ARID1A Expression is Down-Regulated by Oxidative Stress in Endometriosis and Endometriosis-Associated Ovarian Cancer

Abstract

Oxidative stress is considered an important factor in the development of endometriosis, including its malignant transformation. Previous studies have found that AT-rich interactive domain 1A (ARID1A), a tumor suppressor gene, is frequently mutated and inactivated in endometriosis-associated ovarian cancer (EAOC), and such a change in this gene is considered an early event in malignant transformation. We observed oxidative stress status by measuring the activity of the antioxidant enzyme manganese superoxide dismutase (MnSOD), malondialdehyde (MDA), and ARID1A gene expression in tissue samples from patients with endometriosis, EAOC, or non-endometriosis-associated ovarian cancer (non-EAOC). We also induced oxidative stress in the cultured cells from patients with primary endometriosis by adding H₂O₂ and tested for any alteration of ARID1A gene expression based on different H₂O₂ concentrations. The results showed that MnSOD activity in endometriosis and EAOC was lower than in non-EAOC, but MDA levels were higher. This study also showed that oxidative stress reduced ARID1A gene expression.

Keywords: ARID1A; endometriosis; malignant transformation; ovarian cancer; oxidative stress; tumor suppressor gene.

Figure 1.

Endometriotic tissues exhibited decreased ARID1A mRNA relative expression, protein level, and MnSOD activity but increased MDA level. Normal endometrial tissues were used as control. (A) ARID1A mRNA relative expression was significantly lower in the EAOC and endometriotic tissues. Relative expression shown in y-axis is average of gene expression, measured by Livak formula (2^−ΔΔCt). mRNA quantification was done using a 1-step reverse transcription polymerase chain reaction. Statistical significance difference was determined using 1-way ANOVA (*P = .035, **P = .019). (B) Endometriosis and ovarian cancer tissue samples had lower protein level. Extracted protein was analyzed using ELISA and optical density was measured at 450 nm length (expressed in pg/mg). Statistical significance difference was determined using 1-way ANOVA (*P = .092). (C) MnSOD activity in endometriotic tissues was the lowest. MnSOD activity, depicting antioxidant enzyme, was measured using xanthine and xanthine oxidase. The wells were incubated at 37°C for 5 minutes, and their absorbance was read at 505 nm wavelength. Statistical significance difference was determined using 1-way ANOVA (*P = .049, **P = .035, ***P = .044, ****P = .041). (D) Increased MDA level in endometriotic and ovarian cancer tissues. MDA level, reflecting the extent of oxidative stress, was measured using thiobarbituric acid reactive substances method with various concentrations of Aqua Dest and trichloroacetic acid. Its absorbance was read at 530 nm wavelength. Statistical significance difference was determined using Mann-Whitney test (*P < .011 and **P < .02). ANOVA indicates analysis of variance; ARID1A, AT-rich interactive domain 1A; EAOC, endometriosis-associated ovarian cancer; ELISA, enzyme-linked immunosorbent assay; MDA, malondialdehyde; MnSOD, manganese superoxide dismutase; mRNA, messenger RNA.

Figure 2.

Oxidative stress suppressed ARID1A mRNA relative expression and protein level. Normal endometrial and endometriotic cells were cultured using Dulbecco’s Modified Eagle Medium with a density of 25 000 cells/mL. Tissue samples were stored in those media at 4°C for less than 24 hours. (A) Fluorescence absorption was significantly higher in endometriotic cells, even with the absence of H₂O₂ induction. Fluorescence absorption, depicting reactive oxygen species level, was measured using a dichlorodihydrofluorescein diacetate assay. Samples were incubated at 37°C for 30 minutes and read by cytometer. Statistical significance difference was determined using t-test (*P < .05; RFU, relative fluorescence unit). (B) Both normal endometrial and endometriotic cells responded to increased concentration of H₂O₂ induction by increasing cell proliferation. H₂O₂ induction was done using various concentrations of H₂O₂ (100 and 1000 nM; 0 nM as control) for 48 hours. Viable cells were manually counted using hemocytometer under microscope with ×40 magnitude. Statistical significance difference was determined using paired t-test (*P = .010, **P = .001, ***P = .002, ^P = .001, ^^P = .04). (C) H₂O₂ suppressed relative mRNA expression of ARID1A with dramatic reduction in endometriotic cultured cells. Relative expression was measured by Livak formula (2^−ΔΔCt) and mRNA quantification was performed using a 1-step reverse transcription polymerase chain reaction. Statistical significance difference was determined using t-test (*P < .05). (D) ARID1A protein level was dose dependently suppressed by increasing amount of H₂O₂. Extracted protein was analyzed using enzyme-linked immunosorbent assay, and optical density was measured at 450 nm length (expressed in picogram per milligram). Statistical significance difference was determined using 1-way analysis of variance (*P < .05). ARID1A indicates AT-rich interactive domain 1A; EAOC, endometriosis-associated ovarian cancer; mRNA, messenger RNA.