Pleuromutilin Inhibits Proliferation and Migration of A2780 and Caov-3 Ovarian Carcinoma Cells and Growth of Mouse A2780 Tumor Xenografts by Down-Regulation of pFAK2.

BACKGROUND Pleuromutilin is a natural tricyclic, derived from the fungus, Pleurotus mutilus. This study aimed to investigate the effects of pleuromutilin on migration and proliferation of A2780 and Caov-3 human ovarian carcinoma cells and the growth of A2780 tumor xenografts in mice and the molecular mechanisms involved. MATERIAL AND METHODS A2780 and Caov-3 human ovarian carcinoma cells were cultured with and without 40, 160, and 200 μM of pleuromutilin. The Edu fluorescence assay, the wound-healing assay, and Matrigel were used to measure A2780 and Caov-3 cell proliferation, migration, invasion, and adhesion in vitro, respectively. Western blot measured protein levels of FAK, p-FAK, MMP-2, and MMP-9. A2780 cells were injected subcutaneously into mice to determine the effects of pleuromutilin on the growth of tumor xenografts. RESULTS Pleuromutilin significantly reduced A2780 and Caov-3 cell proliferation at 48 h in a dose-dependent manner (P<0.05), and at 200 μM, pleuromutilin reduced cell proliferation by 21.43% and 23.65%, respectively. Treatment of A2780 cells with pleuromutilin significantly reduced cell migration, invasion, and adhesion and the expression of p-FAK, MMP-2, and MMP-9 compared with untreated controls. In the mouse tumor xenograft model, treatment with pleuromutilin significantly reduced tumor size compared with the untreated group and inhibited tumor metastasis to the intestine, spleen, and peritoneal cavity. CONCLUSIONS In A2780 and Caov-3 human ovarian carcinoma cells, pleuromutilin inhibited cell proliferation, migration, invasion, and adhesion in a dose-dependent manner, and reduced tumor growth and metastases in a mouse A2780 cell tumor xenograft model.

Background

Worldwide, ovarian carcinoma has a high mortality rate and is the seventh most commonly diagnosed malignancy in women [1]. The incidence of ovarian cancer has increased during the past few decades [1]. The mortality rate from ovarian cancer remains high due to the lack of early diagnosis and the lack of effective treatments for late-stage malignancy [2]. Women who present with late-stage ovarian carcinoma continue to have a poor prognosis [1]. Clinically, ovarian carcinoma is divided into several subtypes and high-grade and low-grade carcinoma based on tumor histopathology [3].

Ovarian carcinoma commonly metastasizes to the peritoneal cavity and results in 5-year survival of less than 30% [4]. Worldwide, ovarian carcinoma has the highest patient mortality rate of all gynecological malignancy [3]. Advances in treatment, including chemotherapy, has resulted in the development of effective treatment approaches based on targeted molecular therapy [5-7]. There have been recent developments in targeted therapies and immunotherapy in ovarian carcinoma [8]. Recent studies have investigated the role of bevacizumab and nivolumab in ovarian carcinoma, with the aim of developing effective treatment [9,10].

Pleuromutilin is a natural tricyclic, derived from the fungus, Pleurotus mutilus. Pleuromutilin was first isolated in 1951 and consisted of a tricyclic 5-6-8 member diterpenoid structure [11]. Pleuromutilin has shown significant activity against Gram-positive bacteria in vitro, but these findings have not been supported clinically [12]. Mechanistic studies have shown that pleuromutilin interacts with the ribosomes of prokaryotic organisms and inhibit the synthesis of proteins [13,14]. Changes in the structure of pleuromutilin by chemical modification has been undertaken to develop C-14 acyloxy-derived compounds (Figure 1) [15,16]. The modification at C-14 of pleuromutilin has resulted in the development of an oral antibiotic, tiamulin, which is currently used in veterinary practice [17,18]. However, there have been no previous studies on the effects of pleuromutilin on human malignant cells in vitro.

Figure 1

The chemical structure of pleuromutilin.

Therefore, this study aimed to investigate the effects of pleuromutilin on migration and proliferation of A2780 and Caov-3 human ovarian carcinoma cells and the growth of A2780 tumor xenografts in mice and the molecular mechanisms involved.

Material and Methods

Cell culture

A2780 and Caov-3 cells were obtained from the Cell Bank, Chinese Academy of Sciences, Shanghai, China. The cells were cultured in RPMI-1640 (Gibco, Thermofisher Scientific, Waltham, MA, USA) medium containing 10% fetal bovine serum (Gibco, Thermofisher Scientific, Waltham, MA, USA) at a temperature of 37°C under an atmosphere of 5% CO2.

The Edu fluorescence cell proliferation assay

A2780 and Caov-3 cell proliferation after 48 of treatment with pleuromutilin was determined using Edu proliferation assay in accordance with the manufacturer’s instructions. The cells were distributed at 3×104 cells/well density in 96-well plates and cultured for 24 h. The medium was changed by fresh medium mixed with 10, 20, 40, 80, 160, and 200 μM of pleuromutilin, and incubation was performed for 48 h. Cell proliferation was determined using an Edu proliferation assay kit (Guangzhou RiboBio Co., Ltd., Guangzhou, China). An Olympus IX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan) was used to observe the stained cells.

Cell cycle and apoptosis analysis

Flow cytometry was used for the analysis of apoptosis in A2780 and Caov-3 cells. Briefly, the cells were put into 6-well plates at 2×105 cells/well density and exposed for 48 h to 40, 160, and 200 μM pleuromutilin. The cells in log phase were harvested by centrifugation and subsequently washed two times with ice-cold PBS. Then the cells were resuspended in PBS followed by fixing with cold 75% ethyl alcohol at 4°C overnight. The cells were stained with Annexin-V/propidium iodide (PI) and examined using a BD FACSVantage SE flow cytometer (Becton-Dickinson, San Jose, CA, USA) was used to identify cell apoptosis.

Cell adhesion assay

Matrigel (BD Biosciences, Shanghai, China) coated 96-well culture plates were treated for 1.5 h with 1% bovine serum albumin (BSA) at room temperature to block the non-specific binding sites. A2780 and Caov-3 cells were incubated for 48 h with 40, 160, and 200 μM of pleuromutilin. The cells at 3×104 density were cultured in the plates at 37°C and allowed to adhere for 2 h to the Matrigel-coated wells. The cells which did not adhere to the wells were removed by washing the plates with cold PBS three times. The cell adhesion was determined by comparing the adherent cell population in pleuromutilin treated plates with the control.

Analysis of cell invasion

The invasion of A2780 cells was determined using a Boyden chamber. The cells at a density of 3×104 cells/ml were collected by trypsinization and then resuspended in culture medium without serum. The cells at 2×105 cells/ml concentration were then put into the upper compartment of the Boyden chamber. Pleuromutilin at increasing concentrations was added to the inner well of the Boyden chamber pre-coated with 45 μl of Matrigel (at dilution of 1: 6 in serum-free medium). The lower chamber contained RPMI-1640 medium mixed with 20% fetal bovine serum (FBC). The invasion potential of the cells into the Matrigel was assessed following 48 h of incubation at 37°C. The invaded cells were fixed with methyl alcohol and stained using hematoxylin and eosin (H&E). Cotton swabs removed the non-invaded cells in the upper chamber. Cell invasion was determined using light microscopy.

Cell migration assay

The migration potential of A2780 cells after treatment with pleuromutilin was assessed using the wound-healing assay. The cells were cultured in the six-well plate at a density of 2×105 cells/well, and incubated with 40, 160, and 200 μM of pleuromutilin for 48 h. The monolayer of cells was scratched using a plastic cell scraper, followed washing three times with PBS to remove the non-adherent cells. The adhered cells were incubated for 48 h in a humid atmosphere of 5% CO2 at 37°C. Cell migration was determined by assessing the cell counts in the scratched zone.

Western blot

A2780 cells were treated with 40, 160, and 200 μM of pleuromutilin for 48 h, and then harvested after washing in cold PBS. The cells were treated with RIPA lysis buffer (Roche, Shanghai, China) for 45 min on ice. The lysate was centrifuged at 4°C at 12,000 rpm for 15 min to isolate the supernatant. The concentration of proteins in the supernatant was determined by the Bradford method. The proteins were transferred onto the membrane (Merck Millipore, Burlington, MA, USA) and separated on an 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Non-specific binding sites were blocked by a 2 h incubation with 10% dried skimmed milk powder in TBST at room temperature. Incubation of the membranes was performed overnight at 4°C with primary antibodies to p-FAK, MMP-2, FAK, MMP-9, and β-actin (Cell Signaling Technology, Shanghai, China). After washing the membranes three times with TBST, the secondary antibodies were applied, and incubation was performed at room temperature for 1 h. The protein expression levels were detected using an electrochemiluminescence (ECL) kit (Merck Millipore, Burlington, MA, USA) and the bands were visualized using autoradiography.

The mouse A2780 cell tumor xenograft model

Sixty 6-week-old female nude mice were supplied by the Centre for Experimental Animals, Wuhan University, Wuhan, China. The mice were housed in sterilized, air-filtered, pathogen-free cages in the animal center. The temperature was maintained at 23±2°C with a humidity of 65%. The mice had access to water and food ad libitum and were maintained in a 12-hour light and dark cycles. The mice were divided randomly into the untreated control group, and groups treated with 50, 100, 150, and 200 mg/kg of pleuromutilin. The mice were inoculated with A2780 human ovarian carcinoma cells at a cell density of 2×106 cells in 100 μl of PBS on the dorsal side of the body under anesthesia. The solid tumors that formed in the mice were sliced into 1 mm3 thin sections, which were implanted in the ovarian capsule of the mice using laparotomy under anesthesia. The incision in the abdomen was sutured with 3-0 silk after returning the ovary to its original position. On the second day of tumor implantation, the mice were treated with 50, 100, 150, and 200 mg/kg doses of pleuromutilin through the intraperitoneal route. The mice were euthanized on day 30 after tumor implantation to excise the ovarian tumors, and the volume was measured using calipers.

Statistical analysis

Data were presented as the mean±standard deviation (SD). Student’s t-test and two-way analysis of variance (ANOVA) were used. Data were analyzed using GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA, USA). A P-value <0.05 was considered to be statistically significant.

Results

Pleuromutilin inhibited A2780 and Caov-3 cell growth

Pleuromutilin treatment significantly (P<0.05) suppressed the proliferation of A2780 and Caov-3 cells in a dose-dependent manner (Figure 2). The different concentrations of pleuromutilin tested against A2780 and Caov-3 cells ranged from 0–200 μM (10, 20, 40, 80, 160, and 200 μM). Pleuromutilin showed an IC50 of 40 μM against A2780 and Caov-3 human ovarian carcinoma cells. The suppression of A2780 and Caov-3 cell proliferation was maximum at 48 h of pleuromutilin treatment in the range of 20–200 μM. At 200 μM of pleuromutilin, the proliferation of A2780 and Caov-3 cells was reduced to 21.43 and 23.65%, respectively.

Figure 2

A2780 and Caov-3 human ovarian carcinoma cell proliferation were reduced by pleuromutilin. The cells were treated with 0–200 μM concentrations of pleuromutilin. The Edu proliferation assay was used to evaluate cell proliferation and cell cytotoxicity. * P<0.05, ** P<0.02 and *** P<0.01 vs. the untreated cells.

Pleuromutilin induced apoptosis in A2780 and Caov-3 cells

Exposure of A2780 and Caov-3 cells to pleuromutilin for 48 h significantly increased apoptosis (Figure 3). The apoptosis induction in A2780 and Caov-3 cells was significant from 40 μM pleuromutilin.

Figure 3

The effect of pleuromutilin on A2780 and Caov-3 human ovarian carcinoma cell apoptosis. The cells after treatment with pleuromutilin at different concentrations were analyzed by flow cytometry. Cell apoptosis was quantified. * P<0.05 and ** P<0.01 vs. untreated cells.

Pleuromutilin resulted in A2780 and Caov-3 cell cycle arrest

Pleuromutilin treatment significantly increased the A2780 and Caov-3 cell populations in the G1 phase of the cell cycle when compared with the control (Figure 4). However, the percentage of A2780 and Caov-3 cells in the S-phase and G2/M phase was significantly reduced following treatment with pleuromutilin for 48 h.

Figure 4

The effect of pleuromutilin on A2780 and Caov-3 human ovarian carcinoma cell cycle progression. The cells were treated with increasing concentrations of pleuromutilin and then analyzed by flow cytometry.

Pleuromutilin reduced A2780 and Caov-3 cell adhesion

Treatment of A2780 and Caov-3 cells with pleuromutilin for 48 h caused a significant reduction in cell adhesion in a dose-dependent manner (Figure 5). A2780 and Caov-3 cell adhesion was suppressed by 18% and 23%, respectively, on treatment with 200 μM of pleuromutilin when compared with the untreated cells.

Figure 5

The effect of pleuromutilin on A2780 and Caov-3 human ovarian carcinoma cell adhesion. (A) The cells after treatment with pleuromutilin at increasing concentrations were analyzed for adhesion. Magnification ×200. (B) The population of cell adhesion was quantified. * P<0.05, ** P<0.02 and *** P<0.01 vs. the untreated cells.

Pleuromutilin reduced A2780 cell invasion

The cell invasion assay showed a significant (P<0.02) reduction in A2780 cell invasion by treatment with pleuromutilin (Figure 6). An ncrease in pleuromutilin concentration from 40 to 200 μM resulted in reduced A2780 cell invasion compared with the untreated cells.

Figure 6

The effect of pleuromutilin on A2780 human ovarian carcinoma cell invasion. (A) The cells treated with pleuromutilin for 48 h were examined for invasion by the transwell assay. Magnification ×200. (B) The results were quantified. *P<0.05 and **P<0.02 vs. the untreated cells.

Pleuromutilin reduced A2780 cell migration

The migration of A2780 cells was suppressed significantly on treatment with pleuromutilin (Figure 7). The wound-healing assay showed that pleuromutilin treatment at 200 μM almost completely inhibited A2780 cell migration compared with the untreated cells.

Figure 7

The effect of pleuromutilin on A2780 human ovarian carcinoma cell migration. (A) The cells were treated with increasing concentrations of pleuromutilin and examined by phase-contrast microscopy. Images were taken at a magnification of ×200. (B) Cell migration was quantified. * P<0.05 and ** P<0.02 vs. the untreated cells.

Regulation of p-FAK, MMP-2 and MMP-9 expression by pleuromutilin

In A2780 cells, pleuromutilin treatment decreased the expression of p-FAK in a dose-dependent manner (Figure 8). The reduction of p-FAK expression was significant from 20 μM of pleuromutilin at 48 h. The expression of MMP-2 and MMP-9 were also suppressed in pleuromutilin treated A2780 cells compared with the control. Although the reduction of MMP-2 and MMP-9 expression by pleuromutilin was significant from 20 μM, but the effect was maximum at 200 μM.

Figure 8

The effect of pleuromutilin on p-FAK and matrix metalloproteinases in A2780 human ovarian carcinoma cells. (A) The expression of p-FAK, MMP-2, and MMP-9 in A2780 cells at 48 h of pleuromutilin treatment assessed by Western blot. (B) Relative expression of p-FAK, MMP-2, and MMP-9 in A2780 cells. The level of β-actin used as control. * P<0.05 and ** P<0.01 vs. the untreated cells.

Inhibition of in vivo tumor metastasis by pleuromutilin

In the mouse ovarian tumor xenograft model treated with pleuromutilin, the tumor size was significantly less when compared with the untreated group (Figure 9). The tumor size in untreated, 50 mg/kg, 100 mg/kg, 150 mg/kg and 100 mg/kg pleuromutilin treatment groups was 1621.5±400.6, 1543.8±360.3, 1476.2±305.7, 1225.6±280.5 and 814.9±190.6 mm3, respectively. In the untreated mouse group, ovarian xenograft tumor metastasis occurred to the intestine, spleen, and peritoneal cavity. Treatment of the mouse xenograft tumor model with pleuromutilin inhibited metastases to the intestine, spleen, and peritoneal cavity. All the mice in the 200 mg/kg pleuromutilin treatment group survived during the study. however, the survival rate of mice in the treated, 50 mg/kg, 100 mg/kg and 150 mg/kg treatment groups were 60%, 80%, 90%, and 100%, respectively.

Figure 9

The effect of pleuromutilin on A2780 mouse xenograft tumor growth and metastasis. The mice were inoculated with A2780 cells and treated with increasing doses of pleuromutilin. The volume of tumors excised from the mice was measured on day 31 after A2780 cell inoculation. * P<0.05 and ** P<0.02 vs. the untreated mice.

Discussion

The aim of this study was to investigate the effects of pleuromutilin on migration and proliferation of A2780 and Caov-3 human ovarian carcinoma cells and the growth of A2780 tumor xenografts in mice. The findings showed that pleuromutilin significantly reduced ovarian cancer cell proliferation in vitro by decreasing the proliferation, metastasis, and adhesion through the down-regulation of FAK activation and MMP-2/9 expression. A2780 tumor xenograft growth and metastasis were also inhibited by pleuromutilin treatment in mice.

Tumor cell adhesion, metastasis, and angiogenesis play a vital role in the development and progression of malignancy [19,20]. Detached tumor cells adhere to sites of metastasis by receptors present in the cell membranes [19,20]. The high rate of proliferation of these cells is the basis of the pathogenesis of cancer [19]. In the present study, pleuromutilin treatment significantly reduced A2780 and Caov-3 cell proliferation in a dose-dependent manner. Treatment with pleuromutilin significantly suppressed A2780 and Caov-3 cell adhesion when compared with the untreated cells. The migration and invasion of A2780 cells treated with pleuromutilin were significantly reduced compared with the control cells. These findings suggested that pleuromutilin had tumor suppressor effects in A2780 human ovarian carcinoma cells in vitro.

Carcinoma cell metastasis and adhesion is regulated by several pathways through the expression of molecules that include MMPs, TIMPs, and FAK [21,22]. FAK has a vital role in the regulation of interactions between cells and the extracellular matrix (ECM) [20,21]. Phosphorylation of FAK promotes the migration of malignant cells, increases cell proliferation, and cell survival [22,23]. The results from the present study showed that pleuromutilin had an inhibitory effect on the phosphorylation of FAK in A2780 cells. These findings suggest that pleuromutilin suppressed the metastatic potential of A2780 cells by targeting FAK activation.

Carcinoma cell metastasis through the basement membrane and extracellular matrix (ECM) is increased by the overexpression of matrix metalloproteinases (MMPs) [19,21,24]. Previous studies have shown a direct relationship between the expression of MMPs and carcinoma cell metastasis [19,25,26]. In the present study, pleuromutilin treatment significantly suppressed the expression of MMP-2/9 in A2780 cells at 48 h. Therefore, pleuromutilin treatment inhibited the proliferation and metastasis of A2780 cells by targeting MMP overexpression. Ovarian carcinoma metastasizes to distant organs that include the lungs [27], pancreas [28], peritoneum [29] and hepatic tissues [30]. The present study investigated the effect of pleuromutilin on the development of mouse A2780 tumor xenografts and metastasis in vivo. The results showed that pleuromutilin treatment of mouse tumor xenografts inhibited tumor growth and metastasis in a dose-dependent manner. The in vivo effects of pleuromutilin in this study were not associated with toxicity.

Conclusions

This study aimed to investigate the effects of pleuromutilin on migration and proliferation of A2780 and Caov-3 human ovarian carcinoma cells and the growth of A2780 tumor xenografts in mice and the molecular mechanisms involved. In A2780 and Caov-3 human ovarian carcinoma cells, pleuromutilin inhibited cell proliferation, migration, invasion, and adhesion in a dose-dependent manner, and reduced tumor growth and metastases in a mouse A2780 cell tumor xenograft model.