Sensitization of Carboplatinum- and Taxol-Resistant High-Grade Serous Ovarian Cancer Cells Carrying p53, BRCA1/2 Mutations by Emblica officinalis (Amla) via Multiple Targets

Abstract

Background: Ovarian cancer (OC), the most lethal gynecologic malignancy, is highly resistant to current treatment strategies. High-grade serous epithelial ovarian cancer (HGSOC) cells with increased somatic mutations and genomic instability and the resulting heterogeneous mutant phenotypes are highly resistant to therapy. Plant-derived natural products, including Amla (Emblica officinalis) extract (AE), have demonstrated potent anti-neoplastic properties. Recently we demonstrated that AE inhibits cell growth and the expression of angiogenic factors in OVCAR3 and SKOV3 OC cells in vitro as well as in xenografts in vivo. The goal of this study was to determine the anti-proliferative, anti-angiogenic and anti-metastatic effects of AE on carboplatinum- and taxol-resistant HGSOC cells carrying p53, BRCA1/2 mutations. Methods: Anti-proliferative and anti-metastatic effects of AE on recently characterized carboplatinum- and taxol-resistant HGSOC cells (TOV3041G, OV866(2), OV4453 and, OV4485) was determined using the MTT, migration, invasion and spheroid assays in vitro. To understand the mechanism of AE-induced changes in angiogenesis-related hypoxia-inducible factor 1α (HIF-1α) and insulin growth factor receptor 1 (IGF1R), and EMT-associated SNAIL1 and E-cadherin proteins were studied using immunostaining and Western blotting. In vivo effects of AE were determined using mouse xenograft tumor model of OC developed by subcutaneous injection of OV4485 cells that carry mutant p53 and BRCA1, most aggressive and resistant among HGSOC cell lines used in this study. Tumor growth was measured using morphometry. Immunostaining and Western blotting were used to determine changes in Ki67 (proliferation marker), CD31 (angiogenesis marker) as well as changes in HIF-1α, IGF1R, SNAIL1 and E-cadherin proteins. Results: AE significantly attenuated migration and invasiveness properties of all tested HGSOC cell phenotypes (P≤0.001), significantly reduced the expression of HIF-1α, IGF1R, and SNAIL1 and increased the expression of E-cadherin in all tested HGSOC cell lines (P=<0.05). Oral administration of AE for 4 weeks caused a significant regression of mouse xenograft tumor (>60%) that derived from OV4855 cells and decreased the expression of endothelial cell antigen-CD31, HIF-1α, IGF1R and SNAIL1 and increased the expression of E-cadherin in tumor tissues. Conclusions: AE sensitizes platinum- and taxol-resistant heterogenous HGSOC cells carrying mutations in p53, BRCA1/2 genes, and attenuates their malignant characteristics through targeting key signaling mechanisms of angiogenesis and metastasis. AE is a potential adjunct therapeutic agent for treating resistant, mutant, heterogenous OC.

Keywords: Amla; angiogenesis; high-grade serous ovarian cancer; metastasis; mutation; resistant; sensitize.

Figure 1

AE treatment inhibited cell proliferation, invasiveness and spheroid formation in highly aggressive, resistant, metastatic mutant high-grade serous ovarian cancer cells. (A-D) Time- and dose- dependent effects of AE on proliferation of (A) TOV3041, (B) OV4453, (C) OV866(2) and (D) OV4485 cells. Cells were treated with different doses of AE for 24-96 hours as shown and proliferation was assesses using the MTT assay. Results are presented as percent of untreated control cells (mean ± S.E.M, n=6 independent observations, *, P<0.05). (E-H) AE inhibited invasiveness of OV866(2) and OV4485 cells. (E-F) AE (400 µg/ml) inhibited the invasiveness of OV866(2) after incubation for 6 h and (G-H) AE (500 µg/ml) inhibited the invasiveness of OV4485 cells for 24 h determined using the Transwell assay. Representative images (20x magnification) show OV866(2) and OV4485 cells that crossed to the basal side of the membrane indicating invasiveness. (F, H) Number of migrated cells from randomly chosen five areas of crystal violet-stained cells (mean ± SEM of 3 independent experiments each in duplicate. *, P≤0.05 compared with control). (I-L) AE decreased the formation of compact spheroid formation by TOV3041G, OV866(2), OV4453 and OV4485 cells. Representative images show loosened spheroids after AE treatment. C=control, AE=Amla extract.

Figure 2

AE inhibited wound-healing by HGSOC cells in a time- and dose-dependent manner. (A-H) TOV3041, OV4453, OV866(2) and OV4485 cells were scratch-wounded at 90% confluency and treated with 100-800 µg/ml AE and further incubated for 48-72 h. Gap distances were measured, normalized using an untreated control. (A, C, E) Representative images showing the inhibitory effect of AE on wound-healing migration of (A) TOV3041, (C) OV4453, and (E) OV866(2) cells captured at 0, 6, 24, and 48 h. (G) Representative images showing the inhibitory effect of AE on wound-healing migration of OV4485 cells captured at 0, 24, 48 and 72 h. B, D, F and H Quantitative analysis of the results presented under (A, C, E, G). Results are shown as mean + S.E.M. from 6 independent experiments. *, P≤0.05 compared with 0 hour was considered significant.

Figure 3

AE downregulates the expression of HIF-1α, IGF1R, and SNAIL1, and upregulates the expression of E-cadherin in HGSOC cells - (A, E, I, M) TOV3041G, (B, F, J, N) OV4453, (C, G, K, O) OV866(2) and (D, H, L, P) OV4485. Confluent cells were incubated with AE (400 µg/ml for TOV3041G, OV4453, OV866(2) and 500 µg/ml OV4485) for 48 hours and analyzed using immunocytochemistry. Representative photomicrographs of 4 experiments are shown. Number immuno-stained cells were counted and calculated as percent of total cells. Results show decreased immuno-staining for HIF-1α protein (A-D), (E-H) IGF1R protein and (I-L) SNAIL protein. In contrast immuno-staining for E-cadherin protein was increased (M-P). Results are presented as bar graphs showing Mean ± SEM, *, P≤0.05 compared with control group. Bar=25 µm. Ctrl=control, AE=Amla extract.

Figure 4

Incubation with AE downregulated the expression of HIF-1α, IGF1R and SNAIL1 and increased the expression of E-cadherin in HGSOC cells- TOV3041G, OV4453, OV866(2) and OV4485. Confluent cells were incubated with AE for 48 hours and analyzed using SDS-PAGE and Western blotting as described. (A-D) Representative images in upper panels show that treatment with AE resulted in decreased expression of HIF-1α, IGF1R and SNAIL1 proteins. In contrast, AE-treated cells showed increased expression of E-cadherin protein. (E-H) Densitometry ratios of HIF-1α/β-actin, IGF1R/β-actin, SNAIL1/β-actin and E-cadherin/β-actin. Results are presented as Mean ± SEM of 3 experiments. *, P<0.05 vs. control. β-actin was used as the loading control. Ctrl=control, AE=Amla extract.

Figure 5

AE attenuated OV4485 xenograft tumor growth in athymic nude mice and downregulated HIF-1α, IGF1R and SNAIL1 protein expression but upregulated E-cadherin protein expression. (A) Left upper: Untreated Nude mouse bearing tumors, left lower: tumor from untreated mouse (control), Right upper: AE treated mouse. Right lower: tumor from AE-treated mouse. (B) Line graph showing change in tumor volume with time. Black line: untreated (control) tumor, Gray line: AE treated. (C-N) Immuno-histochemical analysis of xenograft tumor in untreated control and AE-treated mice. Results presented as bar graphs show immuno-positive cells as percent of total cell. (C-D) HIF-1α expression was decreased by AE treatment. Bar=10 µm. (E-F) IGF1R expression was decreased after AE treatment. Bar=10 µm. (G-H) Decreased expression of SNAIL1 in xenograft tumors after AE treatment. Bar=10 µm. (I-J) increased immuno-staining for E-cadherin in xenograft tumors after treatment with AE. Bar=10 µm. (K-L) Decreased immuno-histochemical expression of Ki67 positive cells in mouse tumor xenograft after AE treatment. Bar=10 µm. (M-N) AE treatment decreased the immuno-histochemical expression of CD31 positive cells in mouse xenograft tumors Bar=25 µm. (O) Representative Western blots showing a decreased expression of HIF-1α, IGF1R, SNAIL1 and increased expression of E-cadherin. β-actin was used as the loading control. (P-S) Western blotting results presented as densitometric ratios of, (P) HIF-1α/β-actin, (Q) IGF1R/β-actin, (R) SNAIL1/β-actin and (S) E-cadherin/β-actin. All results were obtained using tumor tissues from 5 mice in each group. Values are Mean ± SEM, *, P≤0.05 compared with control group. Ctrl=control, AE=Amla extract.