6-27 treated and control wells (0, 24 and 48 h) are shown here. W: wound space, WE: wound edge (magnification- 4X, scale bar-200 μm).

Figure 3. Cisplatin sensitive and resistant ovarian cancer cells arrest in G2 and M phase following SP-6-27 treatment

Cisplatin sensitive A2780 or cisplatin resistant cis-A2780 ovarian cancer cells were treated with 0.5μM SP-6-27 or DMSO vehicle control for 24 hours. The cells were evaluated for effects on cell cycle using PI staining and analyzed by flow cytometry using ModFit software. (Ai) Representative cell cycle micrographs of cisplatin sensitive cells depicting G1, S and G2/M cell populations in control and SP-6-27 treated cells. (Aii) Stacked bar graph illustrating the phase distribution of cisplatin sensitive cells in control and SP-6-27 treated groups determined as percentage of the total number of cells in cycle. (Bi) Representative cell cycle micrographs of cisplatin resistant cells depicting G1, S and G2/M cell populations in control and SP-6-27 treated cells. (Bii) Stacked bar graph illustrating the phase distribution of cisplatin resistant cells in control and SP-6-27 treated groups. The data indicates ovarian cancer cell cycle arrest in G2/M phase upon SP-6-27 treatment. Average of three experiments is shown.

Figure 4. SP-6-27 induces apoptotic cell death in chemo-resistant and sensitive ovarian cancer cells

(A) Cisplatin sensitive (A2780) and cisplatin resistant (cis-A2780) ovarian cancer cells were treated with 0.5 μM SP-6-27 (24, 48 h) followed by Annexin V/7-AAD assay. Early apoptotic [first (Q1) quadrant], late apoptotic [second (Q2) quadrant] and dead cells [(Q3) quadrant] are shown. (B) Confocal microscopy analysis for cleaved caspase-3 in SP-6-27 treated (0.5μM, 24h) ovarian cancer cells. Images show active caspase-3 expression in (i) SP-6-27 treated cells compared to (ii) control cells. Nuclei staining was performed using DAPI (blue). Merged phase-contrast-fluorescence microscopy images displaying cellular expression of active Caspase-3 and DAPI are shown here. Original magnification-× 80; Scale bars-10μm. (C) Q-RT-PCR array was performed for identification of the cell death pathway associated genes altered in ovarian cancer cells upon SP-6-27 treatment. mean ±SD of the mRNA fold change using two endogenous controls (BACT and HPRT) is depicted here. Data are the average of triplicate experiments [mean ± (SD)]. (D) Western blot analysis of the intrinsic apoptotic molecules in SP-6-27 treated A2780 ovarian cancer cells show enhanced expression compared to control cells.

Figure 5. SP-6-27 and cisplatin combination treatment exhibits enhanced cytotoxicity towards ovarian cancer cells

(A) The cell viability of cisplatin sensitive cells (A2780) following simultaneous treatment with a combination of SP-6-27 and cisplatin. Blue columns, cells treated with cisplatin only (0-50μM, 48h); red columns, cells treated with combination of cisplatin (0-50μM) and 0.5 μg SP-6-27 for 48h; green column shows cells treated with SP-6-27 alone (0.5 μM, 48h). (B) Sequential combinatorial treatment with SP-6-27 (0.5μM; 24h) followed by cisplatin (0- 50μM; 24h) enhanced cytotoxicity in the of cisplatin resistant cells (cis-A2780) cells. Gray columns, cells treated with cisplatin only; orange column, cells treated with a combination of cisplatin and SP-6-27; yellow column shows cells treated with SP-6-27 (0.5 μM, 48h) alone. The experiments were performed twice in triplicate.

Figure 6. SP-6-27 inhibits capillary tube formation by endothelial cells

HUVECs endothelial cells were grown on matrigel-coated 96-well plates for 24 h. The cells were treated with SP-6-27 (0.5, 1μM) or DMSO vehicle, in the (A) absence/(B) presence of tumor conditioned media from ovarian cells. The ability of the HUVEC cells to form capillary tube like structures was analyzed using phase contrast microscopy at a magnification of ×4, scale bars- 200μm. Images shown are representative of three independent experiments.

Figure 7. Proposed model illustrating the effects of SP-6-27 mediated microtubule inhibition on ovarian cancer cells and endothelial cells

Schematic of the pleotropic effects of SP-6-27 on ovarian cancer growth and angiogenesis. Tubulin de-polymerization caused by SP-6-27 leads to (1) G2-M phase cell cycle arrest in these cells. (2) Enhanced apoptotic cell death- The elevated expression of Bax contributes to the mitochondrial cytochrome c mediated activation of caspase -9 involving Apaf-1. The activated caspases-9 further cleaves procaspase-3 and subsequently leads to apoptosis of the cell. (3) Reduced cancer cell migration- SP-6-27 mediated tubulin de-polymerization suppresses ovarian cancer cell migration. (4) Vascular disruption- SP-6-26 causes vascular disruption of the endothelial cells, thus inhibiting angiogenesis. The endothelial vascular disruption is evident in the presence of ovarian cancer tumor conditioned medium.