Glutathione as a Prognostic Biomarker and a Potential Therapeutic Target for Ovarian Cancer

Aim: Glutathione (GSH) is an antioxidant, protecting cell against toxicity of reactive oxygen species (ROS). Data showed that GSH might play roles in malignancy including ovarian cancer (OC), and, thus, we attempted to determine the clinical significance of GSH and effects of erastin (an inhibitor of GSH synthesis) in OC. Methods: OC tissues were taken from 41 OC patients, and cancer-tissue GSH level was measured with GSH Assay Kit. Survival curves were carried out by the Kaplan-Meier method and evaluated using the log-rank test. Multivariable Cox proportional hazard risk regression model was performed to screen the independent factor affecting the prognosis of OC patients. In vitro effect of erastin was studied using OC cell lines. Cell viability, GSH levels and whole (cytosolic and lipid) ROS production were assessed. Results: Patients with high OC-tissue-GSH levels had an apparently lower progression free survival (PFS) and overall survival (OS) compared with those with low GSH levels. The GSH levels were independent factors for predicting the PFS and OS. The basal ROS level was inversely proportional to GSH levels in OC cell lines. The basal GSH levels were important for estimating the sensitivity to erastin. Reduction of intracellular GSH levels increased whole ROS, which caused cell deaths. Conclusions: Data suggested that the GSH levels could be a candidate of prognostic biomarkers and that erastin might be worth studying as a new therapeutic drug in OC.

• Glutathione
• Reactive Oxygen Species
• Ovarian Cancer
• Erastin

1. Introduction

The number of patients with ovarian cancer (OC) is increasing and deaths due to this disease are also increasing in Japan [1]. Platinum compound is one of the most potent chemotherapy drugs widely used for OC [2] [3]. Although OC is generally sensitive to chemotherapy, most patients ultimately recur and develop resistance to chemotherapy [4] [5]. Survival rates are estimated based on previous outcomes [6], but it is not easy to predict what will happen in OC patients.

Glutathione (GSH), an antioxidant, is used to mitigate the damage of reactive oxygen species (ROS) [7]. They have roles in cell cycle progression and cell death pathway. Erastin is a classical inhibitor that can lead to the depletion of GSH [8].

In this study, we focused on a potential of GSH as a new prognostic biomarker and erastin as a new strategy in anti-tumor therapies for ovarian cancer.

2. Materials & Methods

2.1. Patients

A total of 41 OC tissues were obtained from patients who underwent operation between 2009 and 2018 at Gifu university hospital. The OC specimens were subjected to histological examination by 2 expert pathologists for confirmation of World Health Organization (WHO) classification of the tumor, and staging according to the tumor-node-metastasis (TNM) system. The ethics committee of the Gifu University Graduate School of Medicine approved the experiments. Written informed consent was obtained from all patients.

2.2. GSH Analysis in Human Ovarian Cancer Tissues

We performed GSH analysis using the specimen prepared by freezing the tissue separated. We took central and deep into the OC tissues as samples from the patient during operation. The tissues were lysed in 5% 5-sulfosalicylic acid dihydrate (Wako Pure Chemical Industries). The lysate was centrifuged (1000 *g) and the supernatant was collected. The supernatant was used to determine the amount of GSH in the sample. We used the GSH and GSSG Assay Kit (product No. S0053, Beyotime) and followed the product instructions to determine GSH levels. Briefly, GSH assay buffer, GSH reductase, 5,5’-dithio-bis 2-nitrobenzoic acid solution and supernatant sample were mixed together and incubated at 25˚C for 5 minutes, then NADPH was added into this system to trigger the reaction. The increase in the absorbance of 5-thio-2-nitrobenzoic acid was measured at 412 nm, and the GSH levels were calculated following the product instructions.

2.3. Cell Culture

OC cell lines, TOV-21G, KOC-7C (clear cell carcinoma), CaOV3 and SKOV3ip1 (serous carcinoma) were used. KOC-7C cells were provided by the Gynecological and Obstetrics Department, Kurume University and others were provided by the Gynecological and Obstetrics Department, Osaka University [9]. These cells were cultured in Dulbecco’s modified medium (Wako) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Wako) under 5% CO₂ at 37˚C.

2.4. Drug Treatments

Treatments of OC cell lines with 2.5 - 20 µM erastin, an inhibitor of GSH synthesis, or 0.5% DMSO as control were performed in Dulbecco’s modified medium containing 10% FBS and 1% penicillin/streptomycin.

2.5. Cell Viability Analysis

Cell viability was evaluated using a Premix WST-1 Cell Proliferation Assay System (TaKaRa Bio Incorporated) according to the manufacturer’s instructions. Briefly, cells (1 × 10⁴ cells/well) were seeded in a 96-well plate and treated with different drugs at various concentrations for the indicated times. After addition of 10 μl Premix WST-1 solution to each well, cells were incubated at 37˚C for another 1 hour and the absorbance was determined at 440 nm using a microplate reader.

2.6. Intracellular GSH Analysis

Cells were plated in 6-well plates at a density of 3.0 × 10⁵ cells/well and cultured overnight. Cells were received different treatment for 4 hours followed by harvesting to determine cell number. Nearly 6 × 10⁴ live cells from each sample were transferred to new tubes, washed in phosphate buffered saline (PBS) and centrifuged at 1200 rpm at 4˚C for 5 minutes twice. The cell pellet was resuspended in 80 μl protein removal solution, thoroughly incorporated, and placed in −70˚C and 37˚C sequentially for fast freezing and thawing, then placed in 4˚C for 5 minutes and centrifuged at 10,000 *g for 10 minutes. The supernatant was used to determine the amount of GSH in the sample. We used the GSH and GSSG Assay Kit and followed the product instructions to determine GSH levels.

2.7. Analysis of ROS Production

Cells were plated in 10 cm dishes at a density of 1.0 × 10⁵ cells/well and cultured overnight. After treated with test compounds for 15 hours, harvested in 5 ml Dulbecco’ s modified medium containing Deep Red Reagent (5 µM) (Molecular Probes, Invitrogen) and incubated for 30 min at 37˚C in a tissue culture incubator. After trypsinized, cells were resuspended in 3% FBS in PBS and strained through a 40 mM cell strainer (BD Falcon). Cells were analyzed using a flow cytometer (FACS Aria, BD Biosciences) equipped with 488 nm laser for excitation. Data were collected from the Deep Red Reagent channel (MitoSOX). A minimum of 1.0 × 10⁴ cells were analyzed per condition.

2.8. Statistical Analysis

Data are expressed as means ± SD of 3 independent experiments and were evaluated using an ANOVA LSD test. Student t test was conducted for intergroup comparison. Pearson correlation test was used for correlation analysis. Survival curves were generated with Kaplan-Meier plots. The results are presented with P values from a log-rank test. Multivariable Cox proportional hazard risk regression model was performed to screen the independent factor affected the prognosis of OC patients. All tests were 2-sided, and P values < 0.05 were considered statistically significant.

3. Results

3.1. Patient Characteristics

The GSH levels in 41 OC tissues were measured. The GSH levels were considered as either low (n = 34) or high (n = 7) according to the cut-off value, which was defined as the median of the cohort. The GSH levels were 73.1 ± 74.1 and 669.1 ± 339.5 µmol/l in each low and high group (Table 1). There was no significant difference in all parameters (e.g., age, type of pathology, stage, type of surgery, neoadjuvant and adjuvant chemotherapy).

Table 1

Characteristics of ovarian cancer patients who received GSH levels assay

	GSH levels
	low	high	P-value
Cases (n)	34	7
GSH levels (average ± SD)	73.1 ± 74.1	669.1 ± 339.5	<0.01
age (average ± SD)	60.0 ± 11.0	60.0 ± 16.5	0.55
type of pathology			0.26
HGSC	13	5
LGSC	1	0
CCC	17	1
EC	1	1
MC	2	0
Stage			0.73
I + II	17	3
III + IV	17	4
Type of surgery			0.85
complete and optimal	30	6
Others	4	1
NAC			0.82
+	13	3
-	21	4
adjuvant chemotherapy			0.41
+	31	7
-	3	0

HGSC = high grade serous carcinoma, LGSC = low grade serous carcinoma, CCC = clear cell carcinoma, EC = endometrioid carcinoma, MC = mucinous carcinoma, NAC = neoadjuvant chemotherapy.

3.2. High GSH Levels Predict Poor Prognosis in OC

To investigate the clinical significance of GSH, we assessed the association GSH levels and prognosis in OC. The Kaplan-Meier analysis was performed to determine the prognostic value of GSH in OC patients. We performed a log-rank test which compared patients with higher GSH levels with those with lower levels. Patients with higher GSH levels had significantly shorter progression free survival (PFS) than those with a lower GSH levels (P < 0.01) (Figure 1). It also showed that the overall survival (OS) was shorter in OC patients with higher GSH levels (P < 0.001) (Figure 2).

3.3. GSH Levels as Independent Factors for Predicting the Prognosis of OC

We performed the multivariate Cox regression analysis to show that GSH levels were independent factors predicting the prognosis. GSH levels were independent factors for both PFS (HR = 5.330, 95% CI: 1.334 - 21.291; P = 0.018) and OS (HR = 7.174, 95% CI: 1.572 - 32.738; P = 0.011) (Table 2 and Table 3).

3.4. The Sensitivity to an Inhibitor of GSH Synthesis, Erastin, in OC Cell Lines

Next, we demonstrated in vitro investigation using OC cell lines focused on GSH. Erastin is identified as an inhibitor of GSH synthesis. To determine whether erastin induces growth inhibition in OC cells, TOV-21G, KOC-7C, CaOV3 and SKOV3ip1 cells were treated with erastin for 24 hours and cell viability was assayed using Premix WST-1 assay (Figure 3). Comparatively, TOV-21G and SKOV3ip1 cells were more sensitive to erastin than CaOV3 and KOC-7C cells.

Table 2

Multivariate analysis for progression free survival by Cox regression model

Variable	Hazard ratio	95% CI			P-value
GSH levels	7.174	1.572	-	32.738	0.011
age	0.976	0.926	-	1.028	0.354
type of pathology	3.834	0.972	-	15.126	0.055
stage	4.440	0.541	-	36.439	0.165
type of surgery	0.170	0.030	-	0.974	0.047
NAC	3.191	0.660	-	15.437	0.149
adjuvant chemotherapy	1.049	0.028	-	39.331	0.979

CI = confidence interval, NAC = neoadjuvant chemotherapy.

Table 3

Multivariate analysis for overall survival by Cox regression model

Variable	Hazard ratio	95% CI			P-value
GSH levels	5.330	1.334	-	21.291	0.018
age	0.992	0.953	-	1.032	0.683
type of pathology	3.244	0.839	-	12.539	0.088
stage	2.526	0.374	-	17.061	0.342
type of surgery	0.233	0.045	-	1.198	0.081
NAC	2.833	0.675	-	11.882	0.155
adjuvant chemotherapy	1.632	0.066	-	40.081	0.764

CI = confidence interval, NAC = neoadjuvant chemotherapy.

3.5. The Accumulation of ROS Induced by GSH Depletion

We compared erastin sensitive OC cell line, TOV-21G, with less sensitive cell line, KOC-7C, focused on the basal GSH and ROS level. Although the GSH levels were reduced by erastin both in TOV-21G and KOC-7C cell, the basal GSH levels in KOC-7C cells were higher than that in TOV-21G cells under control condition (Figure 4). We observed that treatment with erastin resulted in increase of ROS in both cell lines but the basal ROS level in KOC-7C cell was lower than that in TOV-21G cell (Figure 5).

Erastin decreased intracellular GSH levels and increased ROS resulting in cell death in OC cells. The sensitivity to erastin was depended on intracellular basal GSH levels.

4. Discussions

Oxidative stress has long been implicated in cancer development and progression [10], suggesting that antioxidant treatment may provide protection from cancer [11]. Among the enzymatic systems involved in the maintenance of the intracellular redox balance, a main role is played by GSH that participates not only in antioxidant defense systems but also in many metabolic processes [12] [13]. GSH is a tripeptide formed by glutamic acid, cysteine, and glycine. In many normal and malignant cells, increased GSH levels are associated with a proliferative response and essential for cell cycle progression [14] [15]. On the other hand, GSH depletion regulates the activation of cell death pathways [16].

Thus, several studies about biochemical function of GSH were published. However, their potential utility in clinical practice as prognostic biomarkers is unknown. To investigate the clinical features of the GSH status, we assessed the associations between GSH levels and prognosis of OC patients. The GSH levels

in OC tissues of 41 patients were measured. Interestingly, OC patients with high GSH levels had an evidently lower PFS and OS compared with those with low GSH levels. Notably, GSH levels were independent factors for predicting the PFS and OS of OC patients. We investigated that elevated GSH levels lead antioxidant defense systems resulting in defense to evade cancer cell death and develop drug resistance. Our data indicated that GSH might be a potent biomarker for predicting the prognosis of OC patients.

Next, we demonstrated in vitro investigation to assess the oxidant system associated with GSH levels in OC. Recently, Qi Cheng et al. [17] reported that erastin works synergistically with cisplatin to inhibit OC cell growth. Erastin induced the depletion of GSH resulting in increase of lipid ROS [18] [19]. In detail, erastin inhibits system Xc resulting in cysteine starvation and ferroptotic cell death [20] [21].

Our data showed that the basal GSH levels were lower in erastin sensitive OC cell lines than less sensitive cell lines and the basal ROS level was inversely proportional to GSH levels in both cell lines. Although the depletion of GSH induced by erastin leads to ROS in both cell lines, cell death could not be lead in less sensitive OC cell lines because the basal ROS level was too low. We showed that the basal GSH levels are important for estimating the sensitivity to erastin.

5. Conclusion

Our study demonstrated that high GSH levels were significantly associated with a poor prognosis independently of other factors in OC. Erastin leads OC cell death depend on intracellular GSH levels. GSH could be a prognostic biomarker and erastin might be worth studying as a new drug of OC treatment.

Acknowledgements

We gratefully acknowledge the work of past and present members of our laboratory.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

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