Oxymatrine inhibits the proliferation of CaSki cells via downregulating HPV16E7 expression

  • Authors:
    • Zhijun Pei
    • Jing Zeng
    • Yan Gao
    • Fuyan Li
    • Wei Li
    • Hong Zhou
    • Yi Yang
    • Ruimin Wu
    • Yijia Chen
    • Jie Liu
  • View Affiliations

  • Published online on: May 10, 2016     https://doi.org/10.3892/or.2016.4800
  • Pages: 291-298
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Abstract

Treatment of recurrent and metastatic cervical cancer remains a challenge, especially in developing countries, which lack efficient screening programs. HPV16E7 has been reported to play an important role in the development of cervical cancer. In recent years, oxymatrine, which was traditionally used as anti-malarial agent, has been shown to inhibit tumor growth with low toxicity to normal cells. In the present study, we investigated the mechanisms underlying the antitumor effect of oxymatrine in cervical cancer. The CCK-8 assay was used to compare the proliferation of untreated and oxymatrine-treated cervical cancer CaSki cells. Flow cytometry was applied to observe the effect of oxymatrine on apoptosis and the cell cycle distribution of CaSki cells. We used qRT-PCR and western blot analysis to determine the mRNA level and protein level of HPV16E7. The HPV16E7 siRNA inhibition was also performed to confirm the effect of downregulating HPV16E7 on the proliferation in CaSki cells. Our results revealed that oxymatrine-treated cells showed time-dependent and dose-dependent inhibition of proliferation and a significant increase in apoptosis. Oxymatrine arrested CaSki cells in G0/G1 phase and S phase while decreased the cells in G2/M phase. The expression of HPV16E7 was significantly downregulated in oxymatrine-treated cells compared with control cells. Knock-down of HPV16E7 effectively inhibited the proliferation of CaSki cells. In conclusion, our data suggest that oxymatrine inhibits cervical cancer growth via downregulation of HPV16E7. Oxymatrine can be considered to be a potential preventive and therapeutic target for cervical cancer.

Introduction

Cervical cancer, which accounts for 8% of all cancer deaths, is the third most common cancer in women after breast and colorectal cancer (1,2). The human papilloma virus (HPV) is known to be an essential cause of cervical cancer, among which 60% or more are with HPV type 16 (HPV16) (3). Carcinogenesis by HPV16 is primarily attributed to the continuous expression of viral protein E6/E7. After infection of HPV, the integration of the viral E6/E7 genes occur to the genome of the cervical epithelium, and the continued expression of E6/E7 not only enhances the neoplastic progression to the cervical epithelium, but also drives the cervical cancer cells to malignant phenotype (46). There has been convincing evidence provided by previous studies that the overexpression of HPV16E7 is associated with the development of cervical cancer (3,7). Therefore, the HPV16E7 is considered to be potential therapeutic target for cervical cancer treatment.

Treatment strategies for cervical cancer today are focused on surgical operation or chemo-radiation. Although a number of chemotherapeutic drugs for treating cervical cancer can be used to control the growth of cancer and have shown certain therapeutic efficacy, the strong side-effects limit their application. Besides, only one-third of women with metastatic cervical cancer respond to chemotherapy and this response is short-lived (1,6). Therefore, to develop novel natural substances with curative selectivity for cervical cancer without showing significant toxic effect to normal cells is becoming much more necessary.

There has been a recent focus on the use of Chinese medicinal herbs to treat a number of diseases. Oxymatrine, which is a quinolizidine alkaloid extracted from the root of traditional Chinese herbal medicine Sophora japonica (Sophora flavescens Ait), has been found to possess several biological effects such as anti-inflammation, inhibiting immune reaction, anti-virus and antitumor (813). Different from the traditional chemotherapy medicine, oxymatrine has been reported to inhibit the proliferation of a number of cancer cells in vitro and also inhibited viral-induced tumor formation in mice, with little influence to some normal cells (14,15).

As far as we know, there are few studies on the application of oxymatrine in the treatment of cervical cancer (10,16). Besides, we found no report concerning the putative relationship between oxymatrine and HPV16E7 in anticancer study. In the present study, we investigated the effect of oxymatrine on cervical cancer cell line CaSki by evaluating cell proliferation, cell apoptosis, cell cycle, the mRNA and protein expression levels of HPV16E7 gene in vitro. The aim of this study was to explore the mechanisms underlying the antitumor effect of oxymatrine on cervical cancer cells and support experimental data for the application of oxymatrine in the prevention and treatment of cervical cancer.

Materials and methods

Oxymatrine

We purchased oxymatrine from Chia Tai Tianqing Pharmaceutical Group Co., Ltd. (Nanjing, China). Before the application, we tested this oxymatrine and found that its purity is >99% indicated by SDS-PAGE analysis.

Cell line and culture

CaSki cells of human cervical carcinoma (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) penicillin (100 μg/ml) and streptomycin (100 U/ml) in 5% CO2 atmosphere at 37°C. The cells were passaged every 2–4 days to keep a proper density and cells in the logarithmic growth phase were used in the experiment.

Proliferation assay

CaSki cells in logarithmic growth phase were seeded in 96-well plates with 1×105 cells each well, and cultured cells with RPMI-1640 complete medium. Twenty-four hours later, the medium was replaced with RPMI-1640 complete medium with various concentrations of oxymatrine (2, 4 and 6 mg/ml) and cultured continuously. In addition, control cells were incubated with medium only. After exposure to oxymatrine, the proliferation of CaSki cells was assessed by using Cell Counting kit-8 assay. After 24, 48 and 72 h, each well of cells were treated with 10 μl of Cell Counting kit-8 solution (Dojindo Laboratories, Kumamoto, Japan) and incubated for 1 h at 37°C. Spectrometric absorbance was measured at 450 nm by an enzyme immunoassay analyzer (Bio-Rad Laboratories, Hercules, CA, USA).

Colony formation assay

Colony formation assay was performed to observe the effect of oxymatrine on colony formation ability of CaSki cells. Cells (1×103) were plated in each well of 6-well plate and cultured with RPMI-1640 complete mediumin a humidified atmosphere with 5% CO2 at 37°C. After 2 weeks, the colonies were fixed by 4% paraformaldehyde (PFA) for 15 min and stained with Giemsa for 20 min. Cell aggregate consisting of 50 or more cells were defined as one colony. Clonies were manually counted by microscope (Olympus, Tokyo, Japan) with x40 field.

Apoptosis assay

Apoptosis assay kit (Multi Sciences, Harbin, China) was used to treat the cell samples and the procedure followed the instructions of the kit. CaSki cells were treated with various concentrations of oxymatrine (0, 2, 4 and 6 mg/ml) for 72 h, and the cells were collected, washed, successively incubated with 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI) for 15 min at room temperature. Before being subjected to flow cytometric analysis, apoptosic ratio of fixed cell sample was measured by flow cytometry (FACSCalibur™; BD Biosciences, San Jose, CA, USA).

Cell cycle analysis

CaSki cells were treated with different concentrations of oxymatrine (0, 2, 4 and 6 mg/ml) for 72 h. The cells were harvested by trypsinization, then were fixed with 70% ethanol and resuspended in 20 mg/Ml PI for 30 min. Flow cytometer (FACSCalibur™; BD Biosciences) was applied to detect DNA content. Based on the flow cytometry data, we determined the relative proportions of cells in the individual cell-cycle phase fraction.

Quantitative real-time PCR

Total RNA was extracted from CaSki cells which had been treated with 4 mg/ml of oxymatrine for 72 h or the control cells using TRIzol RNA reagent (Invitrogen, Carlsbad, CA, USA), and then was treated with DNAse I (Roche, Basel, Switzerland) to remove contaminating genomic and adenoviral DNA. MMLV reverse transcriptase kit (Promega, Madison, WI, USA) was used to convert the prepared total RNA to cDNA, which was then subjected to quantitative real-time PCR (qRT-PCR) using a SYBR-Green Master Mix kit on a Bio-Rad connect real-time PCR platform. Taq DNA polymerase was used. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the mRNA levels. The sequences of primers were as follows: HPV16E7 (157 bp), sense: 5′-ATGCATGGAGATACACCT-3′ and anti-sense: 5′-TTATGGTTTCTGAGAACA-3′; GAPDH (101 bp), sense: 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse, 5′-GCCATCACGCCACAGTTTC-3′. Data were analyzed using the 2−ΔΔCt method.

Western blot analysis

The protein was extracted from CaSki cells treated with 4 mg/ml of oxymatrine for 72 h and the control cells. The cells were lysed by lysis buffer (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40 and 50 mM Tris, pH 8.0), with the addition of 2 mM phenylmethylsulfonyl fluoride, and then centrifuged at 12,000 × g for 30 min at 4°C. Protein concentrations were determined by protein assay kit (Sigma, St. Louis, MO, USA). Equal amounts of proteins (20 μg) were boiled for 10 min, and loaded onto a 15% SDS-PAGE gel and then transferred to the polyvinylidenedifluoride (PVDF) membranes. The membranes were incubated for 1 h with blocking buffer (PBS with 5% skim milk and 0.1% Tween-20) and then with anti-HPV16E7 primary antibody (Abcam, Cambridge, MA, USA) overnight at 4°C. Secondary antibody (Abcam) was used to incubate the membranes for 1 h at room temperature. Before each step, membranes were washed 3 times by PBST. The protein bands were visualized by enhanced chemiluminescence. We used β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as the internal control.

RNA interference

The siRNA for HPV16E7 was designed and synthesized by Sangon Biotech Co., Ltd., (Shanghai, China), and the sequences were as follows (5′-3′): GCACACACGTAGACATTCGdTdT; (3′-5′): dTdTCGUGUGUGCAUCUGUAAGC. A scrambled siRNA which had no homology with human genome was also produced for use as a negative control. The siRNA duplex (50 nM) was transfected using Lipofectamine 2000 reagent (Invitrogen) following the instructions of the manufacturer. The CaSki cells were effected by HPV16E7 siRNA for 72 h before the analysis of cell proliferation and HPV16E7 expression.

Tumorigenicity assay

The ethics approval for this protocol was obtained from The Institutional Animal Care and use Committee at Hubei university of Medicine. CaSki cells treated with 4 mg/ml of oxymatrine for 72 h and control CaSki cells were injected subcutaneously into 4-week old nude mice (3×106 cells/mouse), respectively. Four weeks after the injection, mice were sacrificed by injecting excessive chloral hydrate and tumors were separated and weighed.

Statistical analysis

All experiments were performed for at least three times, and data are shown as the mean ± SD. One-way ANOVA was used to determine statistically significant differences between groups, and P<0.05 was considered statistically significant. Analyses were carried out using GraphPad Prism version 5.0 (Graphpad Software, Inc., La Jolla, CA, USA).

Results

Oxymatrine inhibits proliferation and clonogenicity of CaSki cells

CaSki cells were treated with various concentrations of oxymatrine (0, 2, 4 and 6 mg/ml) for 24, 48 and 72 h. CCK-8 assay was performed to determine the effect of oxymatrine on cell proliferation and found a time-dependent and concentration-dependent inhibition of oxymatrine to the growth of CaSki cells, compared with that in the control cells (P<0.05, n=6; Fig. 1A). Especially when the CaSki cells were treated with oxymatrine for 72 h, the inhibitory effect was much more significant than that treated for 24 h (P<0.05, n=6; Fig. 1A). Therefore, we chose the time-point of 72 h for further investigation.

The inhibitory effect of oxymatrine on CaSki cells was then confirmed by colony formation assay. The results showed that after treated with concentrations of oxymatrine (0, 2, 4 and 6 mg/ml), the colony formation ability of CaSki cells was significantly decreased in a dose-dependent manner, compared with the control group (P<0.05, n=6; Fig. 1B and C), indicating that oxymatrine is able to inhibit the growth of cervical cancer cells.

Oxymatrine induces apoptosis to CaSki cells

The effect of oxymatrine on cell death of CaSki cells was investigated by evaluating the rate of apoptosis with flow cytometry. Results showed that after treatment with 2, 4 and 6 mg/ml of oxymatrine for 72 h, the rate of CaSki apoptosis was 19.62±1.71, 31.84±2.46 and 40.94±2.39%, respectively. The apoptosis rate of the control group was 9.64±0.98%. In contrast with the control group, the apoptosis rate of oxymatrine treated groups increased significantly in a dose-dependent manner (P<0.01, n=6; Fig. 2). As the results show, oxymatrine may suppress CaSki cell proliferation by causing cell death.

Oxymatrine changes the cell cycle distribution of CaSki cells

We performed FCM analysis to evaluate the effect of oxymatrine on the cell cycle of CaSki cells. After treatment with 2, 4 and 6 mg/ml of oxymatrine for 72 h, the proportion of G0/G1 phase was 54.91±2.08, 59.35±2.17 and 65.07±3.22%, respectively, while the percentage of G0/G1 phase control group was 44.35±1.78%. Compared with that in the control group, CaSki cells of the treated group in G0/G1 phase were markedly increased in a dose-dependent manner, while the cells in G2/M phase were decreased in the same way (P<0.01, n=6; Fig. 3). Besides, we found that 6 mg/ml oxymatrine could significantly increase the number of CaSki cells in S phase, compared to the control cells (P<0.01, n=6; Fig. 3). The results demonstrate that oxymatrine arrested CaSki cells in G0/G1 phase and S phase.

Oxymatrine downregulates the mRNA and protein levels of HPV16E7 in CaSki cells

We performed qRT-PCR and western blot analysis to examine the effect of oxymatrine on the expression of HPV16E7 gene. The results showed that after treated with 4 mg/ml of oxymatrine, mRNA levels of HPV16E7 gene in the CaSki cells was significantly decreased by 64.5±2.8% (P<0.01, n=6; Fig. 4A) and protein level was prominently decreased by 52.3±3.2% (P<0.01, n=6; Fig. 4B and C), compared with the control group. These data demonstrate that oxymatrine could markedly downregulate the expression of HPV16E7 in CaSki cells.

HPV16E7 siRNA suppresses the proliferation of CaSki cells

We used HPV16E7 siRNA to knock down HPV16E7 mRNA level to investigate the effect of downregulating HPV16E7 on the proliferation of CaSki cells. Our results show that after treated with HPV16E7 siRNA, the mRNA and protein level of HPV16E7 in CaSki were reduced by 66.4±2.7 and 57.2±6.1% respectively, in contrast with the control group treated with scramble siRNA (Fig. 5A–C). After treated with HPV16E7 siRNA, the proliferation of CaSki cells was suppressed in a time-dependent manner, compared with control siRNA (P<0.05, n=6; Fig. 5D). Our data demonstrate that knocking down HPV16E7 expression could suppress the proliferation of CaSki cells.

Oxymatrine suppressed the growth of CaSki cells in vivo

CaSki cells (3×106) treated with oxymatrine or control cells were injected subcutaneously into each athymic nude mouse. Four weeks after the injection, mice were sacrificed and tumors were separated. As showed in the tumor images, all 3 mice injected with control CaSki cells formed xenografts. In contrast, 2 out of 3 mice injected with oxymatrine-treated CaSki cells formed smaller xenografts. The weight of tumors from oxymatrine treated CaSki cells was significantly lower than those from the control CaSki cells (P<0.05, n=3; Fig. 6B). The results suggest that oxymatrine could suppress tumorigenicity of cervical cancer cells in vivo.

Discussion

Oxymatrine is a major alkaloid component found in the roots of Sophora species (17). It is commonly used for the treatment of liver disorders and other diseases such as arrhythmia, eczema and skin disorders, leukopenia and bronchitis (13,1821). Some studies have also reported oxymatrine showed anticancer activity in human gastric cancer cells, pancreatic, ovarian cancer, and human breast cancer cells (14,16,22). However, to the best of our knowledge, the mechanisms of the antitumor effect of oxymatrine on cervical cancer have not yet been elucidated. In the present study, various concentrations of oxymatrine were used to treat cervical cancer cell line CaSki cells, and we found that oxymatrine treatment could induce inhibition of proliferation and apoptosis in cervical cancer CaSki cells in vitro and in vivo.

One of the main reasons for tumorigenesis is loss of control of cell proliferation, which is involved both in tumor initiation and progression (23). Therefore, we applied CCK-8 assay and found that oxymatrine could obviously inhibit cell proliferation in cervical cancer CaSki cells in a time-dependent and dose-dependent manner in vitro. Besides, our colony formation assay results also showed similar inhibition effect of oxymatrine on the colony forming ability of CaSki cells in a dose-dependent manner. In vivo, we performed analysis of tumorigenesis and found that the weight of tumors resulted from CaSki cells treated with oxymatrine were significantly reduced, compared with the tumors derived from control CaSki cells. These data indicate that oxymatrine can suppress the growth of cervical cancer cells in vitro and in vivo.

Apoptosis is the process of cell death, with a series of cellular and molecular changes, such as phosphatidylserine externalization, chromatin condensation and cell shrinkage. Apoptosis plays important functions in organ development, homeostasis and immune defense. Uncontrolled cell death is another main reason for tumorigenesis (24,25). Therefore, inducing cell apoptosis of tumor cells is always of great importance in developing anticancer treating methods. In the present study, flow cytometry was applied to the effect of oxymatrine on the apotosis of CaSki cells and the results demonstrated that CaSki cells could be induced to apotosis by oxymatrine in a dose-dependent manner.

Since the change of cell proliferation and apotosis is intimately correlated with the change of the cell cycle (23,24). In this study, in order to investigate the cell cycle related mechanism of the effect of oxymatrine on the proliferation and apotosis on CaSki cells, we also performed flow cytometry and found that the oxymatrine treated CaSki cells were significantly blocked in G0/G1 phase. After treated with increasing concentration of oxymatrine, CaSki cells in G0/G1 phase were prominently increased while cells in the G2/M phase were obviously decreased, in contrast with control CaSki cells. Moreover, we found that oxymatrine could increase the number of CaSki cells in S phase to a certain extent, and 6 mg/ml made a significant difference compared to the control cells. When arrested in G0/G1 and S phase, the CaSki cells tend to decrease the proliferation rate with more apoptosis, which is also in accordance with our proliferation and apoptosis assay data. These data suggest that the growth inhibition and apoptosis induction of CaSki cells may result from the effect of oxymatrine on arresting CaSki cells in G0/G1 phase.

According to previous studies, over 60% of all cervical cancers are closely correlated with HPV16, which encodes the essential genes for virus replication, E6 and E7 oncogenes (7). The viral protein E7 can cause destabilization and the disruption of Rb/E2F complexes, which is essential for driving the process of cell cycle into S phase, and upregulating the anti-apoptotic protein Bcl-2 to promote cell survival and proliferation (3,26). Several researches have reported that inhibition of the expression of E7 oncogene was efficient in HPV-associated cancer therapy (15,27,28). It is reported by previous studies that oxymatrine showed anti-hepatitis virus effects (2931). Although no report on the anti-HPV effect was found, our results demonstrated that oxymatrine could decrease both the mRNA and protein levels of HPV16E7. Since the expression of HPV16E7 was closely correlated with the proliferation and apoptosis in CaSki cells, we speculated that oxymatrine could inhibit proliferation and enhance apoptosis in CaSki cells via downregulating HPV16E7. We also used HPV16E7 siRNA to knock-down HPV16E7 expression, and the results indicated that downregulating HPV16E7 expression could suppress the growth of CaSki cells, which was in accordance with our speculation. We also hypothesized that oxymatrine is able to treat cervical cancer through the anti-HPV effect, which is going to be the subject of our further investigation.

In conclusion, our data showed that oxymatrine significantly induced cell apoptosis, and suppressed cell growth of cervical cancer CaSki cells both in vitro and in vivo. Oxymatrine could decrease the CaSki cells in G2/M phase and arrest the CaSki cells in G0/G1 and S phase, which partly mediated proliferation inhibition and apoptosis induction. Besides, we a found that oxymatrine could downregulate the expression of HPV16E7 at the mRNA and protein level, which offered the molecular explanation to the cell growth inhibitory effect of oxymatrine on CaSki cells. Therefore, we believe that oxymatrine can be developed to a potential preventive and therapeutic candidate for cervical cancer treatment. Our data may provide theoretical support for the clinical anticancer application with oxymatrine. However, the toxicity of oxymatrine and its effect on the proliferation of normal cells were not assessed in the present study. In future, we will continue our study on the toxicity of oxymatrine and other effects of oxymatrine in carcinoma cells in vivo and in vitro.

Acknowledgments

The present study was supported by the National Natural Science Foundation of China (grant no. 81401447).

Abbreviations:

HPV

human papilloma virus

FBS

fetal bovine serum

PFA

paraformaldehyde

PI

propidium iodide

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

PVDF

polyvinylidenedifluoride

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July-2016
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Pei Z, Zeng J, Gao Y, Li F, Li W, Zhou H, Yang Y, Wu R, Chen Y, Liu J, Liu J, et al: Oxymatrine inhibits the proliferation of CaSki cells via downregulating HPV16E7 expression. Oncol Rep 36: 291-298, 2016
APA
Pei, Z., Zeng, J., Gao, Y., Li, F., Li, W., Zhou, H. ... Liu, J. (2016). Oxymatrine inhibits the proliferation of CaSki cells via downregulating HPV16E7 expression. Oncology Reports, 36, 291-298. https://doi.org/10.3892/or.2016.4800
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Pei, Z., Zeng, J., Gao, Y., Li, F., Li, W., Zhou, H., Yang, Y., Wu, R., Chen, Y., Liu, J."Oxymatrine inhibits the proliferation of CaSki cells via downregulating HPV16E7 expression". Oncology Reports 36.1 (2016): 291-298.
Chicago
Pei, Z., Zeng, J., Gao, Y., Li, F., Li, W., Zhou, H., Yang, Y., Wu, R., Chen, Y., Liu, J."Oxymatrine inhibits the proliferation of CaSki cells via downregulating HPV16E7 expression". Oncology Reports 36, no. 1 (2016): 291-298. https://doi.org/10.3892/or.2016.4800