br Introduction br Ovarian cancer
Ovarian cancer is one of the most commonly occurring types of cancer and approximately 22,240 new cases of ovarian cancer will be diagnosed in the United States in 2018 (Reid et al., 2017; Siegel et al., 2018). The current standard treatment is a tumorectomy coupled with platinum and/or taxane-based chemotherapy, and while most patients are initially responsive to chemotherapy, the 5-year survival rate of individuals with ovarian cancer is approximately 15–30% (Morgan et al., 2016; Torre et al., 2018). Cancer recurrence and metastasis are the primary reason for treatment failure and a major clinical challenge without a current eﬀective therapy. The cancer stem cell (CSC) has been verified as the primary cause of recurrence and metastasis in cancer (Cornelison et al., 2017; Wang et al., 2014). So it is very urgent and important to find the novel therapies against ovarian cancer.
Natural compounds have been suggested as eﬀective agents for the treatment of cancer and are also considered to have promise for tar-geting for CSC (Moselhy et al., 2015; Pistollato et al., 2017; Taylor and Jabbarzadeh, 2017). Celastrol isolated from Thunder God Vine (Trip-terygium wilfordii; traditional Chinese medicine), has attracted intensive attention due to a broad range of bioactive properties, especially those showing anticancer activities (Chen et al., 2018; Kapoor, 2016; Xiao et al., 2018). Several studies have shown that celastrol can eﬃciently suppress tumor proliferation, migration and induce apoptosis in mul-tiple cancer models (Fan et al., 2014; Fribley et al., 2015; Wang et al., 2015; Wong et al., 2012), but the mechanism of celastrol in treatment of ovarian cancer needs further investigation (Wang et al., 2017; Zhang et al., 2016).
Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) reg-ulates multiple signal transduction pathways that control cell fate
Corresponding author at: Central Laboratory, Liaocheng People's Hospital, Liaocheng, Shandong 252000, PR China. E-mail addresses: [email protected] (X. Li), [email protected] (S. Ren). 1 Contributed equally to this study.
(Zhou and Lu, 2016). The abnormally elevated 372965-00-9 of Pin1 is prevalent in most human cancers, including ovarian, breast, colorectal, and cervical cancers, among others (Angelucci and Hort, 2017; Lu and Hunter, 2014). The expression level of Pin1 has been correlated with poor clinical prognosis (Lu and Hunter, 2014). Overexpression of Pin1 accelerates tumorigenesis by activating over 40 oncogenes and in-activating over 20 tumor suppressors (Zhou and Lu, 2016). Pin1-null mice develop normally and show reduced tumor growth and high re-sistance to tumorigenesis (Fujimori et al., 1999; Girardini et al., 2011). Pin1 inhibition dismantles oncogenic pathway cooperation in both CSC and non-CSC tumor cells and provides a rationale for the development of targeted therapies (Wei et al., 2015). These findings suggest that Pin1 could be as a prognostic cancer marker and a novel target for anticancer therapy (Moore and Potter, 2013; Zhou and Lu, 2016).
Our study aimed to test the hypothesis that celastrol exerted cura-tive eﬀects against ovarian cancer through the inhibition of Pin1. We first investigated whether celastrol can suppress proliferation, migra-tion and induce apoptosis in ovarian cancer cells. The eﬀect of celastrol on cancer stem cell was also evaluated. Furthermore, the underlying mechanism of action concerning Pin1 in human ovarian cancer cells was investigated.
2. Materials and methods
Human ovarian cancer cell lines A2780, SKOV3, and OVCAR3 were obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). A2780 and SKOV3 were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) with 100 U/ml penicillin-streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37 °C with 5% CO2. OVCAR3 was cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS. Celastrol with purity greater than 98% (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved in dimethyl sulfoxide (DMSO).
A2780, SKOV3 and OVCAR3 cells were plated into 96-well plates at a density of 1 × 104 cells/well and cultured for 24 h. After treating samples with varying concentrations of celastrol (0, 0.25, 0.5, 1, 2 and 4 μM) for 48 h, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to determine the cell viability and growth proliferation rate. Then, 50 μl MTT (2 mg/ml) was added to each well and removed after 4 h by pipette. After the MTT was re-moved, 150 μl of DMSO was added to each well for 10 min. The ab-sorbance was measured by a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) at 570 nm. All assays were performed three times.