Guo-qing Chen, Zhen-wei Yao, Wei-ping Zheng, Li Chen,Hong Duan, Yi Shen*

1Research Center of Tissue Engineering and Stem cell, Department of Pathophysiology,Chongqing Medical University, Chongqing 400016, China

2Department of Obsttrics and Gynecology, the First Affiliated Hospital, Chongqing Medical University,Chongqing 400016, China

INTRODUCTION

Recently, many antitumor compounds with new structural features and mechanism of action have been isolated from herbal medicines.Ursolic acid(UA) is a triterpenoid compound that exists widely in food, medicinal herbs, and other plants.Previous studies show that UA has numerous pharmacological activities including antioxidant activity, as well as anti-inflammatory, anti-cancer,and hepatoprotective effects[1-6].Our previous studies show that ursolic acid inhibit the proliferation of human esophageal carcinoma cell Eca-109in vivo[7].To date, however, no study has been conducted in the evaluation of the combination effects of ursolic acid and chemotherapy drugs against human esophageal carcinoma cells.Therefore, the present study was designed to investigate the antitumor effects of combining ursolic acid with 5-fluorouracil (5-FU)on human esophageal carcinoma cell Eca-109in vitro, with special emphasis on the changes in certain key protein expressions relevant to the regulation of cell cycle and apoptotic pathway.

MATERIALS AND METHODS

Materials

Human esophageal carcinoma cell line Eca-109 was supplied by the department of pathophysiology of Chongqing Medical University.Ursolic acid,Hoechst 33258, Propidium iodide (PI) and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) were supplied by Sigma Chemical Co.(St.Louis, MO, USA).5-fluorouracil was obtained from Pharmaceutical factory of Nantong(China).Fetal bovine serum (FBS) and RPMI-1640 culture medium were obtained from GIBCO BRL(USA).The monoclonal mouse anti-human P27kip1,bax, andbcl-2 antibodies were purchased from Santa Cruz Biotechnology, Inc.(USA).Horseradish peroxidase (HPR)-rabit anti-mouse antibody (the second Ab) was purchased from Beijing Zhong Shan Biotechnology Co (China).

MTT Assay

The effect of ursolic acid, 5-fluorouracil and their combination on the proliferation of Eca-109 cells was measured by MTT assay.Briefly 5×103cells/well were grown in 96-well plates overnight.Cells were incubated with 10, 20, 30, 40, and 50μmol/L ursolic acid; 48.0, 96.1, 192.2, 384.4, and 768.8 μmol/L 5-fluorouracil or their combination(The ratio of the two drugs combined was 1:1) for 48 h, then 10 μl of MTT (1 mg/ml) reagent was added to each well, and the cells were further incubated at 37°C for 4 h.The supernatant was removed and 200 μl DMSO in isopropanol was added to each well to solubilize the formazan product.The absorbance at wavelength of 570 nm was measured by a micro ELISA reader.The negative control well contained medium only.The ratios of the absorbance of treated cells relative to those of the control wells were calculated and expressed as percentage of growth inhibition.

Analysis of Cell Cycle Distribution and Cell Apoptosis

Cell cycle and cell apoptosis analyses were carried out using flow cytometry.Eca-109 cells(1×106/well) were seeded in six-well plates and allowed to attach overnight.Growing cells were treated with 10-50 μmol/L ursolic acid, 48.0-768.8μmol/L 5-fluorouracil, or their combination for 48 h.The cells were then washed twice with PBS,centrifuged at 500× g for 5 min, fixed in 70%ethanol at 4°C.Before DNA analyses, the cells were washed again with PBS, treated with 50 μg/ml RNase, and stained with 100 μg/ml PI in the dark.Flow cytometry analysis was performed within 1 h according to the manufacturer’s instructions.

Fluorescent Microscopy

Eca-109 cells (1×106) were grown on coverslips placed into six-well plates.After drug treatment as described above, cells were fixed with cold methanol and acetic acid at 4°C overnight,stained with Hoechst 33258 for 30 min in the dark,washed again in PBS and finally mounted in mounting medium.Processed cells were observed with a fluorescence microscope.

Western Blot

Eca-109 cells (1×106/well) were seeded in 6-well plates.Cells were treated as described above.The cells were scraped and washed twice by centrifugation at 500× g for 5 min at 4°C.The pellet was resuspended in lysis buffer supplemented with proteases and phosphotase inhibitors and incubated for 1 h at 4°C.The lysate was collected by centrifugation at 14,000×g for 40 min at 4°C, and the supernatant was stored at -20°C.50 μg of denatured protein was separated on a sodium dodecyl sulfate-polyacrylamide gel (12%acrylamide) and transferred to PVDF membrane,which was then blocked overnight in 5% skim milk in TBST.For immunoblotting, the membrane was incubated for 1 hour with the antibody againstbax,bcl-2, and P27kip1.Afterward, it was rinsed by TBST and incubated with the second antibody(1:1000) for 1 h.The final blots were assessed with an ECL Western blotting detection system.The protein bands were scanned by computer and the relative expressions of protein were calculated by image anlysis software (Quantity One).

Statistical Analysis

RESULTS

Combined Effects of UA and 5-FU on the Viability of Eca-109 Cells

Cell proliferation was inhibited strongly by ursolic acid in a dose dependent manner.Treating Eca-109 cells with 5-fluorouracil also inhibited cellular proliferation in a dose dependent manner.Compared with single treatment, combination treatment inhibited cell proliferation and viability more significantly (P<0.05) (Table 1).

Combined Effect of UA and 5-FU on Cell Cycle and Cell Apoptosis

Eca-109 cells treated with ursolic acid were arrested in the G0/G1 phase in a typical dose dependent manner, and this effect was more evident when combined with 5-fluorouracil (Table 2).The percentage of apoptotic cells was increased to 14.41%-72.05% when treated with ursolic acid and 5-fluorouracil for 48 h, more than that with single treatment of ursolic acid (2.69%-56.12%) or 5-fluorouracil (12.63%-62.38%) (P<0.05).

Table 1.The inhibitory effects of UA, 5-FU, and their combination on Eca-109 cells for 48 (±s)

Table 1.The inhibitory effects of UA, 5-FU, and their combination on Eca-109 cells for 48 (±s)

Compared with control groups: aP<0.05; Combination effect analysis: bP<0.05.

5-fluorouracil (μmol/L)UA(μmol/L) 0 48.0 96.1 192.2 384.4 768.8 0 0 23.12±1.36a 34.32±1.97a 47.84±2.33a 59.16±1.61a 71.26±1.43a 10 6.26±1.29 32.48±1.50a － － － －20 17.38±1.07a － 41.19±2.01a － － －30 33.82±1.71a － － 56.38±1.42ab － －40 54.39±1.58a － － － 70.75±2.61ab －50 67.04±1.22a － － － － 82.27±1.86ab

Table 2.The effect of UA and 5-FU on the cycle distribution and cell apoptosis of Eca-109 cells (±s)

Table 2.The effect of UA and 5-FU on the cycle distribution and cell apoptosis of Eca-109 cells (±s)

Compared with control groups: aP<0.05; Combination effect analysis: bP<0.05.

UA 5-FU(μmol/L) (μmol/L) G0/G1 phase S-phase G2/M phase Apoptosis Proliferation index 0 0 53.43±1.41 25.72±1.33 20.85±1.45 1.09±0.82 46.57±3.81 10 0 61.30±0.41a 24.19±0.63 14.15±2.01 2.69±0.77 38.34±2.28a 20 0 67.39±1.20a 19.35±1.19a 13.26±0.91 10.06±1.27a 32.61±1.62a 30 0 74.36±2.40a 14.21±0.89a 11.43±0.73 24.31±1.08a 25.64±1.97a 40 0 76.26±1.39a 12.52±0.75a 11.22±0.55 41.36±2.19a 23.74±1.10a 50 0 82.19±0.37a 10.32±0.82a 7.49±1.28 56.12±0.41a 17.81±2.07a 0 48.0 50.51±2.12 21.33±1.04 28.10±3.13 12.63±1.69 49.49±3.12 0 96.1 56.20±1.56a 24.13±2.26 19.80±2.10 21.89±2.56a 44.00±1.01 0 192.2 61.82±2.61a 27.48±1.49a 10.00±0.94 33.91±2.37a 37.55±2.03a 0 384.4 69.36±2.97a 27.13±1.17a 3.50±0.49 47.59±1.40a 30.64±1.18a 0 768.8 75.19±2.81a 22.34±2.50 2.40±0.84 62.38±1.67a 24.81±1.27a 10 48.0 67.83±2.52a 19.78±1.54 12.30±1.04 14.41±2.10 32.17±2.66 20 96.1 73.91±1.36ab 21.39±1.67 4.70±0.79 27.20±1.51ab 26.09±2.54ab 30 192.2 78.43±2.64ab 19.61±1.43 1.90±0.23 40.05±2.67ab 21.57±1.33ab 40 384.4 82.83±1.56ab 15.44±1.01ab 1.70±0.41 58.46±2.44ab 17.17±1.21ab 50 768.8 87.69±2.37ab 10.08±1.19ab 1.50±0.34 72.05±2.87ab 11.59±0.92ab

Hoechst 33258 Staining

After treatment of Eca-109 cells with UA, 5-FU,and their combination for 48 h, marked morphological changes of cell apoptosis such as condensation of chromatin, nuclear fragmentations and apoptotic bodies were found clearly using Hoechst 33258 staining (Figure 1).Compared with single treatment, apoptotic cells significantly increased in combination treatment.

Combined Effect of UA and 5-FU on Expression of bcl-2, bax, P27kip1

Using Western blotting analysis, we assessed the effects of UA, 5-FU, and their combination treatment on the protein expressions ofbcl-2,baxand P27kip1.Treatment of Eca-109 cells with UA(10-50 μmol/L) for 48 h resulted in decrease of the protein expression ofbcl-2 and increase ofbaxand P27kip1.As shown in Figure 2 and Figure 3,compared with single treatment, combination treatment with UA and 5-FU reduced the level ofbcl-2 expression more significantly.The level ofbaxand P27kip1proteins in combination treatment cells were higher than those in single-treated cells.

Figure 2.Western blot analysis of P27kip1.A: β-action;B: UA-treated group; C: Combination treatment group.Lane 1: Control group; Lanes 2-6: UA (10-50 μmol/L or combined with 5-FU (48.0-768.8 μmol/L).

Figure 3.The relative expressions of bcl-2, bax and P27kip1 Eca-109 cells treated with UA, 5-Fu, and their combination for 48 h.A: bcl-2/β-action; B: bax-2/β-action; C: P27kip1/β-action; Drug concentration 1: Control group; Drug concentration 2-6: UA (10-50 μmol/L), 5-FU (48.0-768.8 μmol/L) or their combination.

DISCUSSION

The treatments for human esophageal carcinoma with chemotherapy rarely uses a single drug but usually combines two or more drugs with different mechanisms of action.An optimal treatment of combination chemotherapy may increase the therapeutic efficacy, decrease the toxicity toward the host or non-target tissues, and minimize or delay the development of drug resistance[8,9].In our study, we investigated the viability of human esophageal carcinoma cell Eca-109 under the influence of ursolic acid and 5-fluorouracil and in combination.Our study demonstrated that ursolic acid, when combined with anti-tumor agent 5-fluorouracil, showed adjuvant antiproliferative effects on Eca-109 cellsin vitro.

Apoptosis is essential for the development and maintenance of tissue homeostasis and the elimination of unwanted or damaged cells from multicellular organisms.The aberrant regulation of apoptosis has been observed in many forms of tumors[10].baxandbcl-2 are the apoptosis-related proteins of mitochondrial pathway.Studies have reported thatbcl-2 inhibits apoptosis by preventing cytochrome c release from the mitochondria and inhibiting caspase activation[11], andbaxis a promoter for apoptosis.The ratio ofbcl-2:baxdictates the ultimate sensitivity or resistance of tumor cells to apoptosis.To probe the possible mechanism by which ursolic acid enhances the cytotoxic effects of 5-fluorouracil, we performed some experiments on cell apoptosis.Flow cytometry analysis showed that ursolic acid exhibited strong effects of inducing apoptosis on Eca-109 cellsin vitro.And fluorescent microscopy showed ursolic acid treatments led to the occurrence of typical morphological changes of apoptosis.Western blotting analysis showed that treating Eca-109 cells with ursolic acid significantly inhibited thebcl-2 expression and increasedbaxexpression, indicating that ursolic acid induced apoptosis through mitochondrial pathway in vitro, which was consistent with our previous report[7].Compared with single treatment,combination treatment with ursolic acid and 5-fluorouracil reduced the level ofbcl-2 expression and increased the level ofbaxexpression more significantly, indicating that ursolic acid combined with 5-fluorouracil showed adjuvant effects of inducing apoptosis on Eca-109 cellsin vitro.

P27kip1protein, members of the KIP family,inhibits the activities of cyclin D-, E-, and A-dependent kinases and induce cell-cycle arrest to allow cells to stop and repair DNA lesions before DNA replication occurs in S phase.Low levels of P27kip1are reported to be causally related to tumorigenesis.The level of P27kip1expression decreases during human esophageal carcinoma development and progression[12].In this study, we found that the administration of ursolic acid,5-fluorouracil or their combination resulted in accumulation of cells at G0/G1 phase.Affected cells are unable to successfully enter cell cycle.Ultimately, the prolonged mitotic arrest in most of these cells may lead to apoptosis.In order to get a better understanding of the mechanism by which ursolic acid, 5-fluorouracil and their combination leads to greater G0/G1 arrest, we further investigated the status of a key protein, P27kip1known to regulate G0/G1 transition.We clearly demonstrated that the level of P27kip1protein was increased in Eca-109 cells treated with either ursolic acid or 5-fluorouracil alone.These changes in combination treatment group were significantly greater than that in single treating group,indicating that treating Eca-109 cells with ursolic acid or 5-fluorouracil or their combination resulted in increased expression of P27kip1, leading to G0/G1 arrest.

In summary, we have found that ursolic acid,when combined with 5-fluorouracil, has adjuvant antiproliferative effects, which mainly due to the induction of cell cycle arrest as well as apoptosis.The possible mechanisms underlying the action might be attributed to the changes in certain key proteins relevant to the regulation of cell cycle and/or apoptosis.This investigation suggests an important potential clinical application of the combination regimen for the treatment of esophageal carcinoma patients.

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