Rhein Inhibits the Migration of Ovarian Cancer Cells through Down-Regulation of Matrix Metalloproteinases

Abstract

The root of Rheum officinale BAILL as a traditional Chinese medicine, which main function is removing heat from the blood, promoting blood circulation and clearing toxins away. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is one of the most important active components in the root of Rheum officinale BAILL, which could inhibit the proliferation of tumor cells. However, the study on the mechanism of anti-cell migration capacity of Rhein on ovarian cancer is not yet clear. Here, we demonstrated that Rhein had dose-dependent effects of ovarian tumors on drugs and could inhibit the proliferations and migration of two typical ovarian cancer cell lines, A2780 and OV2008. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that the survival rate of ovarian cancer cells was significantly decreased when treated with Rhein. Rhein inhibited the proliferation of ovarian cancer cells in dose-dependent manner. Moreover, the wound healing assay and transwell assay indicated that the cell migratory potential and expression of matrix metalloproteinases were markedly inhibited by Rhein. Our findings suggested that Rhein could be a potential candidate to be developed as a drug for the prevention of ovarian cancer cell migration.

INTRODUCTION

Ovarian cancer is one of the most deadly gynecologic tumor.¹⁾ For patients with ovarian cancer, first-line clinical treatment is mainly cytoreductive surgery and paclitaxel-based chemotherapy.²⁾ Although most of the patients performed well at the beginning of treatment, there are still many difficulties and challenges about treatment.³⁾ The major clinical issue in the prognosis and management of ovarian cancer is the development of ascites and peritoneal metastases.⁴⁾ Therefore, for the ovarian cancer patients, it is urgent to develop novel and effective anti-metastatic drugs.

Rheum officinale BAILL is a tradition Chinese medicine, which has been normally used for chronic renal failure,⁵⁾ asthma⁶⁾ and chronic kidney disease.⁷⁾ Rhein is one of the active anthraquinone glycoside separated from Rheum officinale BAILL.⁸⁾ Rhein has a wide range of bioactivities, such as inhibiting cell proliferation,⁹⁾ decreasing hypertrophy,¹⁰⁾ suppressing migration and invasion¹¹⁾ and inducing tumor cell apoptosis.¹²⁾ Nevertheless, the effects of Rhein in resistance to ovarian cancer cell migration and its molecular mechanism are not clear.

Matrix metalloproteinases (MMPs) are a kind of endopeptidases family with the ability to resolve extracellular matrix.¹³⁾ There are two important roles of MMPs, one is the function of Pro-angiogenic, another is assistance to transfer cancer cells.¹⁴⁾ These functions of MMPs including MMP-1, MMP-2, MMP-9 make it critical in metastatic disease.¹⁵⁾ MMP-1 plays an important role in the linker region of domain interactions.¹⁶⁾ It has been reported that a kind of bioactivator dryofragin inhibited migration of human osteosarcoma cells by suppressing MMP-2/9.¹⁷⁾ However, little research has been focused on the resistance to ovarian cancer cell migration.

In this study, we found that Rhein could inhibit the proliferation and migration of ovarian cancer cells, and the underlying mechanism was associated with downregulation of MMPs. Rhein might be an effective compound to be developed as a potential drug for treatment of ovarian cancer.

MATERIALS AND METHODS

Materials, Reagents and Chemicals

The root of Rheum officinale BAILL were purchased from the drug store of Yichun Tang in Dalian of Liaoning Province, and the materials were identified by associate professor Lin Zhang in the department of Integrative Medicine of Dalian Medical University. A voucher specimen (No. 20171015) was stored in the institute of Integrative Medicine. Chengdu Must Bio-Technology Co., Ltd. (Sichuan, China) applied Rhein. The chemical formula of Rhein is C₁₅H₈O₆, which purity was >98%.

Extraction and Isolation of Rhein

The dried and powdered root of Rheum officinale BAILL (4 kg) were extracted with 10 times 75% ethanol and heated for two times (3 h each time). The combined filtrate was decompressed and evaporated to obtain the extract (467 g). The extracts were then dispersed in water and extracted with petroleum ether, dichloromethane and n-butanol, respectively. The dichloromethane extraction (36 g) was separated by silica gel column chromatography and eluted with different ratio of ethyl acetate and petroleum ether. Rhein (12.4 mg) was obtained by recrystallization from the forth fraction eluted (1 : 4), the structure of Rhein was shown in Fig. 1A.

Fig. 1. Effect of Rhein on the Proliferation of Ovarian Cancer Cell Lines and Normal Ovarian Cell Line

(A) Chemical structure of Rhein. (B) Ovarian cancer cells A2780, OV2008 and normal ovarian cells IOSE80 were treated with Rhein (0, 50, 100, 150, 200 or 250 µM) for 24 h and 48 h. MTT assay was used to verify cell viability. (C) Rhein inhibited colony formation in a dose dependent manner. * p < 0.01 and ** p < 0.001 as compared to the control group.

Rhein (as orange-yellow needle crystal; ¹H-NMR (Dimethyl sulfoxide ((DMSO), 600 MHz) δ: 13.8 (1H, s), 11.9 (2H, s), 8.10 (1H, br s), 7.82 (1H, t, J = 7.8, 7.8 Hz), 7.74 (1H, br s), 7.71 (1H, d, J = 7.8 Hz), 7.40 (1H, d, J = 7.84 Hz). ¹³C-NMR (DMSO, 150 MHz) δ: 161.9 (s), 124.6 (d), 138.5 (s), 119.2 (d), 119.9 (d), 138.1 (d), 125.1 (d), 161.5 (s), 191.8 (s), 181.4 (s), 134.2 (s), 133.6 (s), 116.6 (s), 119.1 (s), 165.9 (s). The structures of Rhein were identified by comparing the NMR data with those in published paper.¹⁸⁾

Cell Lines and Cell Culture

American Type Culture Collection (ATC C) supplied A2780, OV2008 and IOSE80 cell lines. The cells were maintained in Dulbecco’s modified eagle’s medium (DMEM) with 10% Fetal bovine serum (FBS) at 5% CO₂ in a incubator with 37°C

Cell Proliferation Assays

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examined the effect of Rhein on cell viability. The cells (1 × 104/well) with Rhein (0, 50, 100, 150, 200 or 250 µM) were incubated for 24 h. After MTT solution incubation for another 4 h, 150 µL of DMSO was added to each well. 490 nm wavelength was used to measure the absorbance.

Colony Formation Assay

The cells (500/dish) were culture with Rhein (0, 100, 200 or 300 µM) for 14 d. Afterwards, 70% ethanol and crystal violet (Sigma, St. Louis, MO, U.S.A.) was used to fix and stain cells. Numbers of colonies was counted by Image J software.

Wound Healing Assays

A 200 µL pipette tip was used to scrape the cells in 24 well plate. The cells were incubated with 0, 30, 60 or 90 µM Rhein solution for 24 h. Cell migration was detected by a phase-contrast microscope after 24 h. The Image J software was used to measure and quantify the denuded area.

Transwell Migration Assays

Ovarian cancer cells were treated with 0, 30, 60 or 90 µM Rhein for 24 h. 2 × 10⁴ cells were added to each upper compartment containing serum-free DMEM, while medium with 10% FBS used as a chemical inducer was added to the lower compartment. After 24 h, 0.1% crystal violet was used to stain the cells on the underside of cell membrane. The cells on the underside of transwell membrane were measured and counted by a microscope.

Western Blot

Total protein was extracted from the cells samples by RIPA lysis buffer (Beyotime, China). The same amount of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to the polyvinylidene fluoride (PVDF) membrane. After incubation with the specific primary and secondary antibody, protein expression were detected by ECL kit and measured by Image J software.

Statistical Analysis

The data was collected from three independent assay. The Student’s t-test was used to compare two groups by GraphPad Prism 5.0 (San Diego, CA, U.S.A.). The difference was considered statistically significance when p < 0.05.

RESULTS

Inhibiting Proliferation of Ovarian Cancer Cells by Rhein

For purpose of investigating the anti-proliferation activity of Rhein on ovarian cancer, the ovarian cancer cell lines A2780, OV2008 and the normal ovarian cell line IOSE80 were treated with Rhein in different concentrations (0, 50, 100, 150, 200 or 250 µM) for 24 h and 48 h (Fig. 1B). The activity of ovarian cancer cells was examined at the suitable dose. The result showed that the ability to proliferation and growth of the ovarian cells treated with Rhein was reduced in time-dependent manner (p < 0.001). Nevertheless, the ability to proliferate and grow of the normal ovarian cell line was not significantly inhibited (p > 0.05). In addition, the cytotoxic effects of Rhein were used to examine and verify by colony formation assay. The colony formation was proportional to the treatment concentrations. As shown in Fig. 1C, the colony formation capacity of both the A2780 and OV2008 cells were inhibited by Rhein in dose-dependent manner. As our results showed, Rhein could inhibit the proliferation and had a long-term cytostatic effect on colony formation in ovarian cancer cells.

Suppressing the Migration of Ovarian Cancer Cells by Rhein

The wound healing assay and the transwell assay were performed to evaluate the anti-migration capacity of Rhein, and the inhibition effects of Rhein on proliferation of ovarian cancer cells was not shown at low concentrations (0, 30, 60 and 90 µM). Rhein dose-dependently reduced the migration of A2780 and OV2008 cells in the wound healing assay (Fig. 2). The wound closure rates of Rhein-treated cells were lower than of non-treated cells (p < 0.0001). Furthermore, in the transwell assay, it was shown that Rhein significantly inhibited the migration of ovarian cancer cells (Fig. 3A), and the migration cells of Rhein-treated group were lower than that of control group (Fig. 3B). The results from wound healing assay and transwell assay all indicated that the migration of ovarian cancer cells was suppressed by Rhein.

Fig. 2. The Effect of Rhein on Wound Healing Analysis on Ovarian Cancer Cell Migration

(A) Photographs represent the migration of A2780 cells under A microscope at A magnification of 100 times before and after injury. The migration of A2780 cells was quantified by measuring the closed area of the wound before and after injury. (B) The image represents OV2008 cell migration under a 100-fold magnification microscope before and after injury. The migration of OV2008 cells was quantified by measuring the closed area of the wound before and after the injury. The experiment was repeated three times. * p < 0.01 and ** p < 0.001 as compared to the control group.

Fig. 3. Effect of Rhein on Migration of Ovarian Cancer Cells by Transwell Assay

The cells were treated with Rhein (0, 20 or 30 µM) for 24 h. (A) Photographs show that cells migrate to the lower side of the transwell membrane under A microscope with A magnification factor of 200. (B) Count and average the number of cells in each field. The migrated cells were expressed relative to the control group. The experiment was repeated three times. * p < 0.01 and ** p < 0.001 compared to the control group.

The Effects of Rhein on MMPs Expression

Based on the effects of Rhein on ovarian cancer cell migration, the mechanisms were further investigated. Now that MMPs act important roles in cancer cell migration, the MMP-1, MMP-2 and MMP-9 protein expression were detected in A2780 cells by Western blot (Fig. 4). As our results showed, the inhibitory activity of Rhein on the migration of ovarian cancer may be related to the down regulation of MMP-1, MMP-2 and MMP-9 expression.

Fig. 4. The Effects of Rhein on MMP-1, MMP-2 and MMP-9 Protein Expression

Western blotting was used to detect the levels of MMP-1, MMP-2 and MMP-9 proteins. Bottom-actin is used as a loading control. All the experiments were repeated three times. Image J software is used to quantify the band by measuring the band density. Each column represents the mean calculated from three independent experiments plus or minus standard deviation (S.D.) Compared to the control group. * p < 0.01 and ** p < 0.001 as compared to the control group.

DISCUSSION

Since the chemotherapy resistance and unexpected side effects are the critical challenges for cancer treatments, traditional herbal medicines are increasingly being used to treat a wide variety of cancer.^19,20) The recurrence rate of ovarian cancer caused by drug resistance is one of the main causes of tumor death. Chemotherapy drugs have obvious killing effects on cancer, but they also produce drug resistance for a long time, leading to the re-proliferation of tumor cells. Our results are based on the potential drug candidate Rhein, which has a sustained killing effect on tumor cells treated with cisplatin in many other tumors, such as breast cancer, but the mechanism of anti-migration in ovarian cancer still uncertain.²¹⁾ Rhein, an important chemical component of Rheum officinale has been reported to possess anti-inflammatory,²²⁾ anti-oxidation²³⁾ and anti-tumor effects.²⁴⁾ Studies have showed that MMPs are expressed as proenzymes that are activated in the extracellular space.²⁵⁾ The first step for cancer cell migration was degradation of extracellular matrix by MMPs, MMPs acts an important role in cancer cell migration.²⁶⁾ Moreover, it was difficult to work out prognoses in ovarian cancer patients due to the abnormal MMPs expression in ovarian cancer cells.²⁷⁾ Our results indicated that Rhein could inhibit the expression of MMP-1, MMP-2 and MMP-9 compared with the control groups. These data suggested that Rhein may suppress ovarian cancer migration through down regulation of MMPs expression. However, Further studies in vivo will be done to verify the conclusions.

In conclusion, Rhein could effectively suppress the proliferation and migration of ovarian cancer cells, and the underlying mechanism may be related to the expression of MMPs. The results revealed that Rhein could be a kind of novel and effective drug for ovarian cancer patients.

Acknowledgments

This study was financially supported by National Natural Science Foundation of China (81873195), Natural Science Foundation of Liaoning Province of China (20170540273), the Education fund item of Liaoning province (LQ2017002), The China Postdoctoral Science Foundation (2017M611241), and the project of Dalian Young Star on Science and Technology in 2017 (2017RQ122), and the research fund from Academy of Integrative Medicine Department of Dalian Medical University. The funders had no role in study design, experiment performing, data analysis, or manuscript preparation.

Conflict of Interest

The authors declare no conflict of interest.

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