Enhanced Accumulation of Cisplatin in Ovarian Cancer Cells from Combination with Wedelolactone and Resulting Inhibition of Multiple Epigenetic Drivers

Abstract

Purpose: Cisplatin resistance is a major concern in ovarian cancer treatment. The aim of this study was to investigate if wedelolactone could perform better in resistant ovarian cancer cells when used in combination with cisplatin.

Methods: Growth inhibitory potential of wedelolactone and cisplatin was investigated through MTT reduction assay in ovarian cancer cell lines including A2780 (sensitive), A2780^cisR (cisplatin resistant) and A2780^ZD0473R. Resistance factor (RF) of drugs was determined in these three cell lines. Combination index (CI) was calculated as a measure of combined drug action. Effect of this combination on changes in the cellular accumulation of platinum levels and platinum-DNA binding was also determined in vitro using AutoDock Vina while the effect of wedelolactone on inhibition of possible key culprits of resistance including Chk1, CD73, AT tip60, Nrf2, Brd1, PCAF, IGF1, mTOR1 and HIF2α was investigated in silico.

Results: Cisplatin and wedelolactone showed a dose-dependent growth inhibitory effect. RF value of wedelolactone was 1.1 in the case of A2780^cisR showing its potential to bring more cell death in cisplatin-resistant cells. CI values were found to vary showing antagonistic to additive outcomes. Additive effect was observed for all sequences of administration (0/0, 0/4 and 4/0 h) in A2780^cisR. Enhanced cellular accumulation of cisplatin was observed in parent and resistant cells on combination. Docking results revealed that among the selected oncotargets, Chk1, CD73, Nrf2, PCAF and AT tip60 were more vulnerable to wedelolactone than their respective standard inhibitors.

Conclusion: These findings have shown that additive outcome of drug combination in A2780^cisR and raised levels of platinum accumulation followed a clear pattern. This observation indicates that the presence of wedelolactone might have contributed to sensitize A2780^cisR. However, in silico results point to the possible effects of this compound on epigenetic factors involving tumor microenvironment, epithelial mesenchymal transition, and immune-checkpoint kinases.

Keywords: A2780; chemotherapy resistance; coumestan; growth inhibition.

Figure 1

Percentage of ovarian cancer diagnosed at different stages and its correlation with survival.

Figure 2

(A) Illustrates IC₅₀ (n=7) values while (B) illustrates RF values. ### indicates cisplatin (as control) whereas ** indicates P <0.01 and *** indicates P <0.001.

Figure 3

Cell survival fraction values as calculated in ovarian cancer cell lines including A2780, A2780^cisR and A2780^ZD0473R in response to cisplatin and wedelolactone. Data are expressed as means ± SEM (n = 7); and interactions examined by ANOVA (two-way) pursued by multiple comparison test (post hoc Bonferroni) using the software Graph Pad Prism-6. * indicates P < 0.05, ** indicates P <0.01 and *** indicates P <0.001. # shows significant difference relative to the control.

Figure 4

Intracellular platinum accumulation levels in A2780 and A2780^cisR calculated in response to sequential (0/0, 0/4 and 4/0 h) drug administration; Data are expressed as means ± SEM (n = 3); and analyzed by ANOVA (two-way) pursued by multiple comparison test (post hoc Bonferroni) using the software Graph Pad Prism-6. *** indicates P <0.001. ### shows significant difference relative to the control through two-way ANOVA followed by post hoc Bonferroni multiple comparisons test. ***P <0.001 compared with the cisplatin uptake in untreated control.

Figure 5

Intracellular platinum-DNA binding levels in A2780 and A2780^cisR calculated in response to sequential (0/0, 0/4 and 4/0 h) drug administration; data are expressed as means ± SEM (n = 3); and analyzed by ANOVA (two-way) followed by multiple comparison test (post hoc Bonferroni) using the software Graph Pad Prism-6. ** indicates P <0.01 and # shows significant difference relative to the control through two-way ANOVA followed by post hoc Bonferroni multiple comparisons test.

Figure 6

Illustrates E-values (Kcal/mol) of best docked poses of wedelolactone and standard drugs with hepatocyte growth factor receptor (cMET), checkpoint kinase 1 (Chk1), ecto-5′-nucleotidase (CD73), mammalian target of rapamycin (mTOR1), nuclear factor erythroid 2–related factor 2 (Nrf2), hypoxia-inducible factor 2-alpha (HIF2α), with heat shock protein 60 (HSP60), insulin-like growth factor (IGF1), P300/CBP-associated factor (PCAF), bromodomain protein 1 (Brd1) and acetyl transferase tip (AT tip60).

Figure 7

Shows 2D best pose of (A) hepatocyte growth factor receptor (cMET) with wedelolactone; (B) hepatocyte growth factor receptor (cMET) with crozotinib; (C) checkpoint kinase 1 (Chk1) with wedelolactone; (D) checkpoint kinase 1 (Chk1) with anacardic acid.

Figure 8

Shows 2D interactions of the best pose of (A) bromodomain protein 1 (Brd1) with wedelolactone; (B) bromodomain protein 1 (Brd1) with olinone; (C) acetyl transferase tip60 (AT tip60) with wedelolactone, (D) acetyl transferase tip60 (AT tip60) with anacardic acid. (E) CD73 with wedelolactone; (F) CD73 with Pt2385.

Figure 9

Shows 2D interactions of the best pose of (A) mammalian target of rapamycin (mTOR1) with wedelolactone; (B) mammalian target of rapamycin (mTOR1) with sirolimus; (C) nuclear factor erythroid 2–related factor 2 (Nrf2) with wedelolactone; (D) nuclear factor erythroid 2–related factor 2 (Nrf2) with trigonelline; (E) hypoxia-inducible factor 2-alpha (HIF2α) with wedelolactone; (F) hypoxia-inducible factor 2-alpha (HIF2α) with PT2385.

Figure 10

Shows 2D interactions of the best pose of (A) heat shock protein 60 (HSP60) with wedelolactone (B) heat shock protein 60 (HSP60) with myrtucommulone; (C) insulin like growth factor (IGF1) with wedelolactone (D) insulin like growth factor (IGF1) with PPP; (E) P300/CBP-associated factor (PCAF) with wedelolactone (F) P300/CBP-associated factor (PCAF) with anacardic acid.

Figure 11

Signaling pathways involved in inactivation of apoptosis and resistance to cisplatin which are possibly inhibited by wedelolactone (WDL). Created with BioRender.com.