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Effect of Euonine on the Proliferation and Apoptosis of Human Uterine Leiomyoma Cells

Running title: Role of euonine on uterine leiomyoma

Highlights:

1. The proliferation rate of uterine leiomyoma cells was determined.
2. The apoptosis rate of uterine leiomyoma cells was determined.
3. The phosphorylation levels of EGFR from uterine leiomyoma cells were measured.

Abstract

Objective: To explore the effect of euonine on the proliferation and apoptosis of uterine leiomyoma cells in vitro, and the mechanism of that.

Methods: Human uterine leiomyoma cells were cultured by using collagenase digestion, and were affected by euonine at the levels of 1μg/ml, 3μg/ml, 6μg/ml, then MTT assay was carried out for the proliferation ability of uterine leiomyoma cells, and flow cytometry was used to analyze the apoptosis rate of uterine leiomyoma cells. The phosphorylation levels of ALK, ROS1, EGFR, HER2, FGFR1 and PDGF-alpha from uterine leiomyoma cells were measured by western blot; ELISA assay was performed to detect the expression levels of EGF and TGF-alpha from condition medium of uterine leiomyoma cells. To further identify the mechanism of euonine affecting uterine leiomyoma cells, after affected by euonine for 24h, uterine leiomyoma cells were continued to affect by EGF for 24h. After that, the phosphorylation level of EGFR from uterine leiomyoma cells was examined by western blot.

Result: The proliferation rate was lower and lower and apoptosis rate increased constantly following the rising of dose and affecting time of euonine (P < 0.05). After affected by euonine, only the phosphorylation level of EGFR of uterine leiomyoma cells was downregulated significantly showed by western blot, and the expression level of EGF from condition medium of uterine leiomyoma cells decreased apparently. However, when uterine leiomyoma cells incubated with EGF, the phosphorylation level of EGFR recovered to the previous level.

Conclusion: euonine was workable for the inhibiting of proliferation and the inducing of apoptosis of uterine leiomyoma cells, and whose mechanism may be that euonine downregulated the expression of EGF to decrease the phosphorylation level of EGFR which contribute to this result.

Key words: uterine leiomyoma, euonine, proliferation, apoptosis, EGFR, phosphorylation, EGF

1. Introduction

Uterine leiomyoma appears to be the most common gynecologic benign tumor of the myometrium, occurring in as many as 20.0ï¼… to 40.0ï¼… of womenduring their reproductive years and 40 to 50 years old women accounts for 52.2% to 60.0% [1, 2]. Uterine leiomyoma arises from a single uterine smooth muscle cell [3] which can be confirmed by cytogenetic studies [4, 5]. The etiological factors are involved in cell proliferation, extracellular matrix degradation deposition, local autocrine or paracrine, growth factors, and many other aspects [6]. Although the nature of the initial event is unclear, some studies found that that leiomyomas growth were dependent on estrogen [7-9]. Researches have shown that some neonatal rodents treated with environmental estrogens, such as industrial byproducts and natural plant compounds, have an increased incidence of uterine leiomyoma later in their lives [10-12]. Therefore, the use of anti-estrogen therapy is a main way for the treatment of uterine leiomyoma, however, the effect is not ideal. West et al. (1987 ) used GnRH agonists which could reduce ovarian hormone production in the treatment of uterine leiomyoma, leading to a decrease in the size of leiomyomas, but re-enlargement of leiomyomas occured after therapy with GnRH agonist was discontinued [13]. As a result, looking for a novel and effective treatment is of great significance.

Tripterygium wilfordii Hook. F. is a traditional Chinese medicinal plant grown in the south of China. The pharmacological properties of T. wilfordii have been studied extensively over recent years and the results obtained found that the extracts have some biological activities, such as immunosuppression, anti-inflammatory and antifertility [14]. T. wilfordii extracts were also found to inhibit proliferation and induce apoptosis in lymphocytes and synovial fibroblasts [15]. Therefore it has been widely used to treat various inflammatory and autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, and idiopathic IgA nephropathy [14, 16]. Patients treated with the decoction of T. wilfordii seemed to experience therapeutic benefit at first, but frequently developed adverse effects, as well as, severe toxicity occasionally [17, 18]. Subsequently, efforts were made to maximize therapeutic benefit and minimize toxicity.

The main active constituents of T. wilfordii have been reported to be diterpenes, triterpenes and alkaloids [19-21]. The alkaloids have 22 kinds of monomers and some of them have immunosuppression and antitumor effects while haverelatively lesstoxic [22]. For instance, wilforgine, wilfortrine, wilfordine and euonine have been reported to possess obvious humoral immunosuppressive effects [22], whilst wilfortrine can inhibit leukaemia cell growth in mice [23, 24] and shows anti-HIV activity [25].

Nevertheless, there is few report about the antitumor effects of alkaloids. In the present study, we investigated the roles of euonine in human uterine leiomyoma cells proliferation, apoptosis and explored its mechanism in vitro experiments. This study undoubtedlyprovidesnewinsightsinto the treatment of uterine leiomyoma.

2. Materials and methods

2.1 Preparation and culture of human uterine leiomyoma cells

Uterine leiomyoma tissues were washed 3 times with antibiotic-containing PBS and DMEM then cut into pieces to 1 mm³ and digested in 800 U/mL type I collagenase at 37 ℃ for 6 h. The leiomyoma cells were collected by filtration through a 200 mesh and centrifugation at 13000 rpm/min for 10 min. Then the cells were adjusted itsconcentration to 5 × 10⁵/ml and plated in 24-well tissue culture plates at 1 ml per well.The culture plates were kept in a tissue culture incubator at 37 ℃ and 5% CO₂. The uterine leiomyoma cells identification was determined by immunohistochemistry using α- smooth muscle actin monoclonal antibody (Gene Tech, Shanghai) at a dilution of (稀释倍速待反馈). Cells were passaged to 3-6 generation, and the ones growed stably and at thelogarithmicgrowth phase were used in the experiment. The experiments were divided into two groups by random: test group and control group and each group has three repetitions.

2.2 Proliferation assay of uterine leiomyoma cells

MTT assay was used to detect the cell proliferation inhibition rate. The uterine leiomyoma cells were plated in 96-well tissue culture plates at concentration of 3 × 10⁵/ml at 100 μl per well. Cells were treated either with euonine with final concentration of 1 μg/ml, 3 μg/ml and 6 μg/ml (test groups) or without euonine (control groups). The culture plates were kept in a tissue culture incubator at 37 ℃ and 5% CO2 for 24 h, 48 h and 72 h and the cells were collected by centrifugation. Then 20μl 5 mg/ml MTT solution was added to each well and incubated for 4 h at 37 ℃ and 5% CO2. The cells were collected by centrifugation. Then, 150 μl DMSO solution was added to each well and mixed thoroughly. The absorbance was measured in a microplate reader (BioTek, USA) with a test wavelength of 570 nm. The proliferation rate (%) was calculated as follows :

A_570T represents the 570 nm absorbance of test groups and A_570C represents the 570 nm absorbance of control groups.

2.3 Apoptosis rate assay of uterine leiomyoma cells

Flow cytometry was used to analyze the apoptosis rate of uterine leiomyoma cells. The leiomyoma cells were plated at concentration to 3 × 10⁵/ml in 96-well tissue culture plates at 100 μl per well. In the test groups, euonine with final concentration of 1 μg/ml, 3 μg/ml and 6 μg/ml was added to each well. The culture plates were maintained in culture incubator at 37 ℃ and 5% CO2 for 24 h, 48 h and 72 h and the cells were collected by centrifugation and washed three times with PBS. Cells were then stained with propidium iodide (PI) and incubated at room temperature away from light for 15 min. The cells apoptosis rates were measured by flow cytometry instrument (Elite Esp type).

2.4 Western blot analysis

Phosphorylation levels were determined by western blot. Uterine leiomyoma cells were treated with euonine with final concentration of 3 μg/ml and 6 μg/ml for 24 h, 48 h and 72 h and the control groups were treated without euonine. The lysates were subsequently centrifuged and the cells and supernatants were both collected. Then, the cells collected were treated with RIPA containing protease inhibitors and phosphatase inhibitor, resuspended, shaked in the ice bath for 30 minites and eddied every 10 minites. The supernatants were collected by centrifugation at 13000 rpm/min for 10 min at 4 °C. The protein concentration of uterine leiomyoma cells supernatants was determined by bicinchorinic acid assay (BCA) protein assay. The supernatants of 50 μg were subjected to SDS-PAGE and the proteins band was then excised and electrophoretically transferred from gels to nitrocellulose membranes. Blots were exposed overnight to the ALK, ROS1, EGFR, HER2, FGFR1 and PDGF-alpha phosphorylation antibodies (Life Technologies, USA) at a dilution of ( ) in blocking buffer. After washing, HRP-conjugated goat antimouse secondary antibody that was diluted at ( ) with blocking buffer were added and incubated for 1 h. The antigen-antibody complexes were detected with the chemiluminescence detection system.

2.5 ELISA assay

The supernatants of uterine leiomyoma cells were detected by EGF and TGF-alpha ELISA kits (Raybiotech, USA) according to the manufacturer’s instructions. The 96-well plates coated by primary antibodies were kept at room temperature for 10 min. Seven graded concentrations of protein solution and the supernatants of uterine leiomyoma cells were added into the 96-well plates at 100 μl per well, then incubated at room temperature for 2.5 h. After three washes, the biotin conjugated secondary antibodies at a dilution of ( ) were added at 100 μl per well, then incubated at room temperature for 2 h. After washing, the diluted streptavidin-conjugatedHRP was added at 100 μl per well and incubated at room temperature for 45 min. After washing, TMB was added and incubated for 30 min followed by terminating reaction by adding stop buffer. The OD value of 450 nm was determined by microplate reader.

2.6 EGF affect uterine leiomyoma cells

When the uterine leiomyoma cells were in the logarithmic phase, 3 μg/ml euonine were added to affect cells for 24 h, then 100 ng/ml recombinant EGF proteins (Raybiotech, USA) were added and 24 h later, uterine leiomyoma cells were collected by centrifuging. Then western blot analysis was performed using a standard procedure as described above to assess the phosphorylation levels of EGFR.

2.7 Statistical analysis

The data are presented as the mean ± SD. Statistical analysis was performed using t-test and a probability level of P< 0.05 was considered as statistically significant (SPSS12.0).

3. Results

3.1 Effect of euonine on proliferation rate of uterine leiomyoma cells

Table 1 shows the proliferation rates of uterine leiomyoma cells as determined by MTT assay. Compared with control groups, treatments with the euonine of 3 μg/ml and 6 μg/ml for 24 h, 48 h and 72 h significantly decreased proliferation rates (P < 0.05). Besides, treatment with the euonine of 6 μg/ml for 24 h, 48 h or 72 h significantly decreased proliferation rates (P < 0.05) compared with treated with euonine of 3 μg/ml.

3.2 Effect of euonine on apoptosis rate of uterine leiomyoma cells

Flow cytometry was used to test the apoptosis rate of uterine leiomyoma cells affected by different doses of euonine for different duration. The apoptosis rates of 48 h and 72 h after the addition of euonine of 3 μg/ml and 6 μg/ml were significantly increased (P < 0.05) compared with the control groups and the apoptosis rates increased significantly (P < 0.05) when treated with the euonine of 6 μg/ml for 48 h and 72 h compared with the treatment with 3μg/ml (Table 2).

3.3 Effect of euonine on phosphorylation level of receptor

After affected by euonine, the phosphorylation levels of ALK, ROS1, EGFR, HER2, FGFR1 and PDGF-alpha was detected by western blot which found that among the six receptors, only the phosphorylation levels of EGFR had significant changes (P < 0.05) and its phosphorylation levels decrease significantly with the increase of doses of euonine after 24 h, 48 h and 72 h (Figure 1).

3.4 Effect of euonine onexpression level of EGF and TGF-alpha

ELISA was used to detect the expression levels of EGF and TGF-alpha after affecting by euonine for further investigate the mechanism of euonine. The result indicated that EGF expression level decreased significantly when treated with euonine of 3 μg/ml and 6 μg/ml for 24 h, 48 h and 72 h (Figure 2).

3.5 Effect of EGF on phosphorylation level of EGFR

The phosphorylation level of EGFR after affected by EGF was shown in figure 3. The result indicated that compared with control groups, the phosphorylation level of EGF groups increased obviously. When treated with euonine, the phosphorylation level of EGRF decreased significantly, but the addition of EGF increase the EGRF phosphorylation level (Figure 3).

4. Discussion

Uterine leiomyoma is one of the main causes of women hysterectomy and its treatments are primarily based on surgery. However, this treatment method has adverse effects on the fertility of reproductive women and physical and mental health of the majority of patients, so there are increasing attentions on drug treatments. Euonine whose effective components are diterpenes, triterpenes and alkaloids is commonly advocated as useful for the treatment of various inflammations [19]. The occurrence and development of uterine leiomyoma is closely related to the proliferation, differentiation and apoptosis of uterine leiomyoma cells so the intervention on cell proliferation and apoptosis is a new strategy in present tumor treatment. In the present study we demonstrated that euonine exerts growth inhibitory effects on the uterine leiomyoma cells by decreasing their proliferation and increasing apoptosis and shows an obvious antitumor effect and less toxic. Besides, the effects of euonine on the cultured leiomyoma cells were dose and time dependent.

In addition, we reported a mechanism from the receptors phosphorylation levels of multiple signaling pathways which might contribute to the growth inhibition of uterine leiomyoma cells in the presence of euonine. The receptors are anaplastic lymphoma kinase (ALK), c-ros oncogene 1, receptor tyrosine kinase (ROS1), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor type 2 (HER2), fibroblast growth factor receptor 1 (FGFR1) and platelet derived growth factor alpha (PDGF-alpha). All the above cited belong to the well known receptor tyrosine kinases (RTK) and located in the surface membrane of cells [26]. They will be activated and phosphorylated after exposure to the outside stimulation, and then, the intracellular kinase pathways will be activated to involve in some physiological processes, such as, cells growth, proliferation and differentiation. These signaling pathways are closely related to the tumor cells proliferation, angiogenesis, tumor invasion, metastasis and the inhibition of cells apoptosis. All of the afunction of the protein tyrosine kinase, abnormal phosphorylation levels, abnormal activity of the key factors or cellular localization can induce tumor, diabetes, immune deficiency and cardiovascular disease [26]. Our study found that among the six receptors, only the EGFR phosphorylation was inhibited after treated with euonine which suggests that the mechanism of euonine inhibiting uterine leiomyoma cells may be achieved by inhibiting EGFR phosphorylation. EGFR belongs to ErbB family type I receptor tyrosine kinases which play a critical role in mediating gowth factor signaling and its overexpression has been associated with the oncogenic activity such as proliferation, unregulated cell growth, differentiation and survival [27]. As a result, it becomes an attractive target for the design of novel antitumor drugs [27].

In mammals, the EGFR activation involves the binding of peptide growth factors including EGF, TGF-alpha and heparin-binding EGF (HB-EGF) [28]. EGF is mitogenic for leiomyomas and regulates leiomyoma growth [29, 30]. Yeh et al. (1991) found that EGF may be involved in the autocrine or paracrine regulation of the growth of leiomyoma cells [31]. TGF-alpha expresses less in leiomyoma cells [32]. HB-EGF stimulates leiomyoma cells proliferation and inhibits apoptosis [33]. From the expression levels of EGF and TGF-alpha after affecting by euonine, only the EGF expression was inhibited significantly. The results indicate that the antitumor mechanism of euonine is realized by inhibiting the EGF expression of tumor cells to block the activation of EGFR thereby making its mediated pathway can’t signal to promote cell growth, proliferation and differentiation. Moreover, recombinant EGF proteins were added in vitro to confirm our reasoning above. Shushan et al. (2004) found that the selective EGF receptor blocker AG1478 is able to inhibit leiomyoma cell proliferation [34] which is in accordance with our results.

Our results confirm a potential role of the euonine in inhibiting the growth of uterine leiomyoma cells. However, the selection of minimal toxic concentration needs further researches to maximize the benefit of euonine.

5. Conclutions

Uterine leiomyoma is a extremely common benign tumor, unfortunately, few medical treatments are available for this disease. Our results suggest that certain concentration of euonine exhibits inhibitory effects on the proliferation of uterine leiomyoma cells in vitro. The antitumor mechanism is realized by inhibiting the EGF expression of uterine leiomyoma cells to block the activation of EGFR thereby making its mediated pathway can’t signal to promote cell growth, proliferation and differentiation.