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. 2013 Mar 1;132(5):1201-12.
doi: 10.1002/ijc.27724. Epub 2012 Jul 27.

Plumbagin inhibits tumorigenesis and angiogenesis of ovarian cancer cells in vivo

Sutapa Sinha  1 , Krishnendu Pal, Ahmed Elkhanany, Shamit Dutta, Ying Cao, Gourish Mondal, Seethalakshmi Iyer, Veena Somasundaram, Fergus J Couch, Viji Shridhar, Resham Bhattacharya, Debabrata Mukhopadhyay, Priya Srinivas
Affiliations

Plumbagin inhibits tumorigenesis and angiogenesis of ovarian cancer cells in vivo

Sutapa Sinha et al. Int J Cancer. .

Abstract

Angiogenesis is a hallmark of tumor development and metastatic progression, and anti-angiogenic drugs targeting the VEGF pathway have shown to decrease the disease progression in cancer patients. In this study, we have analyzed the anti-proliferative and anti-angiogenic property of plumbagin in cisplatin sensitive, BRCA2 deficient, PEO-1 and cisplatin resistant, BRCA2 proficient PEO-4 ovarian cancer cells. Both PEO-1 and PEO-4 ovarian cancer cells are sensitive to plumbagin irrespective of BRCA2 status in both normoxia and hypoxia. Importantly, plumbagin treatment effectively inhibits VEGF-A and Glut-1 in PEO-1 and PEO-4 ovarian cancer cells. We have also analyzed the p53 mutant, cisplatin resistant, and BRCA2 proficient OVCAR-5 cells. Plumbagin challenge also restricts the VEGF induced pro-angiogenic signaling in HUVECs and subsequently endothelial cell proliferation. In addition, we observe a significant effect on tumor regression among OVCAR-5 tumor-bearing mice treated with plumbagin, which is associated with significant inhibition of Ki67 and vWF expressions. Plumbagin also significantly reduces CD31 expression in an ear angiogenesis assay. Collectively, our studies indicate that plumbagin, as an anti-cancer agent disrupts growth of ovarian cancer cells through the inhibition of proliferation as well as angiogenesis.

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Figures

Figure 1
Figure 1. Plumbagin inhibits ovarian cancer cell growth in vitro
A. Ovarian cancer PEO-1, PEO-4 and OSE cells were treated with increasing concentrations of plumbagin under both normoxia and hypoxia. The cell viability was measured using MTS assay. B. MTS assay by plumbagin in OVCAR-5 cells. P values were calculated comparing with the respective DMSO treated cells in normoxia using two-way ANOVA followed by Tukey’s post hoc test. The figures are representative of three separate experiments (in quadruplicates) with similar results.
Figure 2
Figure 2. Plumbagin induces apoptosis in vitro: A, B, and C
PEO-1, PEO-4, OSE, and OVCAR-5 cells were treated with DMSO or 1/5/10 μM doses of plumbagin under normoxia and hypoxia. Apoptosis was measured by Annexin-FITC/PI method. Dead cells (PI-positive) were differentiated from late apoptotic cells (Annexin-positive and PI-positive), early apoptotic cells (Annexin-positive and PI-negative) and live cells (Annexin-negative and PI-negative). P values were calculated comparing with the respective DMSO treated cells in normoxia and hypoxia using one-way ANOVA followed by Tukey’s post hoc test. The figures are representative of three separate experiments with similar results. D. Colony formation assay after plumbagin treatment in PEO-1 and PEO-4 cells. The figures are representative of three separate experiments with similar results. E. Quantitation of clonogenic assay was done using adobe photoshop CS3.
Figure 3
Figure 3. Plumbagin modulates different signaling pathways. A, B, and C
PEO-1, PEO-4 and OSE cells were treated with increased concentrations of plumbagin and were kept in normoxic or hypoxic conditions and western blotting was done with antibodies against HIF-2α, p-Akt1/2/3 (ser-473), Akt-1/2/3, p-MAPK-42/44, MAPK-42/44, p53, p-Mdm-2 (ser-166), and Mdm-2 (T-Mdm-2). β-Actin was used as a loading control. Expression levels of proteins have been shown with bar graphs. Adobe photoshop version 7.0 has been used for the western quantitation.
Figure 4
Figure 4. Plumbagin inhibits both VEGF and Glut-1 expressions in ovarian cancer cells
RNA was collected from DMSO or plumbagin (1, 5 or 10 μM) treated PEO-1, PEO-4, and OVCAR-5 cells incubated under normoxic and hypoxic conditions. Real-time PCRs for VEGF-A and Glut-1 were done with the cDNAs from the respective samples, and β-actin was used for cDNA normalization. A and B. Subjecting both PEO cell lines to hypoxic growth conditions resulted in significant VEGF and Glut-1 mRNA accumulation and PEO-4 cells express considerably higher amount of basal level of both VEGF and Glut-1 mRNA compared to PEO-1 cells [***p<0.0001; paired t test, 2 tailed]. C, E and G represent VEGF-A mRNA expression and D, F and H represent Glut-1 mRNA expression in PEO-1, PEO-4 and OVCAR-5 cells respectively. P values were calculated comparing with the respective DMSO treated cells in normoxia and hypoxia using one-way ANOVA followed by Tukey’s post hoc test. The figures are representative of three separate experiments with similar results.
Figure 5
Figure 5. Effect of plumbagin on HUVECs. A. Plumbagin inhibits VEGF- induced proliferation of HUVECs
Serum starved HUVECs were treated with DMSO or 0.5/1/5 μM of plumbagin in the presence or absence of VEGF (10 ng/ml) and thymidine incorporation assay was done. P values were calculated comparing with the respective DMSO treated cells using one-way ANOVA followed by Tukey’s post hoc test. The figures are representative of three separate experiments with similar results. B. Effect of plumbagin on HUVEC cell viability was measured using MTS assay. C. Effect of plumbagin on signaling pathway in HUVEC’s. Serum starved HUVEC cells were pre-treated with DMSO or 1/5 μM of plumbagin and then stimulated with VEGF (10 ng/ml) for 5 min. Protein expression was analysed by western blot. β-Actin was used as a loading control. Expression levels have been shown with bar graphs. Adobe photoshop version 7.0 has been used for western quantitation. D. Relative CD31 mRNA expression in nude mouse ears: 1mg/kg intraperitoneal injection of plumbagin in 25% PEG significantly reduced CD31 mRNA expression [**p<0.001, paired t test, 2 tailed] compared to 25% PEG treatment only in mouse ear pre-treated with Ad-VEGF. E. Plumbagin induces Ca2+ mobilization. Serum starved HUVEC cells were pre-treated with DMSO or 500 nM/1 μM of plumbagin and then used for intracellular Ca2+ mobilization assay as described in ‘Materials and Methods’ section.
Figure 6
Figure 6. Plumbagin inhibits OVCAR-5 tumor growth in SCID mice
A. Images depicting differences in OVCAR-5 tumor growth with and without plumbagin treatment. B. Average OVCAR-5 tumor volume after 3 weeks of treatment in control and treatment groups. C. Significant regression in tumor weight has been observed. Plumbagin inhibits tumor cell proliferation in vivo. Ki67-stained tumor sections: D. Control group received only 25% PEG, E. received plumbagin in 25% PEG. Plumbagin alone demonstrated a significant effect on the inhibition of the Ki67 level. F. Quantitation of the Ki67 positive cells. Plumbagin inhibits angiogenesis in vivo. vWF stained sections: G. Control group received only 25% PEG, H. received plumbagin in 25% PEG, showing no staining of vWF. P value (Paired t test, 2 tailed) was calculated by comparing treated group vs. control group.
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