Oxfendazole Induces Apoptosis in Ovarian Cancer Cells by Activating JNK/MAPK Pathway and Inducing Reactive Oxygen Species Generation

Abstract

Ovarian cancer (OC) is one of the most common and high mortality type of cancer among women worldwide. The majority of patients with OC respond to chemotherapy initially; however, most of them become resistant to chemotherapy and results in a high level of treatment failure in OC. Therefore, novel agents for the treatment of OC are urgently required. Benzimidazole anthelmintics might have the promising efficacy for cancer therapy as their selectively binding activity to β-tubulin. Recent study has shown that one of the benzimidazole anthelmintics oxfendazole inhibited cell growth of non-small cell lung cancer cells, revealing its anti-cancer activity; however, the pharmacological action and detailed mechanism underlying the effects of oxfendazole on OC cells remain unclear. Therefore, the present study investigated the cytotoxic effects of oxfendazole on OC cells. Our results demonstrated that oxfendazole significantly decreased the viability of OC cells. Oxfendazole inhibited the proliferation, induced G2/M phase arrest and apoptotic cell death in A2780 cells. The c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) pathway was activated and reactive oxygen species (ROS) generation was increased in OC cells treated with oxfendazole; oxfendazole-induced apoptosis was notably abrogated when co-treated with JNK inhibitor SP600125 and ROS scavenger N-acetyl-L-cysteine (NAC), indicating that JNK/MAPK pathway activation and ROS accumulation was associated with the oxfendazole-induced apoptosis of OC cells. Moreover, oxfendazole could also induce the proliferation inhibition and apoptosis of cisplatin resistant cells. Collectively, these results revealed that oxfendazole may serve as a potential therapeutic agent for the treatment of OC.

INTRODUCTION

Ovarian cancer (OC) is one of the most common and high mortality type of cancer among women worldwide. A total of 313959 women worldwide were reported to be diagnosed with OC in 2020; and 207252 females succumbed from this gynecologic malignancy.¹⁾ Surgery combined with chemotherapy, which based on platinum salts and taxanes, is still the main treatment option for patients with advanced-stage OC. Though patients with OC respond to chemotherapy initially, most of them become resistant to chemotherapy and results in a high level of treatment failure in OC.²⁾ And the 5-year survival of patients with advanced-stage OC is only 27%.³⁾ Therefore, novel agents are urgently needed in order to increase the treatment efficacy of OC and overcome chemotherapy resistance.

Benzimidazole anthelmintics, which include oxfendazole, albendazole, parbendazole, fenbendazole, mebendazole, oxibendazole, ricobendazole, and flubendazole and have a chemical structure of benzimidazole, have been widely used in both human and veterinary medicine to control internal parasites. Benzimidazole anthelmintics can cause parasitic worm death by selectively binding to their β-tubulin.⁴⁾ As targeting microtubules of cancer cells is a terrific strategy to kill cancers, benzimidazole anthelmintic drugs might have the promising efficacy for cancer therapy and some of its members have been studied as antitumor agents in recent years. For example, fenbendazole could induce non-small cell lung cancer cell apoptosis by inhibiting proteasome function and inducing endoplasmic reticulum stress/reactive oxygen species (ROS).⁵⁾ Additionally, there is only one study reported that oxfendazole inhibited cell growth of non-small cell lung cancer cells by suppressing c-Src activation.⁶⁾ However, the pharmacological effects and detailed mechanisms of oxfendazole against OC are still unclear.

In this study, we showed that oxfendazole induced cell proliferation inhibition, cell cycle arrest and apoptosis in OC cells. Moreover, our results indicated that mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinase (JNK) pathway and ROS accumulation were involved in oxfendazole-induced OC cell apoptosis. Interestingly, platinum-resistant OC cells were sensitive to oxfendazole. Therefore, oxfendazole may be a potential agent for OC treatment.

MATERIALS AND METHODS

Reagents

Oxfendazole was obtained from Selleck Chemicals (#53716-50-0, Houston, TX, U.S.A.). N-Acetyl-L-cysteine (NAC) (#A7250) and SP600125 (#S5567) were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies against poly(ADP-ribose) polymerase (PARP) (#9542), caspase-3 (#9662), cleaved-caspase-3 (#9661), MCL-1 (#4572), BCL-XL (#2764), X-linked inhibitor of apoptosis protein (XIAP) (#14334), phosphorylated JNK (#4668), JNK (#9252), phosphorylated c-Jun (#9261), and c-Jun (#9265) were obtained from Cell Signaling (Danvers, MA, U.S.A.). Anti-β-actin (81115-1-RR), and anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibodies (SA00001-2) were obtained from ProteinTech Group, Inc. (Chicago, IL, U.S.A.).

Cell Culture

SKOV-3 and OVCAR-3 cells were purchased from the American Type Culture Collection (Manassas, VA, U.S.A.). KGN and A2780 cells were obtained from BeNa Culture Collection (Beijing, China). Cisplatin resistant cells (A2780/DDP) were established by our group. All cell lines were cultured in RPMI-1640 (Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mL penicillin and 10 mg/mL streptomycin (Thermo Fisher Scientific, Inc.) in a humidified atmosphere with 5% CO₂ at 37 °C.

Cell Viability Assay

Cell counting kit-8 (CCK8) assay (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used to detect the cell viability. The OC cells were seeded in 96-well microplates (1 × 10⁴ cells/well) and incubated at 37 °C overnight. The next day, cells were treated with oxfendazole (10, 25, 50, and 100 µM) for 48 h at 37 °C. Twenty microliter of CCK8 reagent was finally added into each well and the absorbance was detected by using a multimode reader at 450 nm.

Colony Formation Assay

OC cells were seeded in a 6-well plate (500 cells/well) and treated with 7.5 and 10 µM oxfendazole. After 7 d incubation, cells were fixed in ice-cold methanol for 10 min and then stained with crystal violet solution at room temperature for 10 min. Images of the colonies were captured using an Epson Perfection V370 Photo scanner (Epson America, Inc., Long Beach, CA, U.S.A.)

Cell Cycle Analysis

OC cells were treated with 10 and 25 µM oxfendazole for 12 h. Cells were then collected and fixed with 66% ice-cold ethanol at 4 °C overnight. Cells were stained with 500 µL propidium iodide (PI; BD Biosciences, Franklin Lakes, NJ, U.S.A.) at room temperature for 15 min in the dark before the cell cycle was analyzed by using flow cytometry (BD Biosciences). When analyzed by flow cytometry, forward scatter area height (FSC-H) versus FSC-A were used to select single cells.

Apoptosis Analysis

The oxfendazole-induced apoptosis of A2780 cells was analyzed by using an annexin V-fluorescein isothiocyanate (FITC)/PI staining kit (Nanjing KeyGen Biotech Co., Ltd.). OC cells were treated with 10 and 25 µM oxfendazole for 48 h and then washed with phosphate buffered saline (PBS). OC cells were then resuspended in a solution containing 500 µL binding buffer, 5 µL annexin V-FITC and 5 µL PI for 15 min at 37 °C in the dark and the apoptotic cells were finally analyzed by flow cytometry (BD Biosciences). Annexin V+ and annexin V+/PI+ cells were determined as apoptotic cells.

ROS Analysis

ROS accumulation was measured by using a ROS assay kit (Beyotime Institute of Biotechnology, Shanghai, China). OC cells were treated with 25 µM oxfendazole for indicated time and washed with PBS. OC cells were then loaded with 2′,7′-dichlorofluorescin diacetate (10 µM) and incubated for 20 min at 37 °C in the dark. The ROS generation was measured by flow cytometry (BD Biosciences).

Western Blot Analysis

Radioimmunoprecipitation assay buffer (Cell Signaling Technology, Inc.) containing protease and phosphatase inhibitors (Nanjing KeyGen Biotech Co., Ltd.) was used to lyse OC cells were treated with oxfendazole. The concentration of proteins was detected with a BCA protein assay kit (Pierce; Thermo Fisher Scientifc, Inc.). Equal total cellular protein was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride membranes. After blocked in 5% non-fat dry milk, the membranes incubated with primary antibodies overnight at 4 °C. The next day, the membranes were incubated with the anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h at room temperature and target proteins were determined by using enhanced chemiluminescence (Thermo Fisher Scientifc, Inc.).

Statistical Analysis

All experiments were performed in triplicate, and data are presented as the mean ± standard deviation. GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, U.S.A.) was used to analyze the data via unpaired two-tailed t-tests (Differences between two groups) or one-way ANOVA followed by Tukey’s post hoc test (Differences among 3 or more groups). p < 0.05 was considered to indicate a statistically significant difference.

RESULTS

Oxfendazole Significantly Reduces OC Cell Viability

We firstly determined the effects of oxfendazole on OC cell viability. Various OC cell lines were treated with oxfendazole (10, 25, 50, and 100 µM) for 48 h, and the cell viability was then detected by using the CCK8 assay. As shown in Fig. 1, oxfendazole significantly inhibited the viability of A2780, OVCAR-3, SKOV-3, and KGN cells in a dose-dependent manner, and their half-maximal inhibitory concentrations are 13.88, 19.82, 32.55, and 13.93 µM, respectively.

Fig. 1. Ovarian Cancer Cell Viability Is Significantly Reduced by Oxfendazole

A2780, OVCAR-3, SKOV3, and KGN cells were treated with various concentrations of oxfendazole (10, 25, 50, and 100 µM) for 48 h and cell counting kit-8 assay was used to detect the cell viability. ** p < 0.01, *** p < 0.001 vs. 0 µM.

Oxfendazole Induces the Proliferation Inhibition and G2/M Phase Arrest of OC Cells

We then used colony formation assays to detect the effects of oxfendazole on the proliferation of A2780 cells. After treated with 7.5 and 10 µM oxfendazole for 7 d, the colonies become fewer and smaller when compared with control group (Fig. 2A), suggesting that oxfendazole effectively inhibited the proliferation of A2780 cells. We then wondered whether the proliferation inhibition effects of oxfendazole to A2780 cells was associated with the alterations of the cell cycle. Our results showed that the percentage of cells in the G2/M phase increased from 17.55% in control group to 32.32 and 53.50% in A2780 cells treated with 10 and 25 µM oxfendazole for 12 h, respectively (Fig. 2B). These results indicated that oxfendazole inhibited the proliferation and induced G2/M phase arrest of OC cells.

Fig. 2. Oxfendazole Reduces Colony Formation and Induces G2/M Phase Arrest in A2780 Cells

(A) A2780 cells were treated with oxfendazole for 7 d and the colony formation ability of the cells was then determined. (B) A2780 cells were treated with oxfendazole for 12 h and the cell cycle of A2780 cells were detected by using flow cytometry. ** p < 0.01, *** p < 0.001 vs. control.

Oxfendazole Induces Apoptosis of OC Cells

Flow cytometry was used to analyze whether oxfendazole induced the apoptosis of A2780 cells. As presented in Fig. 3A, oxfendazole significantly increased the percentage of apoptotic A2780 cells, from 0.67% in the control group to 12.70 and 19.95% in the 10 and 25 µM oxfendazole treatment groups, respectively. Moreover, oxfendazole induced the cleavage PARP (an indicator of apoptosis) and its upstream protein caspase-3 (Fig. 3B), indicating that oxfendazole might induce apoptosis of OC cells through caspase pathway. As the apoptosis can be affected by BCL-2 and IAP family proteins, the expressions of anti-apoptosis proteins MCL-1, BCL-XL, and XIAP were measured. Western blot results revealed that MCL-1, BCL-XL, and XIAP expression levels in A2780 decreased after oxfendazole treatment (Fig. 3C), further indicating the apoptosis-induced effects of oxfendazole in OC cells.

Fig. 3. Oxfendazole Induces Apoptosis of A2780 Cells

A2780 cells were treated with 10 and 25 µM of oxfendazole for 48 h. (A) Apoptotic cells were determined by using flow cytometry. *** p < 0.001 vs. control. (B) Expression levels of cleaved-PARP and caspase-3 were measured by Western blot. (C) Expression levels of MCL-1, BCL-XL and XIAP were detected by Western blot.

Oxfendazole-Induced Apoptosis Is Associated with Activation of MAPK/JNK Pathway in OC Cells

The MAPK/JNK pathway has been reported to have an important role in anti-cancer effects of some newly develop drugs. We therefore detected the effect of oxfendazole on MAPK/JNK pathway. As shown in Fig. 4A, oxfendazole treatment of A2780 cells increased the expression levels of phosphorylated JNK (p-JNK) while had no effects to the total JNK. In addition, the expressions of phosphorylated c-Jun (p-c-Jun), one of the downstream targets of p-JNK, was also elevated in oxfendazole treated OC cells. These results indicated that oxfendazole could induced MAPK/JNK pathway activation in OC cells. To identify whether the MAPK/JNK pathway was involved in oxfendazole-mediated apoptosis, OC cells were co-treated with SP600125, a general inhibitor of JNK, and oxfendazole. Surprisingly, SP600125 partly rescued the oxfendazole-induced apoptosis. The percentage of apoptotic cells decreased from 20.10 to 4.60% in A2780 (Fig. 4C). Western blot assay showed that SP600125 inhibited the cleavage of PARP and caspase-3 induced by oxfendazole (Fig. 4B). Together, these results demonstrated that oxfendazol induced OC cell apoptosis via the MAPK/JNK pathway activation.

Fig. 4. JNK/c-Jun Pathway Is Involved in Oxfendazole-Induced Cell Apoptosis

(A) A2780 cells were treated with 10 and 25 µM of oxfendazole for 48 h and phosphorylation-JNK (p-JNK), total JNK, phosphorylation-c-Jun (p-c-Jun) and total c-Jun were analyzed by Western blot. A2780 cells were co-treated treated with 25 µM oxfendazole and 20 µM sp600125 for 48 h. And then (B) expression levels of cleaved-PARP and caspase-3 were measured by Western blot and (C) apoptotic cells were determined by using flow cytometry. *** p < 0.001.

ROS/JNK/c-Jun Axis Is Involved in the Oxfendazole-Induced Apoptosis of OC Cells

Excessive ROS accumulation in cancer cells can bring cancer cell death. Previous studies have reported that oxfendazole could induced ROS accumulation. Therefore, the effects of oxfendazole on the levels of ROS in A2780 cells were determined. From the flow cytometry results, we could detect that the levels of ROS were significantly increased in A2780 cells treated with oxfendazole when comparing with the control group (Fig. 5A), which suggested that oxfendazole induced ROS accumulation in OC cells. To further determine whether ROS accumulation mediated the anticancer activity of oxfendazole against OC cells, the ROS scavenger NAC was used to inhibit the excessive ROS accumulation induced by oxfendazole. Results from Fig. 5B showed that the effects of oxfendazole on the apoptosis of A2780 cells were notably abrogated (Fig. 5B). Moreover, oxfendazole-induce JNK activation was restored after the ROS accumulation in cells was scavenged by NAC (Fig. 5C). These data indicated that ROS/JNK/c-Jun axis was associated with the oxfendazole-induced apoptosis of OC cells.

Fig. 5. ROS Production Plays an Important Role in Oxfendazole-Induced Apoptosis for A2780 Cells

(A) A2780 cells were co-treated with 25 µM of oxfendazole and 5 mM NAC for 6 h and then the ROS production were determined by using flow cytometry. A2780 cells were co-treated treated with 25 µM oxfendazole and 5 mM NAC for 48 h, then (B) apoptotic cells were determined by flow cytometry and (C) expression levels of p-JNK and JNK were measured by Western blot. ** p < 0.01, *** p < 0.001.

Oxfendazole Induces Proliferation Inhibition and Apoptosis in Platinum-Resistant OC Cells

Cisplatin resistant cells (A2780/DDP) were established by subjecting the cells to successively increasing concentrations of cisplatin. And the resistance to cisplatin were confirmed by using the CCK8 assay. The results showed that A2780/DDP cells were not as sensitive as A2780 to cisplatin (Fig. 6A). The effect of oxfendazole in overcoming cisplatin resistance in OC cells was then investigated in this study. CCK8 results showed that oxfendazole inhibited A2780/DDP cell viability with IC₅₀ values of 29.06 µM (Fig. 6B). The colony formation assay showed that the proliferation of A2780/DDP cells was inhibited by oxfendazole (Fig. 6C). Moreover, oxfendazole could induce A2780/DDP apoptosis (Fig. 6D). Taken together, these results demonstrated that oxfendazole could overcome cisplatin resistance in OC cells.

Fig. 6. Oxfendazole Overcomes Platinum Resistance in Ovarian Cancer Cells

(A) A2780 and cisplatin resistant cell line A2780/DDP were treated with various concentrations of cisplatin (5, 10, 25, 50, 75, and 100 µM) for 48 h and cell counting kit-8 assay was used to detect the cell viability. *** p < 0.001. (B) The cell viability of A2780/DDP was tested after being treated with 10, 25, 50, and 100 µM oxfendazole for 48 h. (C) A2780/DDP cells were treated with oxfendazole for 7 d and the colony formation ability of the cells was then determined. (D) A2780/DDP cells were treated 25 µM of oxfendazole for 48 h and apoptotic cells were determined by using flow cytometry. *** p < 0.001 vs. 0 µM.

DISCUSSION

OC is one of the third most common types of gynecological cancer worldwide.¹⁾ The standard chemotherapy treatment option is still the carboplatin or cisplatin combined with paclitaxel for advanced-stage OC patients. However, the 5-year survival of patients with advanced-stage OC is still very low due to the recurrence and resistant to these anticancer agents.^2,7) Therefore, it is urgently required to develop novel effective agents against OC. In the present study, the therapeutic effects of oxfendazole, one of the benzimidazole anthelmintics that have been widely used for controlling internal parasites, were investigated in OC cells. Oxfendazole inhibited OC cell proliferation by inducing G2/M phase arrest. Additionally, Oxfendazole caused the apoptosis of OC cells, which may through the caspase pathway. The activation levels of MAPK/JNK pathway and the levels of ROS were increased in OC cells following oxfendazole treatment, which resulted in oxfendazole-induced apoptosis. The results of this study indicate that oxfendazole may be a potential therapeutic agent for the treatment of OC.

MAPK families play important roles in cell growth, differentiation and death.^8,9) In mammalian cells, the canonical MAPK pathways include extracellular signal-regulated kinase (ERK)1/2, JNK/MAPK, and p38/MAPK. Usually, the activation of ERK1/2 promote cancer growth and resistant to chemotherapy.^10,11) However, various chemical and radiant stress cause cancer cell death by activating the JNK/MAPK and p38/MAPK.^12–14) One of the benzimidazole anthelmintic drugs mebendazole was reported to inhibit hepatocellular carcinoma growth by inactivating ERK1/2.¹⁵⁾ But whether oxfendazole-mediated apoptosis in OC cells through MAPK pathway is still unclear. We then investigated whether JNK/ MAPK pathway was involved in oxfendazole-mediated OC cell apoptosis. After treated with oxfendazole, the expression levels of phosphorylation JNK (p-JNK) in OC cells increased. Moreover, the expression levels of p-c-Jun also increased in OC cells with oxfendazole treatment. These results suggested that oxfendazole could induced MAPK/JNK pathway activation in OC cells. Interestingly, the expression levels of total c-Jun also increased in oxfendazole treated OC cells. After phosphorylated and activated by p-JNK, p-c-Jun translocates into the nuclei where its association with ATF-2 causes transcriptional activation of target genes. And the transcription factor c-Jun can regulate the expression of itself.¹⁶⁾ That is the reason why oxfendazole increased the expression levels of p-c-Jun as well as total c-Jun. To further confirm the MAPK/JNK pathway was involved in oxfendazole-mediated apoptosis, OC cells were co-treated with SP600125, a general inhibitor of JNK, and oxfendazole. Our results showed that SP600125 partly rescued the oxfendazole-induced apoptosis in OC cells. Together, these results highlight the important role of MAPK/JNK pathway in oxfendazole-induced apoptosis in OC cells.

ROS have important roles in cell proliferation, differentiation, development, and apoptosis by affecting various pathways.^17,18) Moderate ROS levels may promote tumor development. Excessive amounts of ROS, however, can bring cancer cell death. Therefore, it may be the effective strategy to kill cancer cells by modulating ROS levels. Indeed, there are many anti-cancer agents have been shown to eliminate cancer cells by increasing ROS levels.^18,19) Albendazole, one of the benzimidazole anthelmintics, has shown to eliminate breast cancer and colon cancer cells by inducing ROS accumulation and DNA damage.²⁰⁾ Previous reports showed that oxfendazole had effects to induce ROS accumulation.²¹⁾ Our results also showed that oxfendazole could significantly increase the ROS levels in OC cells. Moreover, oxfendazole-induced OC cells apoptosis could be abrogated by the ROS scavenger NAC, indicating that ROS accumulation was associated with the oxfendazole-induced apoptosis of OC cells. Many studied have shown that ROS could induce cancer cell death by activating MAPK/JNK pathway.^22,23) Our results showed that oxfendazole could induce ROS accumulation as well as MAPK/JNK pathway activation. Moreover, oxfendazole-induce MAPK/JNK activation was restored after the ROS accumulation in cells was scavenged by NAC. These results suggested that ROS/JNK/c-Jun axis was associated with the oxfendazole-induced apoptosis of OC cells.

Currently, cisplatin, carboplatin, and paclitaxel are still the main chemotherapeutic drugs for treating advanced ovarian cancer patients.^24,25) However, a number of patients are resistant to these chemotherapeutics, either primary or acquired.²⁶⁾ Therefore, newly develop agents should have the ability to overcome these drug resistance. Our group has established a cisplatin resistant OC cell line (A2780/DDP) by subjecting the cells to successively increasing concentrations of cisplatin. In this study, we showed that oxfendazole could not only inhibit the proliferation of A2780/DDP cells, but also induce A2780/DDP apoptosis, indicating that oxfendazole could overcome cisplatin resistance in OC cells.

In summary, our results demonstrate that oxfendazole inhibited OC cell proliferation by arresting cell in G2/M phage, and induced cell apoptosis by activating JNK/MAPK and increasing ROS levels. Moreover, oxfendazole could overcome cisplatin resistance in OC cells. These results suggested that oxfendazole may be a potential candidate for anti-OC treatment.

Acknowledgments

This work was supported by Grants from Guangdong Basic and Applied Basic Research Foundation (2022A1515140001), Dongguan Science and Technology of Social Development Program (2018507150011651, 201950715001208, 202050715001215), and National Medical Science and Technology Foundation (W2016CWGD05).

Conflict of Interest

The authors declare no conflict of interest.

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