Induction of apoptosis in HepG2 cells by solanine and Bcl-2 protein

Available online at www.sciencedirect.com

Journal of Ethnopharmacology 115 (2008) 194–202

Induction of apoptosis in HepG2 cells by solanine and Bcl-2 protein Y.B. Ji, S.Y. Gao ∗ , C.F. Ji, X. Zou Postdoctoral Programme of the Institute of Materia Medica, The Center for Life Sciences and Environmental Sciences, Harbin University of Commerce, 138 Tongda Street Daoli District, Harbin 150076, Heilongjiang Province, PR China Received 13 June 2007; received in revised form 10 September 2007; accepted 22 September 2007 Available online 29 September 2007

Abstract The nightshade (Solanum nigrum Linn.) has been widely used in Chinese traditional medicine as a remedy for the treatment of digestive system cancer. The anti-tumor activity of solanine, a steroid alkaloid isolated from the nightshade has been demonstrated. To observe the effect of antitumor and mechanism of solanine. The MTT assay was used to evaluate the IC50 on the three digestive system tumor cell lines. The effect on the morphology was observed with a laser confocal microscopy; the rate of apoptosis and the cell cycle were measured using flow cytometry (FCM); the expression of Bcl-2 protein was measured by Western blot. The results show that the IC50 for HepG2 , SGC-7901, and LS-174 were 14.47, >50, and >50 ␮g/ml, respectively; the morphology of cells in the negative control was normal; for the treated groups, typical signs for apoptosis were found. The rate of apoptosis in HepG2 cells induced by solanine was found to be 6.0, 14.4, 17.3, 18.9, and 32.2%, respectively. Observation of the cell cycle showed that cells in the G2 /M phases disappeared while the number of cells in the S phase increased significantly for treated groups. Western blot showed that solanine decreased the expression of Bcl-2 protein. Therefore, the target of solanine in inducing apoptosis in HepG2 cells seems to be mediated by the inhibition in the expression of Bcl-2 protein. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Solanine; HepG2 ; Apoptosis; Bcl-2 Protein

1. Introduction Solanine is found mainly in the tuber of the potato (Solanum tuberosum L.) and in the whole plant of the nightshade (Solanum nigrum Linn.) of the Solanaceae family. The whole plant of the nightshade contains many steroid alkaloids, including solamargine, solasonine, and solanine, as well as sponin and other substances. Its total alkaloids can be used for anti-tumor purposes, with a strong inhibitory effect on tumors in animals and a clear cytotoxic effect on tumor cells (Ji, 1995). Its ethanol extract is capable of inhibiting the growth of breast cancer and inducing apoptosis in tumor cells (Son et al., 2003). The anti-tumor effect of solamargine has been reported (Liu et al., 2004), but reports on the anti-tumor effect of solanine are limited to its anti-karyokinetic effect. In this study, the inhibitory effect of solanine on tumors in the digestive system was observed in vitro, and screening for a cell line with the greatest sensitivity was performed. Further, the role of solanine in inducing apopto-

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sis in the sensitive tumor cell line and its effect on Bcl-2 protein were observed. 2. Materials and methods 2.1. Test drugs Solanine Fig. 1 purity 99% extracted from the nightshade, provided by Heilongjiang Institute for Drug Control. 2.2. Reagents RPMI 1640 culture medium (HyClone, US); fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.); pancreatin (GIBCO, US); DMSO (AMRESCO, US); BSA (Sigma, US); trypan blue (BIBCO, US); acridine orange (AO) (AMRESCO); ethidium bromide (EB) (AMRESCO); PI (Sigma); Triton X-100 (Watson Biotechnologies, Inc., Shanghai); RNase A (Watson Biotechnologies, Inc.); mouse anti-human Bcl-2 antibody (Beijing Zhongshan Biotechnology Co., Ltd.); anti-mouse Bcl-2 II labelled with alkaline phosphatase (Beijing Zhongshan Biotechnology Co., Ltd.); Tris

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Fig. 1. The molecular structure of solanine.

(Sigma); NP-40 (Fluka, US); PMSF (AMRESCO); aminoacetic acid (Watson Biotechnologies, Inc.); Tween-20 (AMRESCO); SDS (Sigma); sodium vandate (Beijing Chemical Reagent Co.); EGTA (Sigma); DTT (Sinopharm Group Chemical Reagent Co., Ltd.); sodium dithionite (Fuchen Chemical Reagent Co., Shanghai); DTNB (Sigma); ammonium persulphate (APS) (Wenzhou Chemical Materials Factory); TEMED (Sigma); leupeptin (Sigma); Coomassie brilliant blue G-250 (Kermel Chemical Reagents Co., Ltd., Tianjin); acrylamide (Institute for New and Pure Chemical Reagents, Tianjin); N,N-methylene bisacrylamide (Institute for New and Pure Chemical Reagents). The rest of the reagents used in the experiment were all analytically pure products produced in China. 2.3. Apparatuses CO2 incubator (CO-150, NBS, US); invert microscope (CKX-41-32, OLYMPUS, Japan); superclean table (SW-CJ-2F, Suzhou Purification Equipment Co.); confocal laser scanning microscope (CLSM) (SP-2, Leica, Germany); flow cytometer (EPICS XL-MCL, Beckman-Coulter, US); microplate reader (680, Bio-Rad, US); micromixer (TL-2000MM III) (Jiangyan Medical Equipments Co., Ltd., Jiangsu); electrophoresis analyzer (EC120/CSSU78, Bio-Rad); gel imaging system (GIS2019, Beijing Tianneng Instrument Factory); table refrigerated centrifuge (6R, Beckman-Coulter); electrorotator (DYCZ-40D, Beijing Liuyi Instrument Factory); centrifuge (LX-100, Haimen Qilin Instrument Co., Ltd., Jiangsu; nitrocellulose blotting membrane (NC membrane) (0.45 ␮m, Pall-Gelman, US); Whatman filter (3 mm, Watson Biotechnologies, Inc.). 2.4. Tumor cell lines Human hepatocarcinoma cell line HepG2 , human gastric carcinoma cell line SGC-7901, and human large intestine cancer cell line LS-174, all purchased from American Type Culture Collection, US, were provided by the Institute of Cancer Studies of Heilongjiang Cancer Hospital.

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cells LS-174 of suitable concentrations were inoculated in culture flasks containing RPMI 1640 solution with 10% of fetal bovine serum. The cultures were then incubated at 37 ◦ C, with 5% CO2 and saturated humidity. The cells were made to adhere to the inner wall of the flasks as they grew, and culture transfer was performed once every 2–3 days. The procedure for culture transfer is as follows: the culture medium was dumped from the flasks, which was then rinsed three times with PBS; after the cells were digested with pancreatin, fresh culture medium was added to the flasks, and the cells were blown and beaten to reach an even distribution; after the cell concentration was adjusted to suitable levels, the cells were transferred to a new set of culture flasks, and a suitable amount of culture medium was added to each. 2.6. The cytotoxic effect of solanine on HepG2 cells observed using MTT Human hepatocarcinoma cells HepG2 , human gastric carcinoma cells SGC-7901, and human large intestine cancer cells LS-174 taken in the stage of logarithmic growth were stained with trypan blue and their numbers were recorded. The percentage of cells that resisted staining ought to be above 97%. After the cell concentrations were adjusted to 1 × 104 ml−1 by adding RPMI 1640 culture solution containing 10% fetal bovine serum, the cells were transferred to 96-well culture plates at 100 ␮l/well, with six parallel wells for each kind of cells. The culture plates were then incubated in a CO2 incubator at 5% CO2 and 37 ◦ C for 24 h to make the cells adhere to the inner wall of the flask. The samples were divided into negative control, positive control (with camptothecin), and solanine groups. To the negative control was added RPMI 1640 at 100 ␮l/well; to the positive control was added camptothecin at 100 ␮l/well to reach a final concentration of 2 ␮g/ml; the treated groups were treated with solanine at 100 ␮l/well to reach final concentrations of 0.08, 0.4, 2, 10, and 50 ␮g/ml, respectively. The samples were then incubated in the CO2 incubator at 5% CO2 and 37 ◦ C for 48 h. After 48 h, the cells were taken out, the culture medium was discarded, and 1 mg/ml MTT was added at 100 ␮l/well, after which the cells were incubated for 4 h more. After 4 h, the liquid was discarded, DMSO was added at 150 ␮l/well, after which the samples were mounted on the micromixer for 5 min to make the blue granules in the samples dissolve thoroughly. The culture plate was then placed on the microplate reader, and OD was measured at 570 nm. The inhibition rate was calculated according to the following formula:

2.5. Cell incubation

inhibition rate (%)   average absorbance for treated group = 1− × 100 average absorbance for control group

Human hepatocarcinoma cells HepG2 , human gastric carcinoma cells SGC-7901, and human large intestine cancer

The experiment data were processed using Excell to yield IC50 values.

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2.7. Effect of solanine on the morphology of HepG2 (Peng and Wang, 2000) 2.7.1. Cell incubation and drug administration HepG2 cells taken in the stage of logarithmic growth were digested with pancreatin, and the cell concentration was adjusted to 1 × 104 ml−1 by adding RPMI 1640 culture medium with 10% fetal bovine serum. The sample was then put in Petri dishes at 200 ␮l/dish and incubated in CO2 incubator for 24 h to make the cells adhere to the inner wall of the dishes. The experiment was performed on a negative control, a positive control, and solanine groups. Drugs were administered 24 h after the plating. The solanine groups were treated with solanine of final concentrations of 0.016, 0.08, 0.4, 2, and 10 ␮g/ml, respectively, while the negative control was treated with an equal volume of solvent, and the positive control was treated with camptothecin with a final concentration of 0.08 ␮g/ml. The samples were then incubated in the CO2 incubator at 5% CO2 and 37 ◦ C for 48 h. 2.7.2. Staining and observation Forty-eight hours later, the cells were taken out of the incubator, and culture medium was sucked from the Petri dishes. The cells were rinsed with PBS three times for 5 min/time, and 200 ␮l of AO/EB was added to the samples to reach a final concentration of 5 ␮g/ml. The samples were then incubated at 37 ◦ C for 10 min, after which they were rinsed three times for 5 min/time. Two hundred microlitre of PBS was added to the samples, which were placed under the confocal laser scanning microscope so that the morphology of the cells can be observed. Dual-channel activation was used for the measurement, with an excitation wavelength of 488 nm and a radiation wavelength of 500–520 nm for PMT1 (AO) and an excitation wavelength of 543 nm and a radiation wavelength of 600–700 nm (Bernas et al., 2005) for PMT2 (EB). Objective APO CS40×/1.25 oil, zoom > 1, pinhole 1.5 Airy, mode XYZ, format 512 × 512. Serial scanning was used so as to eliminate interference due to spectral overlap. 2.8. Rate of apoptosis and cell cycle observed using flow cytometry (1) Plating and drug administration: HepG2 cells taken in the stage of logarithmic growth were digested with pancreatin, after which the cell concentration was adjusted to 5 × 105 ml−1 . The experiment was performed on a negative control, a positive control, and solanine groups. Plating was performed in a 50 ml culture flask for each group, and the flasks were incubated in a CO2 incubator at 5% CO2 and 37 ◦ C for 24 h so that the cells could adhere to the inner wall of the flask. Drugs were administered after 24 h. The negative control was treated with an equal volume of solvent, and the positive control was treated with camptothecin with a final concentration of 0.08 ␮g/ml, while the solanine groups were treated with solanine of a final concentration of 0.016, 0.08, 0.4, 2, and 10 ␮g/ml, respectively. The samples were then incubated in the CO2 incubator at 5% CO2 and 37 ◦ C for 48 h.

(2) After being digested by pancreatin, HepG2 cells were rinsed three times with PBS. The supernatant was discarded, and the residue was blown into a small amount of PBS and mixed well with it, after which 2 ml of cold ethanol (70%) was added to fixate the cells. The sample was then left at 4 ◦ C for the night (the time for fixation was to be more than 12 h). (3) The samples were centrifuged the next day, the supernatant was discarded, and the residue was rinsed three times with PBS. Eight hundred microlitre of PI staining solution was added to and thoroughly mixed with the samples, which were then left to be stained in dark for 30 min. After being filtered through a nylon net, the samples were measured with a flow cytometer, with an excitation wavelength of 488 nm (Zhang et al., 2004; Stervbo et al., 2006). 2.9. Effect of solanine on the expression of Bcl-2 protein measured by Western blot 2.9.1. Cell incubation and drug administration HepG2 cells taken in the stage of logarithmic growth were digested with 0.25% pancreatin, and the cell concentration was adjusted to 5 × 105 ml−1 by adding RPMI 1640 containing 10% fetal bovine serum. The cell suspension was then incubated in 100 ml culture flasks. The samples were incubated in CO2 incubator for 24 h at 5% CO2 and 37 ◦ C, after which the solanine groups were treated with solanine of different concentrations, with final concentrations of 0.0032, 0.016, 0.08, 0.4, and 2 ␮g/ml, respectively, while the negative control was treated with solvent of an equal volume. The samples were then incubated in CO2 incubator for 48 h at 5% CO2 and 37 ◦ C. 2.9.2. Preparation of the samples One millilitre of PBS was added to the culture flask. The cells were scraped using a cell scraper, added to a 1.5 ml centrifuge tube, and centrifuged at 1500 rpm for 5 min, after which the sample was rinsed twice with PBS. One hundred microlitre of cell lysate was added to the sample, which was then whirled, after which the sample was lysed from 30 min in an iced waterbath. It was then centrifuged for 10 min at 4 ◦ C and 12,000 × g. The supernatant was collected and refrigerated at −20 ◦ C for later use. 2.9.3. Determination of the protein content The Bradford method (Kruger, 1994) was used to determine the protein content. 2.9.4. Electrophoresis 1) Twelve percent Resolving Gel and five percent Stacking Gel were prepared. 2) Specimens were taken from the samples according to different protein contents, SDS buffer solution was added and mixed with the specimens, after which the specimens were boiled for 5 min in 100 ◦ C water bath. The specimens were mounted after being cooled down.

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3) The gel was run at a voltage of 80 V, which was then changed to 110 V after the specimens had reached the separation gel. 2.9.5. Transfer Following electrophoresis, the gel was peeled and cut off, and then transferred for 2 h at 50 mA. The membrane was taken out, sealed with TTBS containing 3% BSA, and then set under 4 ◦ C for the night. 2.9.6. Coloration and photographing The membrane was rinsed with TTBS three times for 5 min/time, after which murine anti-human Bcl-2 antibody I diluted 1:250 was added. Three hours later, the membrane was again rinsed with TTBS three times for 5 min/time, after which anti-murine IgG antibody II diluted 1:250 and labelled with alkaline phosphatase was added. The membrane was taken out after 2 h and rinsed three times with TBS, 5 min each time. Coloration was conducted after 3.3 ␮l of BCIP and 6.6 ␮l of NBT were mixed with 1 ml of alkaline phosphatase buffer, photographs were taken using the gel imaging system, and the protein content was quantified. With the protein content of the negative control as 100%, the data thus obtained were used to calculate the protein content of each specimen (expressed in percentage) relative to the negative control. 2.10. Data processing All values are expressed as means ± S.E., and all statistical analysis was performed by analysis of variance (ANOVA). Pvalues less than 0.05 were considered to be significant. 3. Results 3.1. IC50 of solanine for the three tumor cell lines

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Table 2 Effect of solanine on the cell cycle and apoptosis rate of HepG2 cells Drug concentration (␮g/ml)

G0 /G1

S

G2 /M

Apoptosis rate (%)

13.36 0

– 21.9

Control CAM

– 0.08

66.80 57.12

19.84 42.88

Solanine

0.016 0.08 0.4 2 10

73.56 55.08 58.67 72.00 64.23

26.44 44.92 41.33 28.02 35.76

0 0 0 0 0

6.0 14.4 17.3 18.9 32.2

and both the window for PMT1 and that for PMT2 stained. For the 0.08, 0.4, 2, and 10 ␮g/ml solanine groups, cell morphology further changed into rather irregular, crumb-like structures, nuclear fragmentation occurred, and apoptotic bodies appeared, all typical signs for apoptosis. 3.3. Rate of apoptosis and cell cycle determined using flow cytometry The results are shown in Fig. 2 and Table 2. For the negative control, there were peaks for the G0 /G1 , S, and G2 /M phases; for the solanine groups, the G2 /M peak disappeared, and a sub-G0 peak of apoptosis appeared prior to the G0 /G1 phase. With increasing dosage, the rate of apoptosis gradually increased, to 6.0, 14.4, 17.3, 18.9, and 32.2%, respectively, for the 0.016, 0.08, 0.4, 2, and 10 ␮g/ml solanine groups. For the positive control, or the 0.08 ␮g/ml camptothecin group, the rate of apoptosis was 21.9%. Analysis of the cell cycle shows that the G2 /M phase disappeared for the solanine groups, and the proportion of the S phase increased, which means that solanine blocked the cell cycle of the tumor cells at the phase of cell division.

MTT measurements show that HepG2 cells are rather sensitive to solanine (Table 1).

3.4. The effect of solanine on the expression of Bcl-2 protein in the HepG2 cells line observed using Western blot

3.2. Effect of solanine on the morphology of HepG2 cells

Western blot analysis shows that with the increase in the dosage of solanine administered, the content of Bcl-2 protein in HepG2 cells gradually decreased.

The results are shown in Table 2. For cells in the negative control, PMT1 showed a green fluorescence, and the cells had a round shape and its DNA structure intact, and there was hardly any florescence for PMT2. The morphology of cells in the solanine groups was significantly different from the negative control. The 0.016 ␮g/ml solanine group showed a slight change in cell morphology, with the periphery being very rough and showing effervescence, the permeability of cell membrane increased, Table 1 The IC50 of solanine for HepG2 , SGC-7901, and LS-174 cells Compound

Solanine CAM

IC50 (␮g/ml) HepG2

SGC-7901

LS-174

14.47 1.03

>50 19.19

>50 13.96

4. Discussion In China, people eat roasted young plants and tender leaves of the nightshade. Ripe fruits of the plant are also edible. For medicinal purposes in folk medicine, people pick young plants or tender leaves of the nightshade, pound them to pieces, and apply it externally to cure sores, carbuncles, swellings, and injuries and factures. Studies on the alkaloids of the plant, such as solasonine and solamargine, have shown that they have an anti-tumor effect (Kuo et al., 2000; Berek et al., 2001; Lee et al., 2004; Liang et al., 2004; Liu et al., 2004), but there have been relatively few reports on solanine. Solanine is a steroid alkaloid. Research has shown that the anti-tumor effect of many steroid alkaloids has to do with their

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Fig. 2. Changes in HepG2 cell morphology caused by solanine. (A) Negative control, (B) 0.016 ␮g/ml solanine, (C) 0.08 ␮g/ml solanine, (D) 0.4 ␮g/ml solanine, (E) 2 ␮g/ml solanine, (F) 10 ␮g/ml solanine, (G) 0.08 ␮g/ml CAM.

induction of apoptosis in tumor cells (Ambrosio et al., 2000; Jordan et al., 2000; Koike and Ninomiya, 2000; Koike et al., 2000). Based on this, we have, in this study, first observed the cytotoxic effect of solanine on several tumor cell lines from the digestive tracts using MTT, screened for a sensitive cell line, and then observed the effect of solanine in inducing apoptosis in tumor cells by measuring the morphological changes, apoptosis rate, and cell cycle of the tumor cells.

In the cytotoxicity experiment, MTT was used to measure the cytotoxic effect of solanine of different concentrations (0.08, 0.4, 2, 10, 50 ␮g/ml, respectively) on 3 tumor cell lines from the digestive system, namely HepG2 , SGC-7901, and LS-174. Results from the experiment show that the IC50 for the three tumor cell lines were 14.47, >50, and >50 ␮g/ml (Table 1), respectively. Therefore, HepG2 cells evidence the greatest sensitivity toward the cytotoxic effect of solanine.

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In the experiment to observe cell morphology, AO/EB double staining and confocal laser scanning microscopy were used to observe the effect of solanine on HepG2 cells. The main principles involved are the following: First, acridine orange (AO) can go through intact cell membrane and get embedded in the nuclear DNA, which then emits a green fluorescence; while ethidium bromide (EB) can go through only damaged cell membrane and get embedded in the nuclear DNA, which then emits a red fluorescence. Secondly, tumor cells have a round shape and an intact DNA structure, while apoptotic tumor cells have irregular shapes, with DNA fragmentation and the appearance of apoptotic bodies. Thus, when observed under a confocal laser scanning microscope, four kinds of cell morphology were observed. (1) Cells in the negative control group: These cells had a normal morphology with a round shape outside, and were stained with AO (which emits a green fluorescence) but not with EB. (2) Dead cells: These cells had a normal morphology with a round shape outside. They were stained with both AO, which emits a green fluorescence, and EB, which emits a red fluorescence. (3) Cells in an early stage of apoptosis: These cells had a slightly pyknotic and crump-like morphology, which increased the permeability of the cells. Both AO and EB had entered these cells, making the cells emit both a green fluorescence and a red fluorescence. (4) Cells in a late stage of apoptosis: The structure of these cells was further damaged. Not only were the cells stained with both AO and EB, but the morphology of the nuclei was similar to that for the cells in the early stage of apoptosis, being either heavily stained fragments (due to nuclear fragmentation) or apoptotic bodies. In this experiment, morphological changes in tumor cells acted upon by solanine with concentrations of 0.016, 0.08, 0.4, 2, and 10 ␮g/ml, respectively, were observed. Both channels (PMT1 and PMT2) were used for the confocal laser scanning microscopy, with PMT1 showing the fluorescence emitted by AO and PMT2 showing that emitted by EB. The results are shown in Fig. 2, where PMT1 shows a green fluorescence for cells in the negative control, the cells are round outside and have an intact DNA structure, while PMT2 shows hardly any fluorescence. The morphology of the cells in the solanine groups differs significantly from that for the negative control. The 0.016 ␮g/ml solanine group showed a slight change in cell morphology, with a pyknotic nucleus and a very rough periphery, showing effervescence. Both the window for PMT1 and that for PMT2 were stained. For the 0.08, 0.4, and 2 ␮g/ml solanine groups, cell morphology changed further with increasing dosage, with nuclear fragmentation and the appearance of apoptotic bodies. For the 10 ␮g/ml solanine group, the cell morphology became completely diffused, and there were even more cell fragments and apoptotic bodies. The morphological study shows intuitively that solanine can induce apoptosis in tumor cells. Flow cytometry is a method for analyzing the physical, chemical, and biological properties of cells and other bioparticles using the flow cytometer. It can show apoptosis of cells quantitatively. In this experiment, flow cytometry with PI staining was used to measure the apoptosis of HepG2 cells after they had been acted on by solanine for 48 h. Fig. 3A shows the sit-

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uation with the negative control, where there are three phases, namely G0 /G1 , S, and G2 /M, and where there is no apoptosis peak. The solanine groups, on the other hand, all show sub-G0 apoptosis peak prior to the G0 /G1 phase; and, with increasing dosage, the apoptosis rate gradually increased from 6.0 to 32.2% in a dosage-dependent manner. In the camptothecin group, an apoptosis peak also appeared, with an apoptosis rate of 21.9%. Because PI stains only cells in the late stage of apoptosis, the actual apoptosis rate should be greater than what was measured. Statistical analysis of cell cycle of the various groups show that the G2 /M phase disappeared in all the solanine groups, while S phase all increased relative to the negative control. Thus, it can be inferred that solanine can stop the cell in the S phase, or it can block the HepG2 cells in the stage of cell division so that they will not be able to enter the next cycle, thus preventing cell division from being completed and achieving the goal of inhibiting tumor development. Bcl-2 is an anti-apoptosis gene (O’Neill and Hockenbery, 2003; Cory and Adams, 2005; Fathi et al., 2006; Zhang et al., 2006; Karlnoski et al., 2007). Located at 18q21, it has three exons and two open-reading frames (promoters), and is closely related to cell apoptosis (Bernas et al., 2005; Yoshida et al., 2006), as well as closely associated with the mitochondrion (Degli Esposti, 2004; Dias and Bailly, 2005). Immunohistochemical study and observation using confocal microscopes and electronic microscopes have shown that Bcl-2 protein is found in various membranes in the cell, such as the nucleus membrane, membrane of the endoplasmic reticulum, and the mitochondrial membrane, with the largest quantity of it being found on the mitochondrial membrane. However, Hockenbery et al. (1990) believe that Bcl-2 protein is a protein on the inner membrane of the mitochondrion (Harris and Thompson, 2000), and that mitochondrial Bcl-2 protein are mostly located at the PT pores, where they are capable of inhibiting changes in the permeability of the mitochondrion and thus inhibiting cell apoptosis. At the same time, Bcl-2 protein can also inhibit the release of cytochrome C (Orrenius, 2004), thus preventing the activation of caspase (Shimizu et al., 1999; Li and Wang, 2006), which would also lead to the inhibition of apoptosis. The analysis of the effect of solanine on the expression of Bcl-2 protein in HepG2 cells using Western blot shows that solanine can inhibit Bcl-2 activity, or, as the dosage of solanine increases, the expression of Bcl-2 protein is lowered, as shown in Fig. 4. Bcl-2 is an anti-apoptosis gene that indirectly inhibits apoptosis by inhibiting the opening of PT channels on the mitochondrion and hence the lowering of mitochondrial potential (Kim et al., 1997; Rosse et al., 1998), and by blocking the release of cytochrome C from the mitochondrion. The inhibitory effect of solanine on Bcl-2 ultimately leads to an increase in the content of cytochrome C, which directly activates the cascade reaction of caspase. From the analysis above, we conclude the following: HepG2 cells are relatively sensitive to the cytotoxic effect of solanine which induces apoptosis in HepG2 cells by inhibiting the expression of Bcl-2 protein. Solanine can inhibit the proliferation of human liver cancer cells HepG2 in vitro, but it has a potent toxicity (LD 50:45 mg/kg

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Fig. 3. The rate of apoptosis in HepG2 cells induced by solanine as measured using flow cytometry with PI staining. (A) Negative control, (B) 0.016 ␮g/ml solanine, (C) 0.08 ␮g/ml solanine, (D) 0.4 ␮g/ml solanine, (E) 2 ␮g/ml solanine, (F) 10 ␮g/ml solanine, (G) 0.08 ␮g/ml CAM.

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Fig. 3. (Continued ).

Fig. 4. Western blot analysis of the expression of Bcl-2 protein in the HepG2 cell line.

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