Background
Ovarian cancer is one of the most lethal gynecological malignancies in women, with more than 60,000 new cases reported annually in the United States and the European Union, and approximately 239,000 new cases worldwide were reported in 2012 [
1,
2]. Due to the difficultly of early diagnosis and the hidden progression of the disease, most patients are diagnosed at the advanced stage of cancer, which carries an extremely poor prognosis [
3]. Although the new era of targeted drugs has made some progress in ovarian cancer treatment, the antitumor efficacies of current therapies remain limited [
4]. Therefore, it is urgent to develop novel and alternative strategies for epithelial ovarian cancer (EOC) treatment.
Proteasomes have already become effective therapeutic targets in some disease models such as inflammation, fibrosis, ischemia-reperfusion injury, and tumors [
5,
6]. Cancer cells have been shown to depend on the ubiquitin proteasome system (UPS) more than normal cells do [
7]. Therefore, proteasome inhibition has become an effective strategy for cancer therapy. Bortezomib is a classic 20S proteasome inhibitor (PI) approved by FDA for the treatment of multiple myeloma and mantle cell lymphoma. Inhibition of the proteasome with bortezomib was the first clinical validation of targeting the UPS in cancer therapeutics and has gotten a huge success. However, PIs have weak effect on solid tumors in humans [
8]. Alternative proteasome inhibitors with different targets for proteasome activity sites were needed to make more satisfying effects on epithelial ovarian cancer (EOC).
Inhibition of proteasome-associated deubiquitinases (DUBs) has emerged as a promising anti-cancer strategy [
9‐
11]. DUBs function to remove the ubquitin conjugates on protein substrates prior to their degradation and thereby regulating multiple cellular processes, which are highly associated with cancer development [
12‐
15]. The human genome encodes approximately 100 putative DUBs, which are subdivided into six families according to their catalytic and structural features. USP14 and UCHL5 belong to DUBs and could be recruited and activated by 19S proteasome. Overexpression of USP14 or UCHL5 was observed in several carcinomas. Selective inhibition of USP14 and UCHL5 by b-AP15 or auranofin has been developed as a promising strategy against some carcinomas [
16‐
18].
Cisplatin is one of the most effective drugs currently used for treatment of ovarian cancer, biliary cancer, lung cancer, etc. [
19,
20]. However, continuous administration of Cisplatin to patients leads to severe side effects and the acquired or intrinsic resistance to Cisplatin, which gradually limits its use [
21,
22]. We previously dedicated to identify a novel inhibitor of 26S proteasome-associated deubiquitinases USP14 and UCHL5. Our previous study revealed that PtPT, a chemically well-characterized synthetic complex of platinum, targets 26S proteasome-associated DUBs rather than DNA in the cell and thereby exerts safer and potent anti-tumor effects [
23]. The current study aims to investigate the anti-tumor effects of PtPT on epithelial ovarian cancer and its underlying mechanisms.
Methods
Materials
Platinum pyrithione was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM and stored at −20 °C, which was synthesized in our lab. Pan caspase inhibitor z-VAD-FMK was purchased from Enzo Life Sciences International, Inc. (Plymouth Meeting, PA). MTS assay (CellTiter 96 Aqueous One Solution reagent) was obtained from Promega Corporation (Madison, WI, USA). Penicillin, Streptomycin, and Cisplatin were from Sigma-Aldrich (St. Louis, MO, USA); Annexin V-FITC/PI apoptosis Detection Kit and cell apoptosis Rhodamine 123 Detection Kit were from Keygen Company (Nanjing, China). Antibodies raised against ubiquitin (Catalog #3936; 1:1000), Cdc2 (Catalog #9116; 1:1000), Cyclin B1 (Catalog #12231;1:1000), caspase-3 (Catalog #9662;1:1000), caspase-9 (Catalog #9508; 1:1000), caspase-8 (Catalog #9746;1:1000), PARP (Catalog #9532;1:1000), K48- linkage specific polyubiquitin (Catalog #12805;1:1000), K63- linkage specific polyubiquitin (Catalog #12930;1:1000), Bcl-2 (Catalog #15071;1:1000), Bax (Catalog #14796;1:1000), eIF2α (Catalog #5324;1:1000), Phospho-eIF2α (Catalog #3398; 1:1000), ATF-4 (Catalog #11815;1:1000), apoptosis-inducing factor (AIF) (Catalog #5318; 1:1000), and Cox-4 (Catalog #4844; 1:1000) were from Cell Signaling Technology (Beverly, MA, USA). Cleaved caspase-8 (Catalog #L0167; 1:1000) was from Assay biotechnology Company (California, USA); Cleaved caspase-9 (Catalog #BS7070; 1:1000), cleaved caspase-3 (Catalog #BS7004; 1:1000), and GAPDH (Catalog #BS60630; 1:1000) were from Bioworld Technology (St. Louis Park, MN, USA). Enhanced chemiluminescence (ECL) reagents were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
Cell culture
Human ovarian cancer cell lines HO8910,OVCAR3, A2780, and SKOV3 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA), 100 units/ml of penicillin and 100 g/ml of streptomycin, cultured at 37 °C with 5% CO2.
Cell viability assay
Cell viability was assessed with the MTS assay as previously reported [
24]. HO8910, OVCAR3, A2780, and SKOV3 cells were seeded into 96-well plates at a concentration of approximately 5000 cells/well in 100 μl overnight. After stimulation with various concentrations of PtPT or cisplatin (0, 2, 4, 6, 8, 10, 12 and 14 μM) for 24 and 48 h, 20 μl MTS was supplemented for another 3 h of incubation. Absorbance was read at 490 nm and IC
50 values were derived.
Flow cytometry analysis of cell cycle
Cell cycle analysis was performed as reported previously [
25]. A2780 or SKOV3 cells were seeded in 6-cm dishes overnight in DMEM supplemented with 10% FBS, then treated with PtPT for 24 h, and the cells were digested by trypsin and washed twice with ice-cold PBS. The cell pellet was suspended with 70% ethanol at −20 °C overnight, then washed by PBS and incubated with 200 μg/mL RNase A at 37 °C for 15 min. For flow cytometry, 50 μg/mL propidium iodide (final concentration) was added for 15 min staining in the dark at 37 °C. Data was analyzed based on the distribution of cell populations at different phases of the cell cycle.
Cell death assay
Apoptosis was assessed by flow cytometry using Annexin V-FITC and PI double staining as we previously reported [
26]. After treatment with PtPT at various concentrations for 48 h, the cells were incubated in the dark for 15 min at room temperature with Annexin V-FITC followed by PI addition just before analysis. In addition, cells were submitted to Annexin V/PI staining in situ for observation by fluorescence microscopy.
Mitochondrial membrane integrity measurement
The mitochondrial membrane potential of PtPT-treated and untreated cells were assayed by using rhodamine-123 staining as previously reported [
27]. In brief, cells were treated with various concentrations of PtPT for 24 h and stained with 1 μM of rhodamine-123 for 30 min at 37 °C. Following the staining, the cells were washed with 4 °C PBS and then harvested for flow cytometry analysis.
Western blot analysis
Western blot was performed as we described previously [
28]. In brief, an equal amount of the total protein extracted from cultured cells was fractionated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were blocked with 5% milk for 1 h. Primary antibodies (1:1000) and horseradish peroxidase-conjugated appropriate secondary antibodies (1:5000) were used to detect the designated proteins. Blots were reacted to the ECL detection reagents and exposed to X-ray films (Kodak, Japan).
Nude mouse xenograft model
Female Balb/c nude mice aged 5 weeks were purchased from Guangdong Animal Center and all animal protocols used were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University. The mice were housed in barrier facilities with a 12 h light dark cycle, with food and water available ad libitum. Approximately 1 × 10
6 of A2780 or SKOV3 cells were inoculated subcutaneously in the left armpit of each mouse. After 5 days of inoculation, mice were treated with either vehicle (10% DMSO, 30% cremophor and 60% NaCl) or PtPT (7.5 mg/kg/day) for 23 days [
23]. Tumors were measured every other day with the use of calipers. Tumor volumes were calculated as previously reported [
29]. Tumor xenografts were removed afterwards, weighed, fixed, and stored. After 23 days of treatment, tumor xenografts were removed, weighed, stored, and fixed. All experiments were performed in accordance with relevant guidelines and regulations.
Immunohistochemical staining
Formalin-fixed xenografts were embedded in paraffin and sectioned according to standard techniques as previously reported [
29]. Tumor xenograft sections (4 μm) were immunostained using the MaxVision kit (Maixin Biol, Fuzhou, Fujian, China) according to the manufacturer’s instructions. The primary antibodies were against proteins as indicated. 50 μl MaxVisionTM reagent was applied to each slide. Color was developed with 0.05% diaminobenzidine and 0.03% H
2O
2 in 50 mM Tris–HCl (pH 7.6), and the slides were counterstained with hematoxylin. A negative control for every antibody was also included for each xenograft specimen by substituting the primary antibody with preimmune rabbit serum.
Statistical analysis
All the experiments were performed at least thrice and were expressed as Mean ± SD where applicable. SPSS (SPSS, USA) was employed for the statistics. Unpaired Student’s t-test or one way ANOVA is used where appropriate for determining statistic probabilities. P <0.05 was considered statistically significant.
Discussion
EOC is the primary reason for death from gynecologic cancer that affects women in the world every year [
30]. Although most patients initially show sensitivity to cisplatin, a few years later the drug-resistant metastatic disease occurs [
31,
32]. Thus, developing novel anti-tumor therapies that can work alone, or in combination with platinum-based therapy is critical [
33].
Bortezomib, the first 20S proteasome peptide inhibitor approved by United States Food and Drug Administration (FDA) for treating multiple myeloma MM , has also been showing its antitumor activity in other malignancies, including colon cancer, prostate cancer, breast cancer, and ovarian cancer [
34,
35]. It was reported that ovarian cancer cells may have a greater requirement for proteasomal activity than their untransformed counterpart [
36]. Therefore, identifying novel agents that target proteasome, both 19S and 20S, is of great research and clinical value.
Up to date, numerous metals, such as copper, zinc, and gold, have shown their antitumor activity in several carcinomas as proteasome inhibitors. Our previous study also found that PtPT is a specific proteasome inhibitor with low toxicity. However, the effect of PtPT (proteasome-associated deubiquitinase inhibitor) on ovarian cancer cells has not been unraveled. In the current study, we showed that PtPT is an effective chemical against ovarian cancer cells, including A2780 and SKOV3 cells. Additionally, we suggested that PtPT-mediated proteasome inhibition and caspase activation is responsible for PtPT-induced cytotoxicity in ovarian cancer cells. Most importantly, PtPT induced typical proteasome inhibition in both A2780 and SKOV3 cells in vitro and in vivo.
Firstly, we observed that PtPT dramatically suppressed the proliferation of EOC cells, indicating the potential activity for EOC treatment. We further investigated the pattern that PtPT inhibits the EOC proliferation. The ubiquitin-proteasome system is essential in the degradation of several proteins that are involved in the cell cycle control and thereby regulate the growth of cancer cells. Dysregulation of ubiquitin-proteasome system may promote carcinogenesis. We found that PtPT arrested cell cycle progression at G2/M phase in both A2780s and SKOV3 cells via down-regulating Cdc2 and CyclinB1 protein expressions (Fig.
2b and
c). It is known that Cyclin B1 is involved in the early events of mitosis by interacting with Cdk1; and Cdc2 (p34) is an M-phase promoting factor that induces entry into mitosis [
37‐
39].
Cell fate is tightly regulated by mitochondria and caspases. Li et al have reported that proteasome inhibition-induced Bax accumulation plays an important role in proteasome inhibition-mediated caspase activation and cell apoptosis [
40]. In the current study, we found that PtPT increases pro-apoptotic protein Bax while decreasing anti-apoptotic protein Bcl-2 in a dose- and time-dependent manner. We also observed that various concentrations of PtPT led to the decrease of mitochondrial membrane integrity, thereby inducing the release of cytochrome C and AIF. Similar to previous study, the released apoptotic factors, either directly or by forming a caspase-9 complex, induced caspase activation (Fig.
4). To determine whether caspase activation is required for PtPT-induced cell death, cells were treated with PtPT (6 μM) for 48 hours with pan-caspase inhibitor z-VAD, then PARP was detected by western blot. It was found that PARP cleavage was almost completely rescued in the presence of z-VAD, consistent with the previous reports [
23]. PI staining results also showed that z-VAD could reverse PtPT induced cell death (Fig.
4b and
c). These results suggest that PtPT-induced A2780 and SKOV3 cell death is associated with caspase activation. Endoplasmic reticulum stress-associated pathways are critical in the treatment of Ovarian Carcinoma [
41]. Previous studies have shown that agents including bortezomib, bAP-15, and auranofin that inhibited proteasome and induced Ub-proteins accumulation also induced ER-stress, which may represent a mutual pattern [
17,
18]. The current study also found that PtPT significantly induced ER-stress in EOC cells (Fig.
5c and
d), suggesting ER-stress may be involved in the PtPT-induced apoptosis.
In addition to our in vitro studies, we also estimated anticancer activity of PtPT in EOC xenografts. We found that PtPT significantly inhibits ovarian cancer growth of EOC xenografts. Importantly, compared with vehicle control, PtPT treatment has no apparent side toxicity, including the reduction of body weight which is commonly seen in CDDP treatment. These results strongly suggest that PtPT is a better anticancer reagent than CDDP and could be used in the treatment of ovarian cancers. As expected, further results confirming that PtPT inhibits the UPS in vivo via targeting proteasomal DUBs. Briefly, PtPT may be an ideal antitumor agent and could be of great value in the treatment of ovarian cancers.
Conclusion
EOC, an increasing threat to women, remains an incurable carcinoma. Novel chemicals with lower side toxicity are needed for EOC treatment. PtPT significantly inhibited USP14 and UCHL5, thereby accumulating Ub-conjugates. The Ub-conjugates accumulation led to ER stress and caspase activation, which may be the pathway by which PtPT induces apoptosis. Additionally, PtPT induced G2/M phase arrest and thereby dramatically suppressed the proliferation of EOC. In summary, we identified that PtPT, a novel chemical that selectively inhibits 19S proteasomal DUBs USP14 and UCHL5, has potent antitumor activity in EOC without obvious side effect, and could be of great clinical value in the future.
Acknowledgement
We thank Huabo Su (Augusta University) for language editing.