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Combination of Sodium Cantharidinate with Cisplatin Synergistically Hampers Growth of Cervical Cancer

Background Sodium cantharidinate (SC) has been broadly applied in lung cancer treatment in China, while its specific function in cervical cancer (CC), a great contributor to death of female reproductive system cancers, remains unclear. Our research evaluated the anti-tumor effects of SC in CC and the mechanism involved. Methods First, cisplatin (DDP)-resistant Caski-1 and ME180 cell lines were developed and treated with SC. The effects of SC on CC cell growth were then evaluated. Subsequently, the genes targeted by SC were predicted via the bioinformatics website. The correlations between PTPN1 expression and tumor stage, lymph node metastasis and tumor differentiation were examined. We further conducted rescue experiments by overexpressing PTPN1 in CC cells, followed by SC and cisplatin treatments. The activation of the PI3K/AKT pathway in CC cells, and the effect of SC on the growth and drug resistance of Caski-1 cells in vivo were investigated. Results The sensitivity of Caski-1 and ME180 cells to DDP was increased after SC treatment, which also enhanced the inhibitory effect of DDP on the cell growth. By prediction, we found that SC could target PTPN1. Patients with high expression of PTPN1 had higher clinical stage, lymph node metastasis and lower tumor differentiation. SC inhibited PTPN1 expression. Overexpression of PTPN1 attenuated the effect of SC. Furthermore, PTPN1 activated the PI3K/AKT pathway. Moreover, SC treatment inhibited the growth and drug resistance of Caski-1 cells in vivo. Conclusion SC promotes drug sensitivity of CC cells to DDP by targeting PTPN1, thereby impairing the PI3K/AKT pathway.


Introduction
According to global cancer statistics in 2018, cervical cancer (CC) ranks the fourth place for both incidence (3.2% for all cases) and mortality (3.3% for all cases). 1 Despite progresses in screening, vaccination, and management of CC at early stage, a proportion of patients are diagnosed at advanced stage, or with recurrent CC, for whom systemic chemotherapy represents the cornerstone of treatment regimens. 2 As one of the most powerful anticancer drugs, cisplatin (DDP) is a small-molecule platinum that was accidentally exposed to constrain the growth of Escherichia coli and later was discovered to kill tumor cells. 3 However, resistance to DDP may develop, which considerably compromised the effectiveness of DDP to treat advanced or recurrent CC. 4 As a consequence, exploring the theory and mechanism behind the chemosensitivity of CC cells to DDP is an urgency.
Cantharidin is a terpenoid extracted from Chinese blister beetles that illustrates substantial toxicity against cancer cells, and its pharmacological function includes the suppression of xanthine oxides. 5 Cantharidin, the inhibitor of protein phosphatases type 1 and type 2A, has been exhibited to alter cell cycles, including enhancing protein synthesis by facilitating into S phase and cell apoptosis. 6 For instance, sodium cantharidinate (SC) has the potency to induce HepG2 cell apoptosis via the LC3 autophagy pathway. 7 Nevertheless, no study exists regarding the role of SC in CC to date. Protein-tyrosine phosphatases (PTPs) eliminate phosphate groups from tyrosine residues, thus promoting or repressing signal transduction to impact cellular processes such as cell proliferation. 8 Moreover, the significance of PTPs has been underscored in the mediation of human health, and PTP dysfunction is causal to various human diseases, including cancers. 9 Among them, PTP nonreceptor type 1 (PTPN1, also known as PTP1B) has been revealed to be overexpressed in esophageal carcinoma, which was closely associated with incidence. 10 PTPN1 has been reported to be strongly associated with the development of various tumors, such as non-small cell lung cancer, 11 breast cancer 12 and glioma. 13 However, it has not been intensively studied in CC. Furthermore, it has been noted that overexpression of PTPN1 was associated with Erlotinib resistance in lung adenocarcinoma. 14 Therefore, we wondered if PTPN1 was similarly associated with drug resistance in CC. The present study investigated whether PTPN1 also exerts important functions in CC and whether SC may be used to inhibit the expression of PTPN1. The protein and mRNA expression of PTPN1 in the CC cell lines (Caski-1 and ME180) were examined using Western blot and reverse transcription-quantitative polymerase chain reaction analysis (RT-qPCR), respectively. The function of SC in inducing the chemosensitivity of CC cells to DDP was also probed. This research may provide attracting targets for the treatment of CC and further improve the outcomes of patient with CC.

Clinical Sample Collection
The tissue collection and analyses were approved by the Ethics Committee of The First Affiliated Hospital of Anhui Medical University, and all the participants provided written informed consent. The studies were performed as per the Declaration of Helsinki. A total of 59 pairs of CC tissues and adjacent tissues were harvested from January 2016 to January 2019 in The First Affiliated Hospital of Anhui Medical University. All patients had not received radiotherapy or chemotherapy before surgery. Samples from patients with other major diseases were excluded. Samples were selected based on the diagnosis of CC determined by at least two pathologists. The clinical characteristics of the patients were obtained from medical records. Clinical staging and clinicopathological classification were determined in accordance with the International Federation of Gynecology and Obstetrics (FIGO). Tissues were collected at the time of surgery and immediately frozen in liquid nitrogen until use.

Establishment of DDP-Resistant Cell Lines
Caski-1 (#CDC0111, China Center for Type Culture Collection, Wuhan, Hubei, China) and ME180 cells (#HTB-33, American Type Culture Collection, Manassas, VA, USA) resistant to DDP were established by sustained exposure to DDP (#P4394, Sigma-Aldrich Chemical Company, St Louis, MO, USA). Briefly, Caski-1 and ME180 cells were exposed to increasing DDP concentrations for 48 hours, once a week. The initial DDP dose was 1.52 μM, which was the 80% inhibition concentration (IC80) for the sensitive cell lines. Once the cells recovered and grew in the exponential growth phase, the DDP concentration gradually increased to as high as 15.20 µM. After 6 cycles of DDP treatment for 6 months, the resulting resistant lines Caski-1/R and ME180/R were subcultured and treated monthly with 15.20 μM DDP to maintain chemoresistance. Caski-1/R and ME180/R cells were cultured without DDP for approximately 14 d prior to subsequent experiments.

Cell Survival Assay
Cells were plated at 5 × 10 3 cells/well in 96-well microtiter plates and allowed to adhere overnight. Then, a gradient concentration of DDP (0.001-100 nmol/mL) was added and incubated for another 72 h. Cell viability was tested using the CellTiter-Glo ® (CTG) assay. Each value was normalized to cells treated with dimethylsulfoxide, and IC50 values were calculated using Graphpad Prism software.

Colony Formation Assay
Cells were plated into 12-well plates at 1 × 10 3 cells/well. The next day, the cells were incubated with added drugs for 12 d. The medium was renewed every 3 days. Afterward, the cells were fixed with methanol and stained with 0.1% crystal violet (both for 10 min at room temperature) and photographed.

Flow Cytometry
The percentage of apoptosis was determined using the Annexin V/propidium iodide (PI) kit (#C1062, Beyotime Biotechnology Co., Ltd., Shanghai, China). Cells were resuspended in 1 × binding buffer. The cell suspension (1 × 10 5 cells in 100 μL) was transferred to a 5-mL culture tube and mixed with 5 μL Annexin V-fluorescein isothiocyanate (FITC) and 5 μL PI. The mixture was gently vortexed and incubated at 25°C in darkness for 15 min. Then, 400 μL 1 × binding buffer was loaded to each tube. Samples were analyzed by a flow cytometer within 1 h. The green fluorescence of Annexin V-FITC was measured at 530 nm, and red fluorescence of PI was measured at 585 nm. The results were analyzed using FlowJo software (version 7.6.1, BD Biosciences, San Jose, CA, USA).
Following fixation with 4% paraformaldehyde for 0.5 h at ambient temperature, the cells were stained with Hoechst 33258 (5 µg/mL) for 20 min at 37°C in darkness. Images of apoptotic cells exhibiting Hoechst 33258 fluorescence were taken under a fluorescence microscope (Nikon Instruments Inc., Melville, NY, USA). The percentage of apoptotic cells was semi-quantified based on Image-Pro Plus software 6.0 (Media Cybernetics, Bethesda, MD, USA).

Lactate Dehydrogenase (LDH) Release Assay
Parental or drug-resistant Caski-1 and ME180 cells (1.5 × 10 5 cell/well) were cultured overnight in 24-well cell culture plates. The supernatant was collected and added into a 96-well black culture plate (200 μL/well). LDH release levels were measured using the LDH cytotoxicity assay kit (#C0016, Beyotime) as per the manufacturer's instructions. The optical density (OD) value at 450 nm in each well was read using a microplate reader.

RT-qPCR
After transfection, cells or spheres were harvested and RNA was isolated using TRIZOL reagent (Sigma-Aldrich). RNA (10 ng) was used for reverse transcription with the TaqMan RNA RT kit (Applied Biosystems, Life Technologies, Madison, WI, USA). PCR was performed in a quadruple reaction in Rotor-Gene Corbett Q 6000 PCR. Data were collected and analyzed using Rotor-Gene quantitation software. RT-PCR primers (Cat no 204337) were purchased from Exiqon (Woburn, MA, USA).

Xenograft Assay in Nude Mice
All animal experiments were approved by the Research Animal Resource Center of The First Affiliated Hospital of Anhui Medical University. All animal procedures were implemented according to the NIH guidelines. Caski-1 and Caski-1/R cells (3 × 10 6 cells/mL) were injected subcutaneously into the axilla of 5-week-old female nude mice (n = 6, 20-22g, Hunan SJA Laboratory Animal Co., Ltd., Changsha, Hunan, China). The mice were treated by gavage with SC or PBS at a dose of 100 mg/kg per day. Meanwhile, the mice were also treated with DDP every 5 days. The volume of tumors formed by Caski-1 cells was calculated every five days starting from the fifth day after cell implantation and determined by the formula volume = (length × width 2 )/2. All procedures were performed under intravenous anesthesia with pentobarbital sodium at a concentration of 100 mg/kg, and every effort was made to minimize the suffering of mice.

TUNEL Assay
The percentage of apoptosis in tissues was measured using the TUNEL kit (#G7360, Promega). Tissue sections were waxed and rehydrated. Following antigen retrieval with proteinase K working solution and permeabilization, the slides were incubated at 37°C for 2 h with a mixture of TdT and dUTP (1:9). Then, the activity of endogenous peroxidase was inactivated. The tissue was covered with reagent 3 (converter-peroxidase), reacted with DAB color development reagent and counterstained with hematoxylin for 3 min. Finally, the tissues were continuously dehydrated in 70%, 80%, 95%, and 100% ethanol, cleared with xylene, followed by resin mounting. Nuclei stained with hematoxylin were in blue, and positive cells were in brownish yellow. Viewed under a microscope, the tissues were analyzed using Image-Pro Plus 6.0 (Media Cybernetics).

Statistical Analysis
All the experiments were performed with at least three repetitions. The data were analyzed with SPSS 20.0 (IBM Corp. Armonk, N.Y., USA) and GraphPad Prism Software 8.0 (GraphPad Software, San Diego, CA, USA). Statistical analysis was performed utilizing the one-way or two-way ANOVA test combined with Tukey's test or t-test. Data are displayed as mean ± standard deviation (SD). Differences between means were considered significant for p < 0.05.

DDP-Resistant CC Cell Lines are Successfully Developed
We first used gradient concentrations of DDP to treat Caski-1 and ME180 cells for the development of DDP-resistant CC cell lines (Caski-1/R and ME180/R). To determine the success of resistant cell line construction, we examined the survival of parental and resistant Caski-1 and ME180 cells at various concentrations of DDP using the CTG kit, and we observed that Caski-1/R and ME180/R cells had significantly higher IC50 values for DDP ( Figure 1A). A dose of 10 μM DDP was used to treat parental and drug-resistant cells for 6 h. Apoptosis rate and activity were measured using flow cytometry and EdU staining. We found that the apoptosis rate of parental cells was significantly higher and the number of proliferating cells was significantly reduced after 10 μM DDP treatment compared to drug-resistant cells ( Figure 1B and C). Moreover, we further used immunofluorescence staining to detect the expression of stem cell markers CD133, CD44, and SOX2 in the cells. The expression of stem cell markers was significantly increased in drugresistant cells ( Figure 1D). Moreover, the size of the tumor spheres formed by DDP-resistant cells was also significantly larger than that of the parental cells ( Figure 1E). The above results indicate that DDP-resistant CC cell lines were successfully constructed and that the resistant cells exhibited stem cell properties.

SC Inhibits the Growth of CC Cells
SC was found to cause apoptosis in hepatocellular carcinoma cells in a previous study. 7 We anticipated that SC may have a similar role in CC cells. Thus, we treated parental and DDP-resistant Caski-1 and ME180 cells with different concentrations of SC, and assayed cell activity using the CTG kit. It has been revealed that as the concentration of SC increased, the activity of Caski-1 and ME180 cells decreased significantly (Figure 2A and B), and the apoptosis rate increased significantly ( Figure 2C). We subsequently used immunofluorescence to assess the changes in the expression of stem cell markers in resistant cells in the presence of SC. SC significantly inhibited the expression of CD133, CD44, and SOX2 in the cells ( Figure 2D). Sphere formation assay also showed a significant decline in the size and number of spheres formed by Caski-1/R and ME180/R cells after SC treatment ( Figure 2E).

SC Promotes Sensitivity of CC Cells to DDP
Now that we established that SC could inhibit the growth of CC cells and the stem cell properties of drug-resistant cells, we hypothesized that SC treatment promotes drug sensitivity of cells to DDP. To validate our conjecture, we treated parental and resistant Caski-1 and ME180 cells with 50 mM SC and with a gradient concentration of DDP for the IC50 value assessment. We were surprised to find that the IC50 values of Caski-1 and ME180 parental and resistant cells to DDP were significantly reduced after SC treatment ( Figure 3A). Moreover, our treatment of Caski-1 and ME180 cells with SC significantly augmented the inhibitory effect of DDP on the growth of both parental and drug-resistant cells ( Figure 3B and C), and the proportion of apoptosis was significantly higher ( Figure 3D and E). We further examined the release of LDH from Caski-1 and ME180 cells, where SC treatment significantly promoted the release of LDH from both parental and drug-resistant cells after DDP treatment ( Figure 3F), and the activity of caspase-3 was similarly increased ( Figure 3G).

SC Promotes Drug Sensitivity of CC Cells to DDP via Inhibition of PTPN1
To clarify the molecular mechanism of SC, we used the SWISS Prediction (http://www.swisstargetprediction.ch/) website to predict the target of SC, and we noted that the targets of SC are distributed in the phosphatase class ( Figure 4A and B). We found that SC can target PTPN1 (Supplementary Table 1). Thus, we determined PTPN1 expression in Caski-1 and ME180 cells and found that the expression of PTPN1 was significantly increased in DDP-resistant cells, but had a significant decrease after further treatment with SC ( Figure 4C and D). Moreover, we further analyzed the correlation between PTPN1 expression and the clinical stage, lymph node metastasis, and tumor differentiation of the 59 CC patients and found that patients with overexpression of PTPN1 had higher clinical stage and higher risks for lymph node metastasis, and the poorer tumor differentiation. However, PTPN1 expression was independent of the age and tumor size of patients ( Table 1).

Overexpression of PTPN1 Reverses the Enhancer Effect of SC on DDP Sensitivity of CC Cells
To further validate that SC promotes DDP drug sensitivity in CC cells through targeting PTPN1, we transfected PTPN1 overexpression plasmids in parental and drug-resistant Caski-1 and ME180 cells and verified successful transfection by RT-qPCR and Western blot ( Figure 5A and B). We found  Figure 5C). Besides, the inhibition of cell growth by DDP and SC ( Figure 5D and E) was significantly alleviated, and the percentage of apoptosis after DDP and SC treatments was also inhibited ( Figure 5F). Similarly, the release of LDH from cells as well as the activity of caspase-3 was significantly inhibited following PTPN1 upregulation ( Figure 5G-I).

PTPN1 Activates the PI3K/AKT Pathway in CC Cells
In a study by Jin et al, it was noted that PTPN1 promotes the malignant biological behavior of gliomas through activation of the PI3K/AKT pathway. 15 Thus, we examined the extents of PI3K and AKT phosphorylation in Caski-1 and ME180 cells by Western blot. The extents of PI3K and AKT phosphorylation were significantly increased in DDP-resistant cells but were significantly decreased after further treatment with SC. In addition, overexpression of PTPN1 partially counteracted the inhibitory effect of SC on the extents of PI3K and AKT phosphorylation ( Figure 6A and B). Subsequently, we treated both parental and drug-resistant Caski-1 and ME180 cells with the PI3K/AKT-specific agonist Recilisib in combination with SC. After activation of PI3K/AKT, we found a significant increase in IC50 values for DDP in both parental and drugresistant Caski-1 and ME180 cells, suggesting that CC cells were less sensitive to DDP after activation of the PI3K/AKT signaling pathway ( Figure 6C). Moreover, the inhibitory effect of DDP on CC cell growth promoted by SC was largely counteracted by Recilisib, which mainly manifested by the potentiated apoptosis and suppressed proliferation of CC cells caused by SC treatment was significantly attenuated after activation of the PI3K/AKT signaling pathway ( Figure 6D-H).

SC Inhibits the Growth and Drug Resistance of CC Cells in vivo
To determine the effect of SC on the growth and drug resistance of CC cells in vivo, we implanted parental and resistant Caski-1 cells into the axilla of nude mice and treated the mice with SC or PBS by gavage at a dose of 100 mg/kg per day and DDP every 5 days ( Figure 7A). The volume of xenograft tumors formed by Caski-1 cells was calculated every five days starting from the fifth day after inoculation. SC treatment significantly inhibited the growth and resistance of Caski-1 cells to DDP in vivo ( Figure 7B and C). Moreover, we further used immunohistochemistry to detect the number of positive cells for KI67, PTPN1, phos-PI3K, and phos-AKT1 in tumor tissues and found that the staining intensities of KI67, PTPN1, phos-PI3K, and phos-AKT1 in tumor tissues were significantly reduced ( Figure 7D-G) and the number of TUNEL-positive cells was remarkably enhanced ( Figure 7H) after SC treatment. In summary, SC can promote the drug sensitivity of CC cells to DDP by targeting PTPN1 and inhibiting the activation of the PI3K/AKT pathway.

Discussion
DDP, a renowned chemotherapeutic drug, is widely used for management of plentiful human malignancies, involving bladder, head and neck, lung, ovarian, as well as testicular cancers. 16 A major contributor of the DPP treatment failure is drug resistance, which may ultimately affect treatment efficacy, survival and prognosis of CC patients and lead to distant metastasis and relapse. 17 In this investigation, we showed that SC treatment hampered the growth of CC cells and further reinforced the growth inhibitory effect of DDP to CC cells. Mechanistically, SC significantly repressed PTPN1 expression in CC cells,   while restoration of PTPN1 expression conferred chemoresistance to CC cell. Finally, PTPN1-mediated PI3K/AKT pathway activation was accomplished to suppress chemosensitivity in CC.
Although some reports have concentrated on the antitumor activity of SC in clinical practice in recent years, the activity of SC in CC has not been elucidated to date. For instance, SC improved tumor responses and quality of life with reduced risk of hepatotoxicity and gastrointestinal toxicity relative to chemotherapy alone in non-small-cell lung cancer. 18 Moreover, SC possesses the therapeutic ability to lower the expression of S100A3 in hepatocellular carcinoma cells and to induce cellular apoptosis. 19 In the present work, we firstly treated parental and drug resistance cells with SC and provided evidence that SC could remarkably diminish the expression patterns of cancer stem cell markers and spheres formed. Additional DDP treatment disclosed that SC is able to reduce the IC50 value of CC cells to DPP and to expedite apoptosis and LDH release. Consistently, disodium cantharidinate, which is a ramification of cantharidin, contributed to a significant elevation of caspase-3 and PARP protein expression in dendritic cells of patients with bladder carcinoma. 20 In our studies, we confirmed that SC is able to decrease the viability of DPP-resistant CC cells to enhance chemosensitivity.
Mechanistically, we revealed that PTPN1 was targeted and repressed by SC. Also, our study underlined that PTPN1 enhanced the activation of the PI3K/AKT pathway. These findings might offer an explanation for why acquired chemoresistance develops in CC. PTPN1 inhibitors could be regarded a prospective anticancer treatment, no matter as monotherapy and in coordination with other anticancer drugs. 21 For instance, PTPN1 protein expression was dramatically elevated in breast cancer tissues and PTPN1 promoted the proliferation, and suppressed the apoptosis of HER2positive and triple-negative breast cancer cells. 22 Our in vitro evidence displayed that overexpression of PTPN1 reduced the LDH release and apoptosis in the presence of SC.
Dysfunction of the PI3K pathway is frequently observed in many different types of malignancies, and the combination of the PI3K inhibitors with chemotherapy and/or other targeted therapy may be beneficial for optimizing the efficacy of therapies. 23 Amirani et al also suggested that inhibition of the PI3K/AKT pathway restored sensitivity of cancer cells to hormonal, chemical and radioactive agents. 24 In addition, the positive correlation between PTPN1 expression and the PI3K/AKT pathway activation has been corroborated in glioma. 15 By contrast, SC suppressed osteosarcoma cell proliferation and stimulated cell cycle arrest by impairing the PI3K/AKT activation. 25 In the same vein, we noted that SC reduced the extents of PI3K and AKT phosphorylation in parental and DDP-resistant CC cells, which was partially restored by PTPN1 overexpression. To further substantiate the involvement of the PI3K/AKT pathway deficit in SCmediated DDP chemosensitivity, we delivered the PI3K/ AKT pathway agonist Recilisib into CC cells treated with DDP and SC. Recilisib was found to lead to acquired chemoresistance to DDP by preventing CC cells from apoptosis. Also, in cardiomyocytes, Recilisib-activated PI3K/AKT pathway restrained apoptosis induced by miR-23a-5p overexpression. 26 More specifically, KRAS knockdown blocked the activation of PI3K/AKT pathway to inhibit epithelial-mesenchymal transition and proliferation of breast cancer cells. 27

Conclusion
In conclusion, using in vitro and in vivo models, we confirmed that SC can decrease the viability of Caski-1 and ME180 CC cells, and the DDP chemoresistance. Our studies also revealed that SC could suppress the expression of PTPN1 and block the induction of the PI3K/AKT pathway. We therefore demonstrated that SC could be regarded as a possible pharmacological agent in the treatment or prevention of CC. A potential pitfall of this work may be the lack of in vivo experiments using the ME180 cell line. Still, we believe that the existing results already demonstrated that SC can inhibit the growth and drug resistance of CC cells in vitro and in vivo.

Funding
There is no funding to report.

Disclosure
The authors report no conflicts of interest in this work.