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Antihepatoma activity of Physalis angulata and P. peruviana extracts and their effects on apoptosis in human Hep G2 cells
Life Sciences 74 (2004) 2061 – 2073 www.elsevier.com/locate/lifescie
Antihepatoma activity of Physalis angulata and P. peruviana extracts and their effects on apoptosis in human Hep G2 cells Shu-Jing Wu a,b, Lean-Teik Ng c, Ching-Hsein Chen d, Doung-Liang Lin e, Shyh-Shyan Wang e, Chun-Ching Lin a,* a
Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC b Chia-Nan Pharmacy University, Health Nutrition Department, Taiwan, ROC c Department of Food Science and Technology, Tajen Institute of Technology, Pingtung, Taiwan, ROC d Department of Medical Technology, Fooyin University, Kaohsiung, Taiwan, ROC e Tainan District Agricultural Improvement Station, Taiwan, ROC Received 31 March 2003; accepted 24 September 2003
Abstract Physalis angulata and P. peruviana are herbs widely used in folk medicine. In this study, the aqueous and ethanol extracts prepared from the whole plant of these species were evaluated for their antihepatoma activity. Using XTT assay, three human hepatoma cells, namely Hep G2, Hep 3B and PLC/PRF/5 were tested. The results showed that ethanol extract of P. peruviana (EEPP) possessed the lowest IC50 value against the Hep G2 cells. Interestingly, all extracts showed no cytotoxic effect on normal mouse liver cells. Treatment with carbonyl cyanide m-chlorophenyl hydrazone, a protonophore, caused a reduction of membrane potential (Dcm) by mitochondrial membrane depolarization. At high concentrations, EEPP was shown to induce cell cycle arrest and apoptosis through mitochondrial dysfunction as demonstrated by the following observations: (i) EEPP induced the collapse of Dcm and the depletion of glutathione content in a dose dependent manner; (ii) pretreatment with the antioxidant (1.0 Ag/ml vitamin E) protected cells from EEPP-induced release of ROS; and (iii) at concentrations 10 to 50 Ag/ ml, EEPP displayed a dose-dependent accumulation of the Sub-G1 peak (hypoploid) and caused G0/G1-phase arrest. Apoptosis was elicited when the cells were treated with 50 Ag/ml EEPP as characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane. The results conclude that EEPP possesses potent antihepatoma activity and its effect on apoptosis is associated with mitochondrial dysfunction. D 2004 Elsevier Inc. All rights reserved. Keywords: Physalis angulata; P. peruviana; Reactive oxygen species; Dcm; Apoptosis
* Corresponding author. Tel.: +886-7-3121101x2122; fax: +886-7-3135215. E-mail address: [email protected] (C.-C. Lin). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.09.058
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Introduction Apoptosis is a highly organized process, defined by a number of morphological and biochemical changes including membrane bubbling, cell shrinkage, chromatin condensation, DNA fragmentation and appearance of phosphatidylserine in the outer side of the plasma membrane (Schulze-Osthoff et al., 1998; Kim et al., 1999; Park et al., 2002). Reactive oxygen species (ROS) such as superoxide anion (.O2 ), hydrogen peroxide (H2O2), hydroxyl radical (.OH), singlet oxygen, organic peroxide radicals, and nitric oxide generation are recognized as mediators of the apoptotic signaling pathway (Li et al., 2003). Mitochondria are known to be a major physiological source of ROS, which are generated during mitochondrial respiration and have been implicated as mediators of apoptotic signaling pathway. The cellular generation of ROS has been associated with, or contributes to, human disease states such as inflammatory diseases, neurodegenerative diseases, ischemia-reperfusion injury, cancer and aging (Zhu et al., 1994; Nakamura et al., 2002; Siraki et al., 2002). Mitochondrial dysfunction causes energy impairment and/or oxidative stress, and also contributes to the early and common process of apoptotic cell death. The diverse pro-apoptosis stimuli converging on mitochondria cause mitochondrial permeability transition, mitochondrial depolarization, intracellular glutathione depletion and cytochrome c release, are critical events provoking a caspase cascade and eventually lead to cell death (Kroemer and Reed, 2002; Chang et al., 2002; Mari et al., 2002; Wu et al., 2002; Yang et al., 2003). In addition, the collapse of mitochondrial membrane potential and the alteration of oxidation-reduction status have also been noted in a wide variety of cell types (Nakamura et al., 2002; Siraki et al., 2002; Yang et al., 2003). Physalis angulata and P. peruviana have been widely used in folk medicine as anticancer, antimycobacterial, antileukemic, antipyretic, immunomodulatory, and for treating diseases such as malaria, asthma, hepatitis, dermatitis, diuretic and rheumatism (Chiang et al., 1992a, 1992b; Lin et al., 1992; Pietro et al., 2000; Ismail and Alam, 2001; Soares et al., 2003). Antitumor activities of purified compounds, physalins (ex. physalins A, B, D and F) and glycosides (ex. myricetin-3-O-neohesperidoside), have been isolated from the organic fractions of P. angulata (Chiang et al., 1992a, 1992b; Ismail and Alam, 2001; Soares et al., 2003). They were shown to exert anticancer activity on HA 22T (hepatoma), HeLa (cervix uteri), leukemia, lung adenocarcinoma, and epidermoid carcinoma of the nasopharynx KB-16 cells lines (Chiang et al., 1992a, 1992b; Ismail and Alam, 2001). However, the mechanism responsible for the anticancer effect remains unknown. The aims of this study were: (i) to evaluate the antihepatoma activity of aqueous and ethanol extracts of P. angulata and P. peruviana in three human hepatoma cell lines, namely Hep G2, Hep 3B and PLC/ PRF/5; (ii) to identify the mechanistic effects of ethanol extract of P. peruviana induced apoptosis in human Hep G2 cells; and (iii) to study the mitochondrial permeability transition induced by carbonyl cyanide m-chlorophenyl hydrazone and its mediation of the mitochondrial depolarization in cells.
Materials and methods Materials The plants of Physalis angulata L. and Physalis peruviana L. of the family Solanaceae were obtained from Tainan District Agriculture Improvement Station, Taiwan. The authenticity of the plant species was
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confirmed by Professor CC Lin of Graduate Institute of Natural Products, Kaohsiung Medical University, Taiwan. The whole plants were dried and ground to sawdust form, which was then kept in air-tight brown bottle until use. Preparation of extracts Aqueous extract One hundred gram (100 g) of each sample was extracted with 1 L of boiling water for 1 h. The extracts were filtered while the residue was re-extracted under the same conditions twice. The filtrates collected were combined and evaporated to dryness under vacuum. The yield of aqueous extract for P. angulata and P. peruviana were 25.60% and 20.73%, respectively. Ethanol extract One hundred gram (100 g) of P. angulata and P. peruviana sawdust was soaked with 95% ethanol (1 L) at room temperature for 3 days. The samples were filtered with filter paper (Advantec No. 1, Japan) while the residue was further extracted under the same conditions twice. The filtrates collected from three separate extractions were combined and evaporated to dryness under vacuum. The yield obtained for P. angulata and P. peruviana were 19.68% and 24.00%, respectively. Chemicals 5-Fluorouracil (5-FU), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), Dulbecco’s modified Eagle’ medium (DMEM), dimethyl sulfoxide (DMSO), sodium 3V-[1-(phenyl-amino-carbonyl)-3,4tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid (XTT), penicillin, propidium iodide, ribonuclease A (RNase A), sodium dodecyl sulfate, streptomycin and trypsin-EDTA were purchased from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS) was obtained from GIBCO BRL (Gaithersburg, MD). 2V,7V-dichlorodihydrofluorescein (H2DCFDA), rhodamine 123 (Rh-123) and chloromethyl derivatives of fluorescein diacetate (CMFDA) were from Molecular Probes (Eugene, OR). Cell cultures The human hepatoma Hep G2, Hep 3B, PLC/PRF/5 cells and the mouse BALB/C normal liver cells were obtained from the American Type Culture Collection (Rockville, MD). All cells were grown in 90% DMEM supplemented with 10% FBS, 100 units/ml penicillin and 100 Ag/ml streptomycin. The cultures were maintained at 37 jC in a humidified atmosphere of 5% CO2. Antihepatoma activity assay For XTT assay, each cell was seeded at a density of 1 105/well onto 96-well culture plates, and left to adhere to the plastic plates overnight before being exposed to 5-FU (a positive control), 0.1% DMSO (control) and various concentrations (10, 30 and 50 Ag/ml) of P. angulata and P. peruviana extracts. After 48 h of treatment, 150 Al of XTT solution were added to each well and incubated for 4 h. The
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absorbance was measured with an ELISA reader (Multiskan EX, Labsystems) at a test wavelength of 492 nm and a reference wavelength of 690 nm (Goodwin et al., 1995; Scudiero et al., 1998). Cytotoxicity assay The effect of P. angulata and P. peruviana extracts on the viability of mouse BALB/C normal liver cell was measured by the XTT method (Scudiero et al., 1998). The cytotoxic concentration of each extract toward BALB/C normal liver cell lines was calculated by the following formula: % Survival Cell ¼ ODT =ODC 100% The ODT and ODC indicate the absorbance of the test extract-treated group and the control group (0.1% DMSO), respectively. The 50% cytotoxic concentration (CC50), which was expressed as the concentration that achieved 50% cytotoxicity against BALB/C normal liver cell lines, was calculated from the regression line. With the IC50 and CC50 data, the selectivity index for each extract against the different hepatoma cells was calculated as follows: Selectivity Index ¼ CC50 =IC50 Measurement of mitochondrial membrane potential (Dwm) Dwm was measured with Rh-123 using flow cytometry (Coulter Epics Elite ESP, FL, USA). With its high negative charges, Rh-123 will accumulates in normal mitochondria whereas the reduction of Dwm will lead to the release of Rh-123 and the reduction of its fluorescence intensity (Castedo et al., 1996). After 48 h of 10~50 lg/ml EEPP treatment, cells were collected using cell scraper and washed with phosphate-buffered saline (PBS) twice. Cells (5 105/ml) were incubated with Rh-123 (5 mg/ml) for 30 min at 37C, they were then washed with PBS once, followed by subjecting to analysis by an air-cooled argon 488 nm laser with a 525 nm band pass filter and a 550 nm dichroic mirror as detectors. Carbonyl cyanide m-chlorophenyl hydrazone, at a concentration of 10 AM, was used as a positive control for membrane depolarization. Each group has at least 10,000 cells subjecting to treatment with 0.1% DMSO (control) and various concentrations of EEPP (10 and 50 Ag/ml). After 48 h of treatment, cells were washed once with PBS, then re-suspended in PBS, they were then stained with 50 Ag/ml of propidium iodide for 30 min at 37jC and left in the dark for reaction at 4jC. At the end of reaction, the samples were analyzed by a flow cytometer. Data obtained from flow cytometry were analyzed using WinMDI 2.7 software (Scripps Research Institute, La Jolla, CA, USA). Flow cytometry detection of glutathione The cell density was adjusted to 5 105/dish on a 60-mm culture dish. Details of the methods have been previously described (Chang et al., 2002). In brief, cells were detached from
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dishes by treatment with trypsin. They were then collected by centrifugation and re-suspended with PBS. The cells were stained with 25 AM CMFDA at 37jC for 10 min in the dark and the fluorescence was measured by flow cytometry. Based on the criteria of acquisition, a minimum of 10,000 cells in each sample was collected, and the mean fluorescence was estimated by WinMDI 2.7 software. Flow cytometry detection of cell cycle After each treatment, both floating and adherent cells were collected. The cells in suspension were fixed with 70% ice-cold methanol and then transferred to the freezer until use, followed by washing with PBS. Cells were stained with 50 Ag/ml propidium iodide in the presence of 25 Ag/ ml RNase A at 37jC for 30 min. A minimum of 10,000 cells per sample was collected, and the DNA histograms were further analyzed by a flow cytometer. Multicycle software (Phoenix Flow Systems, San Diego, CA) was used to estimate the percentage of each phase in cell cycle. The effect on apoptosis was determined by the degree of increase in the proportion of sub-G1 (hypodiploid) cells. Measurement of annexin V binding The measurement of phosphatidylserine redistribution in a plasma membrane was conducted according to the protocol outlined by the manufacturer in the Annexin V-FITC Apoptosis Detection kit (Sigma Chemical Co, USA). In brief, 1 106 cells were washed and re-suspended in HEPES buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 5 mM CaCl2) containing annexin V-FITC (1:50) and 1 Ag/ml of propodium iodide for 15 min, before being analyzed with a flow cytometer. Intracellular ROS determination Briefly, the cells under confluency were treated with 0.1% DMSO, 50 Ag/ml EEPP, 1.0 Ag/ml vitamin E, and 1.0 Ag/ml vitamin E added to 50 Ag/ml EEPP for 48 h at 37jC. After washing with PBS twice, 10 AM of H2DCFDA was applied to the cells, which were then incubated for 30 min. A flow cytometer was used to detect the fluorescent dichlorofluoresein. Morphological detection of apoptotic cells Cells were untreated or treated with 50 Ag/ml EEPP for 48 h, they were then fixed with 3.0% (w/v) paraformaldehyde at room temperature for 20 min. After washing with PBS, cells were stained with 0.12 Ag/ml Hoechst 33258 for 15 min. Morphological changes were then observed under fluorescent microscopy (Zeiss Axioskop, Mikron Instruments, NY). Statistical analysis Results are expressed as mean F SD from at least three independent experiments. Statistical analysis was performed according to Student’s t-test by one-way analysis of variance. Significant difference was taken as p < 0.05 or p < 0.01.
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Results Antihepatoma activity The effects of aqueous and ethanol extracts of P. angulata and P. peruviana on Hep G2, Hep 3B and PLC/PRF/5 are shown in Table 1. In general, the ethanol extracts exhibited a lower IC50 value than the aqueous extracts, with the lowest IC50 value (9.43 F 0.30 Ag/ml) from the ethanol extract of P. peruviana (EEPP). With the exception of P. angulata ethanol extract, the IC50 values of other extracts for PLC/PRF/5 cell lines were >100 Ag/ml. For Hep 3B cells, ethanol extract of P. angulata showed the lowest IC50 value (25.40 F 0.25 Ag/ml). Among the different plant extracts and cell lines tested, EEPP exhibited the strongest antihepatoma activity against Hep G2 cells. Cytotoxicity assay Cytotoxicity assay was performed to ensure that the anticancer activity of extracts of P. angulata and P. peruviana and 5-FU were not caused by cytotoxic effects of herbal medicine toward the mouse BALB/C normal liver cells. The results showed that CC50 values of ethanol extracts (1278-2862 Ag/ml) were lower than the aqueous extracts (4297-5800 Ag/ml) of P. angulata and P. peruviana (Table 1). Selectivity index The selectivity index is used to evaluate the safety of a drug when applied in a biological system. As shown in Table 1, the SI values of the tested extracts vary from 119 to 300, suggesting that these extracts exhibit no cytotoxic effect on mouse BALB/C normal liver cells. Flow cytometry detection of cell cycle The effect of EEPP on the cell cycle was determined in Hep G2 cells by flow cytometry. After 48 h of treatment, EEPP induced cell cycle arrest (G0/G1 phase) and decreased in the S phase of Hep G2 cells in Table 1 The IC50 and cytotoxicity values of aqueous and ethanol extracts of P. angulata (PA) and P. peruviana (PP) Plant extracts
IC50 value (Ag/ml)a Hep G2
5-FU PA (water) PP (water) PA (EtOH) PP (EtOH)
1.24 44.19 29.89 10.67 9.43
F F F F F
0.31 0.52 0.60 0.20 0.30
PLC/PRF/5
Hep 3B
Cytotoxicityb CC50(Ag/ml)
1.21 F 0.90 >100.00 >100.00 41.60 F 0.3 >100.00
1.72 F 0.10 >100.00 43.06 F 0.93 25.40 F 0.25 41.25 F 1.40
123.04 5800.69 4297.72 1278.12 2862.00
F F F F F
0.10 0.45 0.09 0.12 0.07
Selectivity indexc (SI) 99.23 131.26 143.78 119.79 303.50
Each value represents the mean F SD of three independent experiments. a Antihepatoma activity was determined by XTT assay. 50% inhibition concentration (IC50) was the concentration of crude extracts of P. angulata and P. peruviana that inhibit 50% growth of the three human hepatoma cell lines. b Cytotixic concentration (CC50) was the concentration of crude extracts of P. angulata and P. peruviana that achieved 50% cytotoxicity against mouse BALB/C normal liver cells.
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Table 2 Effects of the ethanol extract of P. peruviana (EEPP) on cell-cycle progression Test samples
G0/G1
Control (0.1% DMSO) EEPP (10 Ag/ml) EEPP (30 Ag/ml) EEPP (50 Ag/ml)
51.07 66.30 71.47 74.00
S F F F F
0.48 0.50* 0.88** 3.59**
38.00 18.67 17.93 7.73
G2/M F F F F
1.68 1.91** 0.98** 0.39**
10.93 15.03 10.60 18.27
F F F F
1.54 1.23* 0.22 2.54**
Data are mean F SD obtained from three independent experiments. The asterisk indicates a significant difference between control and EEPP-treated cells based on Student’s t-tests (*p < 0.05, **p < 0.01).
a dose response manner (Table 2). In addition, the control group (0.1% DMSO) and 10~50 Ag/ml EEPP displayed a dose-dependent accumulation of the Sub-G1 peak (hypoploid) by increasing from 0.21% to 60.29% (Fig. 1).
Fig. 1. Cell cycle distribution in Hep G2 cells after ethanol extract of P. peruviana (EEPP) treatment. 5 105 cells were treated with different concentrations of EEPP for 48 h. (A) control (0.1% DMSO), (B) 10 Ag/ml, (C) 30 Ag/ml, and (D) 50 Ag/ml. The graph was obtained from representative data of three individual experiments.
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Induction of apoptosis by EEPP To further confirm that EEPP leads to apoptosis, Hep G2 cells were stained with annexin V-FITC and propidium iodide, and subsequently analyzed by flow cytometry. The annexin V assay measures phospholipid turnover from the inner to the outer lipid layer of the plasma membrane, an event typically associated with apoptosis. As indicated in Fig. 2, 10 and 50 g/ml EEPP treatment displayed the population of apoptotic cells (29.33% and 65.19%, annexin V+/PI – ). Using FACS analysis, the proportion of annexin V-staining cells was found to increase in 10 f 50 Ag/ml EEPP-treated cells. The percentage of sub-G1 and annexin V-staining cells were 22.00% and 29.33% respectively in 10 Ag/ ml EEPP-treated cells, and 60.29% and 65.19% respectively in 50 Ag/ml EEPP-treated cells (Fig. 1 and Fig. 2). The apoptotic cell death by EEPP at 50 Ag/ml was stained with Hoechst 33258, and confirmed by nuclei morphology using the fluorescence microscopy. Morphological assessment was used to
Fig. 2. Induction of apoptosis by ethanol extract of P. peruviana (EEPP). Flow cytometric analysis of annexin V-FITC and propidium iodide double stained cells. Cells were untreated or treated with 10 and 50 Ag/ml EEPP for 48 h. Data represent the percentage of aopototic cells from three determinations.
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Fig. 3. Changes in the morphology of Hep G2 cells treated with ethanol extract of P. peruviana (EEPP) under a fluorescence microscopy. Morphological changes in untreated cells (A) and cells treated with 50 Ag/ml EEPP (B) for 48 h were examined by staining with Hoechst 33258. The nuclei feature of control cells showed the round and homogeneous nuclei, the apoptotic cells displayed the condensation and fragmentation of nuclei in the EEPP-treated cells (the arrow point).
display the nuclei fragmentation and compaction of chromatin in EEPP-induced cell death (Fig. 3). The results have demonstrated that 50 Ag/ml EEPP induced apoptosis. Apoptotic-related mitochondrial transmembrane potential (Dwm) To investigate the effect of EEPP on mitochondria, Hep G2 cells were treated with EEPP for 48 h. We measured the membrane potential by staining with Rh-123. As shown in Fig. 4, the percentage of total cell counts exhibited a decrease in Dwm. The mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone (used as a positive control for Dcm disruption) also induced a significant change in Dcm. EEPP remarkably reduced the uptake of the fluorescent dye in Hep G2 cells in a dose-dependent manner (Fig. 4). The loss of Dcm was maximal with 50 Ag/ml EEPP and a slow decline with 10 Ag/ml EEPP.
Fig. 4. Effect of the ethanol extract of P. peruviana (EEPP) on dissipation of mitochondrial transmembrane potential (Dcm) in Hep G2 cells as demonstrated by flow cytometry. Cells were treated with 10 and 50 Ag/ml of EEPP for 48 h. Cells exposed to 10 AM CCCP were used as positive control. The asterisk indicates a significant difference between control and EEPP-treated cells based on Student’s t-tests (*p < 0.05, **p < 0.01).
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Fig. 5. Effect of the ethanol extract of P. peruviana (EEPP) on cellular glutathione content (GSH). Hep G2 cells untreated or treated with EEPP at 10 and 50 Ag/ml for 48 h were collected and stained with CMFDA followed by analyzing with flow cytometry. Data were obtained from three independent experiments and are expressed as mean F S.D. The asterisk indicates a significant difference between control and EEPP-treated cells based on Student’s t-tests (**p < 0.01).
Involvement of redox alteration in EEPP-induced apoptosis It has been suggested that the alterations of mitochondria appeared, at least in part, to be regulated by the oxidation-reduction status such as the depletion of intracellular glutathione (GSH) content and intracellular ROS level in cells. By using a GSH-reactive dye, we examined the effects of the EEPPinduced apoptosis in cellular GSH content. In Hep G2 cells, GSH content was significantly declined after EEPP treatment (Fig. 5). After staining with H2DCFDA (a fluorescent dye for peroxides, H2O2), the results displayed a significant ROS generation at 50 Ag/ml EEPP as compared with the control (Table 3). We further investigated the involvement of ROS by treating the cells with an antioxidant (1.0 Ag/ml vitamin E) before treatment with 50 Ag/ml EEPP or treated with only 1.0 Ag/ml of vitamin E for 48 h. Treatment with 50 Ag/ml EEPP resulted in an increase in the mean fluorescence intensity of 219.76 F 4.92 (a.u), and pretreatment with 1.0 Ag/ml vitamin E before 50 Ag/ml EEPP treatment showed a reduction in intensity to 58.18 F 0.95 (a.u), which was closed to a single 1.0 Ag/ml vitamin E-treated
Table 3 Effect of the ethanol extract of P. peruviana (EEPP) on ROS production Treatments
ROS (Mean fluorescence intensity)
Control EEPP Vitamin E + EEPP Vitamin E
135.04 219.76 58.18 53.70
F F F F
3.91 4.92** 0.95** 1.16**
Cells preincubated for 48 h with 50 Ag/ml EEPP, followed by 30 min incubation with 10 AM H2DCFDA in the pretreatment of 1.0 Ag/ml vitamin E or only pretreatment with 1.0 Ag/ml vitamin E. Data were expressed as mean fluorescence intensity (a.u.) F S.D. of three independent experiments. The esterisk indicates a significant difference between control and treated-groups based on Student’s t-tests (**p < 0.01).
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response, 53.70 F 1.16 (a.u). These results indicated that ROS were the cause of the apoptotic cell death in EEPP treatment.
Discussion Different types of cancer chemopreventive agents, including natural products and pharmaceutical compounds, have been studied for efficacy in vitro and in vivo. The induction of apoptosis is known to be an efficient strategy for cancer therapy. Recently, extracts prepared from a variety of plants were demonstrated to possess the ability in triggering the apoptotic pathway (Liu et al., 2000; Liu et al., 2001). In Taiwan, P. angulata is commonly used for treating hepatitis. The present study demonstrated that EEPP was an effective inducer of apoptosis in Hep G2 cells, suggesting the presence of bioactive compounds in the extracts. Previous studies have isolated a number of physalins, which are believed to be the bioactive compounds of the genus Physalis (Chiang et al., 1992a, 1992b; Ismail and Alam, 2001). Physalin B and physalin F, but not physalin D, inhibit the growth of human leukemia cells in vitro. Physalin F also exhibited a strong activity against human hepatoma cell lines. Our results also showed that all tested extracts did not produce cytotoxic effect towards normal cells, suggesting that the anticancer activity of EEPP might be specific to many other types of tumor cells such as leukemia, hepatoma, cervix uteri etc (Chiang et al., 1992a, 1992b; Ismail and Alam, 2001). Thus, much effort was devoted to the study on the influence of EEPP on mitochondria dysfunction and the understanding of their mechanism of action in cultured Hep G2 cells. Hydrogen peroxide is an important member of ROS and is generated predominantly by mitochondria (Gosslau and Rensing, 2002). Excessive production of hydrogen peroxide in mitochondria will damage lipid, proteins, and DNA, as well as leading to reactions that can cause cells to die due to necrosis or apoptosis (Sakurai and Cederbaum, 1998; Gosslau and Rensing, 2002). Interestingly, EEPP revealed the damaging effects at a higher concentration (50 Ag/ml) as noted by an elevated intracellular ROS generation (Table 3), suggesting EEPP might acts as a prooxidant. This finding was consistent with reported results indicating that that oxidants or prooxidants can induce apoptosis (Ueda et al., 2001; Jin et al., 2002). Addition of the antioxidants such as vitamin E or EEPP pretreatment with vitamin E can inhibit ROS production in cells. ROS can be reduced by antioxidants (e.g. by vitamin C, vitamin E and glutathione) (Gosslau and Rensing, 2002; Jin et al., 2002). Our results have demonstrated that vitamin E possesses a powerful ROS-scavenging activity. Cellular glutathione is a major component of the intracellular reducing factor and a critical determinant of apoptosis (Ueda et al., 2001; Armstrong et al., 2002; Nakamura et al., 2002). GSH in mitochondria originates from the cytosolic compartment by a transport system that translocates GSH into the matrix (Kurosawa et al., 1990). In Fig. 5, the Rh-123 fluorescence intensity was effectively reduced by CCCP-treated cells. The protonophore CCCP-treated cells showed a loss of Dcm and morphological swelling (Minamikawa et al., 1999). Treatment with 10~50 Ag/ml EEPP induced a decrease in GSH contents and a loss of Dcm in dose-dependent pattern (Fig. 4 and Fig. 5). The depletion of mitochondrial GSH, the disruption of Dcm and the subsequent increase in production of ROS are believed to cause cell death. Excessive ROS can impair cells and thus become important regulators of cell cycle arrest and apoptosis (Jin et al., 2002). The results showed that EEPP exhibited a dose-dependent accumulation of
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sub-G1 peak (Fig. 1) and resulted in G0/G1 phase arrest and subsequently reduced the DNA synthesis in S phase (Table 2). Redistribution of membrane phosphatidylserine from the inner leaflet of the plasma membrane to the outer surfaces is common in many apoptotic cells (Martin et al., 1995; Wu et al., 2002). Treatment with EEPP resulted in an increase of cell population that was positive for annexin V staining (Fig. 2). The data exhibited that EEPP induced the exposure of phosphatidylserine on the surface of Hep G2 cells in a concentration-dependent manner. The apoptotic morphological changes such as chromatin condensation and DNA fragmentation were observed in the EEPP-treated cells (Fig. 3). These results indicated that the apoptosis induced by EEPP was possibly mediated through multiple pathway, suggesting many compounds, rather than single component, in P. peruviana may act to induce the apoptosis in human Hep G2 cells. In conclusion, the detailed molecular mechanism for the anticancer effect of P. peruviana and the isolation of its active components still need to be further investigated.
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Mari, M., Bai, J., Cederbaum, A.I., 2002. Toxicity by pyruvate in Hep G2 cells depleted of glutathione: role of mitochondria. Free Radical Biology and Medicine 32, 73 – 83. Martin, S.J., Reutelingsperger, C.P.M., McGahon, A.J., Rader, J.A., Schie, R.C.A.A., LaFace, D.M., Green, D.R., 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. Journal of Experimental Medicine 182, 1545 – 1556. Minamikawa, T., Williams, D.A., Bowser, D.N., Nagley, P., 1999. Mitochondrial permeability transition and swelling can occur reversibly without inducing cell death in intact human cells. Experimental Cell Research 246, 26 – 37. Nakamura, Y., Kawakami, M., Yoshihiro, M.A., Miyoshi, N., Ohigashi, H., Kawai, K., Osawa, T., Uchida, K., 2002. Involvement of the mitochondrial death pathway in chemopreventive benzyl isothiocyanate-induced apoptosis. Journal of Biological Chemistry 277, 8492 – 8499. Park, W.H., Lee, M.S., Park, K., Kim, E.S., Kim, B.K., Lee, Y.Y., 2002. Monensin-mediated growth inhibition in acute myelogenous leukemia cells via cell cycle arrest and apoptosis. International Journal of Cancer 101, 235 – 242. Pietro, R.C., Kashima, S., Sato, D.N., Januario, A.H., Franca, S.C., 2000. In vitro antimy-cobacterial activities of Physalis angulata L. Phytomedicine 7, 335 – 338. Sakurai, K., Cederbaum, A.I., 1998. Oxidative stress and cytotoxicity induced by ferric-nitrilo-triacetate in Hep G2 cells that express cytochrome P450 2E1. Molecular Pharmacology 54, 1024 – 1035. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S., Peter, M.E., 1998. Apoptosis signaling by death receptors. European Journal of Biochemistry 254, 439 – 459. Scudiero, D.A., Shoemaker, R.H., Paull, K.D., Monks, A., Tierney, S., Nofziger, T.H., Currens, M.J., Seniff, D., Boyd, M.R., 1998. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Research 48, 4827 – 4833. Siraki, A.G., Pourahmad, J., Chan, T.S., Khan, S., O’Brien, P.J., 2002. Endogenous and endobiotic induced reactive oxygen species formation by isolated hepatocytes. Free Radical Biology and Medicine 32, 2 – 10. Soares, M.B.P., Bellintani, M.C., Ribeiro, I.M., Tomassini, T.C.B., Santos, R.R., 2003. Inhibition of macrophage activation and lipopolysaccharide-induced death by seco-steroids purified from Physalis angulata L. Euopean Journal of Pharmacology 459, 107 – 112. Ueda, S., Nakamura, H., Masutani, H., Sasada, T., Takabayashi, A., Yamaoka, Y., Yodoi, J., 2001. Baicalin induces apoptosis via mitochondrial pathway as prooxidant. Molecular Immunology 38, 781 – 791. Wu, X., Cao, S., Goh, S., Hsu, A., Tan, B.K.H., 2002. Mitochondrial destabilisation and caspase-3 activation are involved in the apoptosis of Jurkat cells induced by Gaudichaudione A, a cytotoxic xanthone. Planta Medica 68, 198 – 203. Yang, W.L., Addona, T., Nair, D.G., Qi, L., Ravikumar, T.S., 2003. Apoptosis induced by cryo-injury in human colorectal cancer cells is associated with mitochondrial dysfunction. International Journal of Cancer 103, 360 – 369. Zhu, H., Bannenberg, G.L., Moldeus, P., Shertzer, H.G., 1994. Oxidation pathways for the intracellular probe 2V, 7V-dichlorofluorescein. Archives of Toxicology 68, 582 – 587.
Antihepatoma activity of Physalis angulata and P. peruviana extracts and their effects on apoptosis in human Hep G2 cells Shu-Jing Wu a,b, Lean-Teik Ng c, Ching-Hsein Chen d, Doung-Liang Lin e, Shyh-Shyan Wang e, Chun-Ching Lin a,* a
Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan, ROC b Chia-Nan Pharmacy University, Health Nutrition Department, Taiwan, ROC c Department of Food Science and Technology, Tajen Institute of Technology, Pingtung, Taiwan, ROC d Department of Medical Technology, Fooyin University, Kaohsiung, Taiwan, ROC e Tainan District Agricultural Improvement Station, Taiwan, ROC Received 31 March 2003; accepted 24 September 2003
Abstract Physalis angulata and P. peruviana are herbs widely used in folk medicine. In this study, the aqueous and ethanol extracts prepared from the whole plant of these species were evaluated for their antihepatoma activity. Using XTT assay, three human hepatoma cells, namely Hep G2, Hep 3B and PLC/PRF/5 were tested. The results showed that ethanol extract of P. peruviana (EEPP) possessed the lowest IC50 value against the Hep G2 cells. Interestingly, all extracts showed no cytotoxic effect on normal mouse liver cells. Treatment with carbonyl cyanide m-chlorophenyl hydrazone, a protonophore, caused a reduction of membrane potential (Dcm) by mitochondrial membrane depolarization. At high concentrations, EEPP was shown to induce cell cycle arrest and apoptosis through mitochondrial dysfunction as demonstrated by the following observations: (i) EEPP induced the collapse of Dcm and the depletion of glutathione content in a dose dependent manner; (ii) pretreatment with the antioxidant (1.0 Ag/ml vitamin E) protected cells from EEPP-induced release of ROS; and (iii) at concentrations 10 to 50 Ag/ ml, EEPP displayed a dose-dependent accumulation of the Sub-G1 peak (hypoploid) and caused G0/G1-phase arrest. Apoptosis was elicited when the cells were treated with 50 Ag/ml EEPP as characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane. The results conclude that EEPP possesses potent antihepatoma activity and its effect on apoptosis is associated with mitochondrial dysfunction. D 2004 Elsevier Inc. All rights reserved. Keywords: Physalis angulata; P. peruviana; Reactive oxygen species; Dcm; Apoptosis
* Corresponding author. Tel.: +886-7-3121101x2122; fax: +886-7-3135215. E-mail address: [email protected] (C.-C. Lin). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.09.058
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Introduction Apoptosis is a highly organized process, defined by a number of morphological and biochemical changes including membrane bubbling, cell shrinkage, chromatin condensation, DNA fragmentation and appearance of phosphatidylserine in the outer side of the plasma membrane (Schulze-Osthoff et al., 1998; Kim et al., 1999; Park et al., 2002). Reactive oxygen species (ROS) such as superoxide anion (.O2 ), hydrogen peroxide (H2O2), hydroxyl radical (.OH), singlet oxygen, organic peroxide radicals, and nitric oxide generation are recognized as mediators of the apoptotic signaling pathway (Li et al., 2003). Mitochondria are known to be a major physiological source of ROS, which are generated during mitochondrial respiration and have been implicated as mediators of apoptotic signaling pathway. The cellular generation of ROS has been associated with, or contributes to, human disease states such as inflammatory diseases, neurodegenerative diseases, ischemia-reperfusion injury, cancer and aging (Zhu et al., 1994; Nakamura et al., 2002; Siraki et al., 2002). Mitochondrial dysfunction causes energy impairment and/or oxidative stress, and also contributes to the early and common process of apoptotic cell death. The diverse pro-apoptosis stimuli converging on mitochondria cause mitochondrial permeability transition, mitochondrial depolarization, intracellular glutathione depletion and cytochrome c release, are critical events provoking a caspase cascade and eventually lead to cell death (Kroemer and Reed, 2002; Chang et al., 2002; Mari et al., 2002; Wu et al., 2002; Yang et al., 2003). In addition, the collapse of mitochondrial membrane potential and the alteration of oxidation-reduction status have also been noted in a wide variety of cell types (Nakamura et al., 2002; Siraki et al., 2002; Yang et al., 2003). Physalis angulata and P. peruviana have been widely used in folk medicine as anticancer, antimycobacterial, antileukemic, antipyretic, immunomodulatory, and for treating diseases such as malaria, asthma, hepatitis, dermatitis, diuretic and rheumatism (Chiang et al., 1992a, 1992b; Lin et al., 1992; Pietro et al., 2000; Ismail and Alam, 2001; Soares et al., 2003). Antitumor activities of purified compounds, physalins (ex. physalins A, B, D and F) and glycosides (ex. myricetin-3-O-neohesperidoside), have been isolated from the organic fractions of P. angulata (Chiang et al., 1992a, 1992b; Ismail and Alam, 2001; Soares et al., 2003). They were shown to exert anticancer activity on HA 22T (hepatoma), HeLa (cervix uteri), leukemia, lung adenocarcinoma, and epidermoid carcinoma of the nasopharynx KB-16 cells lines (Chiang et al., 1992a, 1992b; Ismail and Alam, 2001). However, the mechanism responsible for the anticancer effect remains unknown. The aims of this study were: (i) to evaluate the antihepatoma activity of aqueous and ethanol extracts of P. angulata and P. peruviana in three human hepatoma cell lines, namely Hep G2, Hep 3B and PLC/ PRF/5; (ii) to identify the mechanistic effects of ethanol extract of P. peruviana induced apoptosis in human Hep G2 cells; and (iii) to study the mitochondrial permeability transition induced by carbonyl cyanide m-chlorophenyl hydrazone and its mediation of the mitochondrial depolarization in cells.
Materials and methods Materials The plants of Physalis angulata L. and Physalis peruviana L. of the family Solanaceae were obtained from Tainan District Agriculture Improvement Station, Taiwan. The authenticity of the plant species was
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confirmed by Professor CC Lin of Graduate Institute of Natural Products, Kaohsiung Medical University, Taiwan. The whole plants were dried and ground to sawdust form, which was then kept in air-tight brown bottle until use. Preparation of extracts Aqueous extract One hundred gram (100 g) of each sample was extracted with 1 L of boiling water for 1 h. The extracts were filtered while the residue was re-extracted under the same conditions twice. The filtrates collected were combined and evaporated to dryness under vacuum. The yield of aqueous extract for P. angulata and P. peruviana were 25.60% and 20.73%, respectively. Ethanol extract One hundred gram (100 g) of P. angulata and P. peruviana sawdust was soaked with 95% ethanol (1 L) at room temperature for 3 days. The samples were filtered with filter paper (Advantec No. 1, Japan) while the residue was further extracted under the same conditions twice. The filtrates collected from three separate extractions were combined and evaporated to dryness under vacuum. The yield obtained for P. angulata and P. peruviana were 19.68% and 24.00%, respectively. Chemicals 5-Fluorouracil (5-FU), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), Dulbecco’s modified Eagle’ medium (DMEM), dimethyl sulfoxide (DMSO), sodium 3V-[1-(phenyl-amino-carbonyl)-3,4tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid (XTT), penicillin, propidium iodide, ribonuclease A (RNase A), sodium dodecyl sulfate, streptomycin and trypsin-EDTA were purchased from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS) was obtained from GIBCO BRL (Gaithersburg, MD). 2V,7V-dichlorodihydrofluorescein (H2DCFDA), rhodamine 123 (Rh-123) and chloromethyl derivatives of fluorescein diacetate (CMFDA) were from Molecular Probes (Eugene, OR). Cell cultures The human hepatoma Hep G2, Hep 3B, PLC/PRF/5 cells and the mouse BALB/C normal liver cells were obtained from the American Type Culture Collection (Rockville, MD). All cells were grown in 90% DMEM supplemented with 10% FBS, 100 units/ml penicillin and 100 Ag/ml streptomycin. The cultures were maintained at 37 jC in a humidified atmosphere of 5% CO2. Antihepatoma activity assay For XTT assay, each cell was seeded at a density of 1 105/well onto 96-well culture plates, and left to adhere to the plastic plates overnight before being exposed to 5-FU (a positive control), 0.1% DMSO (control) and various concentrations (10, 30 and 50 Ag/ml) of P. angulata and P. peruviana extracts. After 48 h of treatment, 150 Al of XTT solution were added to each well and incubated for 4 h. The
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absorbance was measured with an ELISA reader (Multiskan EX, Labsystems) at a test wavelength of 492 nm and a reference wavelength of 690 nm (Goodwin et al., 1995; Scudiero et al., 1998). Cytotoxicity assay The effect of P. angulata and P. peruviana extracts on the viability of mouse BALB/C normal liver cell was measured by the XTT method (Scudiero et al., 1998). The cytotoxic concentration of each extract toward BALB/C normal liver cell lines was calculated by the following formula: % Survival Cell ¼ ODT =ODC 100% The ODT and ODC indicate the absorbance of the test extract-treated group and the control group (0.1% DMSO), respectively. The 50% cytotoxic concentration (CC50), which was expressed as the concentration that achieved 50% cytotoxicity against BALB/C normal liver cell lines, was calculated from the regression line. With the IC50 and CC50 data, the selectivity index for each extract against the different hepatoma cells was calculated as follows: Selectivity Index ¼ CC50 =IC50 Measurement of mitochondrial membrane potential (Dwm) Dwm was measured with Rh-123 using flow cytometry (Coulter Epics Elite ESP, FL, USA). With its high negative charges, Rh-123 will accumulates in normal mitochondria whereas the reduction of Dwm will lead to the release of Rh-123 and the reduction of its fluorescence intensity (Castedo et al., 1996). After 48 h of 10~50 lg/ml EEPP treatment, cells were collected using cell scraper and washed with phosphate-buffered saline (PBS) twice. Cells (5 105/ml) were incubated with Rh-123 (5 mg/ml) for 30 min at 37C, they were then washed with PBS once, followed by subjecting to analysis by an air-cooled argon 488 nm laser with a 525 nm band pass filter and a 550 nm dichroic mirror as detectors. Carbonyl cyanide m-chlorophenyl hydrazone, at a concentration of 10 AM, was used as a positive control for membrane depolarization. Each group has at least 10,000 cells subjecting to treatment with 0.1% DMSO (control) and various concentrations of EEPP (10 and 50 Ag/ml). After 48 h of treatment, cells were washed once with PBS, then re-suspended in PBS, they were then stained with 50 Ag/ml of propidium iodide for 30 min at 37jC and left in the dark for reaction at 4jC. At the end of reaction, the samples were analyzed by a flow cytometer. Data obtained from flow cytometry were analyzed using WinMDI 2.7 software (Scripps Research Institute, La Jolla, CA, USA). Flow cytometry detection of glutathione The cell density was adjusted to 5 105/dish on a 60-mm culture dish. Details of the methods have been previously described (Chang et al., 2002). In brief, cells were detached from
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dishes by treatment with trypsin. They were then collected by centrifugation and re-suspended with PBS. The cells were stained with 25 AM CMFDA at 37jC for 10 min in the dark and the fluorescence was measured by flow cytometry. Based on the criteria of acquisition, a minimum of 10,000 cells in each sample was collected, and the mean fluorescence was estimated by WinMDI 2.7 software. Flow cytometry detection of cell cycle After each treatment, both floating and adherent cells were collected. The cells in suspension were fixed with 70% ice-cold methanol and then transferred to the freezer until use, followed by washing with PBS. Cells were stained with 50 Ag/ml propidium iodide in the presence of 25 Ag/ ml RNase A at 37jC for 30 min. A minimum of 10,000 cells per sample was collected, and the DNA histograms were further analyzed by a flow cytometer. Multicycle software (Phoenix Flow Systems, San Diego, CA) was used to estimate the percentage of each phase in cell cycle. The effect on apoptosis was determined by the degree of increase in the proportion of sub-G1 (hypodiploid) cells. Measurement of annexin V binding The measurement of phosphatidylserine redistribution in a plasma membrane was conducted according to the protocol outlined by the manufacturer in the Annexin V-FITC Apoptosis Detection kit (Sigma Chemical Co, USA). In brief, 1 106 cells were washed and re-suspended in HEPES buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 5 mM CaCl2) containing annexin V-FITC (1:50) and 1 Ag/ml of propodium iodide for 15 min, before being analyzed with a flow cytometer. Intracellular ROS determination Briefly, the cells under confluency were treated with 0.1% DMSO, 50 Ag/ml EEPP, 1.0 Ag/ml vitamin E, and 1.0 Ag/ml vitamin E added to 50 Ag/ml EEPP for 48 h at 37jC. After washing with PBS twice, 10 AM of H2DCFDA was applied to the cells, which were then incubated for 30 min. A flow cytometer was used to detect the fluorescent dichlorofluoresein. Morphological detection of apoptotic cells Cells were untreated or treated with 50 Ag/ml EEPP for 48 h, they were then fixed with 3.0% (w/v) paraformaldehyde at room temperature for 20 min. After washing with PBS, cells were stained with 0.12 Ag/ml Hoechst 33258 for 15 min. Morphological changes were then observed under fluorescent microscopy (Zeiss Axioskop, Mikron Instruments, NY). Statistical analysis Results are expressed as mean F SD from at least three independent experiments. Statistical analysis was performed according to Student’s t-test by one-way analysis of variance. Significant difference was taken as p < 0.05 or p < 0.01.
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Results Antihepatoma activity The effects of aqueous and ethanol extracts of P. angulata and P. peruviana on Hep G2, Hep 3B and PLC/PRF/5 are shown in Table 1. In general, the ethanol extracts exhibited a lower IC50 value than the aqueous extracts, with the lowest IC50 value (9.43 F 0.30 Ag/ml) from the ethanol extract of P. peruviana (EEPP). With the exception of P. angulata ethanol extract, the IC50 values of other extracts for PLC/PRF/5 cell lines were >100 Ag/ml. For Hep 3B cells, ethanol extract of P. angulata showed the lowest IC50 value (25.40 F 0.25 Ag/ml). Among the different plant extracts and cell lines tested, EEPP exhibited the strongest antihepatoma activity against Hep G2 cells. Cytotoxicity assay Cytotoxicity assay was performed to ensure that the anticancer activity of extracts of P. angulata and P. peruviana and 5-FU were not caused by cytotoxic effects of herbal medicine toward the mouse BALB/C normal liver cells. The results showed that CC50 values of ethanol extracts (1278-2862 Ag/ml) were lower than the aqueous extracts (4297-5800 Ag/ml) of P. angulata and P. peruviana (Table 1). Selectivity index The selectivity index is used to evaluate the safety of a drug when applied in a biological system. As shown in Table 1, the SI values of the tested extracts vary from 119 to 300, suggesting that these extracts exhibit no cytotoxic effect on mouse BALB/C normal liver cells. Flow cytometry detection of cell cycle The effect of EEPP on the cell cycle was determined in Hep G2 cells by flow cytometry. After 48 h of treatment, EEPP induced cell cycle arrest (G0/G1 phase) and decreased in the S phase of Hep G2 cells in Table 1 The IC50 and cytotoxicity values of aqueous and ethanol extracts of P. angulata (PA) and P. peruviana (PP) Plant extracts
IC50 value (Ag/ml)a Hep G2
5-FU PA (water) PP (water) PA (EtOH) PP (EtOH)
1.24 44.19 29.89 10.67 9.43
F F F F F
0.31 0.52 0.60 0.20 0.30
PLC/PRF/5
Hep 3B
Cytotoxicityb CC50(Ag/ml)
1.21 F 0.90 >100.00 >100.00 41.60 F 0.3 >100.00
1.72 F 0.10 >100.00 43.06 F 0.93 25.40 F 0.25 41.25 F 1.40
123.04 5800.69 4297.72 1278.12 2862.00
F F F F F
0.10 0.45 0.09 0.12 0.07
Selectivity indexc (SI) 99.23 131.26 143.78 119.79 303.50
Each value represents the mean F SD of three independent experiments. a Antihepatoma activity was determined by XTT assay. 50% inhibition concentration (IC50) was the concentration of crude extracts of P. angulata and P. peruviana that inhibit 50% growth of the three human hepatoma cell lines. b Cytotixic concentration (CC50) was the concentration of crude extracts of P. angulata and P. peruviana that achieved 50% cytotoxicity against mouse BALB/C normal liver cells.
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Table 2 Effects of the ethanol extract of P. peruviana (EEPP) on cell-cycle progression Test samples
G0/G1
Control (0.1% DMSO) EEPP (10 Ag/ml) EEPP (30 Ag/ml) EEPP (50 Ag/ml)
51.07 66.30 71.47 74.00
S F F F F
0.48 0.50* 0.88** 3.59**
38.00 18.67 17.93 7.73
G2/M F F F F
1.68 1.91** 0.98** 0.39**
10.93 15.03 10.60 18.27
F F F F
1.54 1.23* 0.22 2.54**
Data are mean F SD obtained from three independent experiments. The asterisk indicates a significant difference between control and EEPP-treated cells based on Student’s t-tests (*p < 0.05, **p < 0.01).
a dose response manner (Table 2). In addition, the control group (0.1% DMSO) and 10~50 Ag/ml EEPP displayed a dose-dependent accumulation of the Sub-G1 peak (hypoploid) by increasing from 0.21% to 60.29% (Fig. 1).
Fig. 1. Cell cycle distribution in Hep G2 cells after ethanol extract of P. peruviana (EEPP) treatment. 5 105 cells were treated with different concentrations of EEPP for 48 h. (A) control (0.1% DMSO), (B) 10 Ag/ml, (C) 30 Ag/ml, and (D) 50 Ag/ml. The graph was obtained from representative data of three individual experiments.
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Induction of apoptosis by EEPP To further confirm that EEPP leads to apoptosis, Hep G2 cells were stained with annexin V-FITC and propidium iodide, and subsequently analyzed by flow cytometry. The annexin V assay measures phospholipid turnover from the inner to the outer lipid layer of the plasma membrane, an event typically associated with apoptosis. As indicated in Fig. 2, 10 and 50 g/ml EEPP treatment displayed the population of apoptotic cells (29.33% and 65.19%, annexin V+/PI – ). Using FACS analysis, the proportion of annexin V-staining cells was found to increase in 10 f 50 Ag/ml EEPP-treated cells. The percentage of sub-G1 and annexin V-staining cells were 22.00% and 29.33% respectively in 10 Ag/ ml EEPP-treated cells, and 60.29% and 65.19% respectively in 50 Ag/ml EEPP-treated cells (Fig. 1 and Fig. 2). The apoptotic cell death by EEPP at 50 Ag/ml was stained with Hoechst 33258, and confirmed by nuclei morphology using the fluorescence microscopy. Morphological assessment was used to
Fig. 2. Induction of apoptosis by ethanol extract of P. peruviana (EEPP). Flow cytometric analysis of annexin V-FITC and propidium iodide double stained cells. Cells were untreated or treated with 10 and 50 Ag/ml EEPP for 48 h. Data represent the percentage of aopototic cells from three determinations.
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Fig. 3. Changes in the morphology of Hep G2 cells treated with ethanol extract of P. peruviana (EEPP) under a fluorescence microscopy. Morphological changes in untreated cells (A) and cells treated with 50 Ag/ml EEPP (B) for 48 h were examined by staining with Hoechst 33258. The nuclei feature of control cells showed the round and homogeneous nuclei, the apoptotic cells displayed the condensation and fragmentation of nuclei in the EEPP-treated cells (the arrow point).
display the nuclei fragmentation and compaction of chromatin in EEPP-induced cell death (Fig. 3). The results have demonstrated that 50 Ag/ml EEPP induced apoptosis. Apoptotic-related mitochondrial transmembrane potential (Dwm) To investigate the effect of EEPP on mitochondria, Hep G2 cells were treated with EEPP for 48 h. We measured the membrane potential by staining with Rh-123. As shown in Fig. 4, the percentage of total cell counts exhibited a decrease in Dwm. The mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone (used as a positive control for Dcm disruption) also induced a significant change in Dcm. EEPP remarkably reduced the uptake of the fluorescent dye in Hep G2 cells in a dose-dependent manner (Fig. 4). The loss of Dcm was maximal with 50 Ag/ml EEPP and a slow decline with 10 Ag/ml EEPP.
Fig. 4. Effect of the ethanol extract of P. peruviana (EEPP) on dissipation of mitochondrial transmembrane potential (Dcm) in Hep G2 cells as demonstrated by flow cytometry. Cells were treated with 10 and 50 Ag/ml of EEPP for 48 h. Cells exposed to 10 AM CCCP were used as positive control. The asterisk indicates a significant difference between control and EEPP-treated cells based on Student’s t-tests (*p < 0.05, **p < 0.01).
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Fig. 5. Effect of the ethanol extract of P. peruviana (EEPP) on cellular glutathione content (GSH). Hep G2 cells untreated or treated with EEPP at 10 and 50 Ag/ml for 48 h were collected and stained with CMFDA followed by analyzing with flow cytometry. Data were obtained from three independent experiments and are expressed as mean F S.D. The asterisk indicates a significant difference between control and EEPP-treated cells based on Student’s t-tests (**p < 0.01).
Involvement of redox alteration in EEPP-induced apoptosis It has been suggested that the alterations of mitochondria appeared, at least in part, to be regulated by the oxidation-reduction status such as the depletion of intracellular glutathione (GSH) content and intracellular ROS level in cells. By using a GSH-reactive dye, we examined the effects of the EEPPinduced apoptosis in cellular GSH content. In Hep G2 cells, GSH content was significantly declined after EEPP treatment (Fig. 5). After staining with H2DCFDA (a fluorescent dye for peroxides, H2O2), the results displayed a significant ROS generation at 50 Ag/ml EEPP as compared with the control (Table 3). We further investigated the involvement of ROS by treating the cells with an antioxidant (1.0 Ag/ml vitamin E) before treatment with 50 Ag/ml EEPP or treated with only 1.0 Ag/ml of vitamin E for 48 h. Treatment with 50 Ag/ml EEPP resulted in an increase in the mean fluorescence intensity of 219.76 F 4.92 (a.u), and pretreatment with 1.0 Ag/ml vitamin E before 50 Ag/ml EEPP treatment showed a reduction in intensity to 58.18 F 0.95 (a.u), which was closed to a single 1.0 Ag/ml vitamin E-treated
Table 3 Effect of the ethanol extract of P. peruviana (EEPP) on ROS production Treatments
ROS (Mean fluorescence intensity)
Control EEPP Vitamin E + EEPP Vitamin E
135.04 219.76 58.18 53.70
F F F F
3.91 4.92** 0.95** 1.16**
Cells preincubated for 48 h with 50 Ag/ml EEPP, followed by 30 min incubation with 10 AM H2DCFDA in the pretreatment of 1.0 Ag/ml vitamin E or only pretreatment with 1.0 Ag/ml vitamin E. Data were expressed as mean fluorescence intensity (a.u.) F S.D. of three independent experiments. The esterisk indicates a significant difference between control and treated-groups based on Student’s t-tests (**p < 0.01).
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response, 53.70 F 1.16 (a.u). These results indicated that ROS were the cause of the apoptotic cell death in EEPP treatment.
Discussion Different types of cancer chemopreventive agents, including natural products and pharmaceutical compounds, have been studied for efficacy in vitro and in vivo. The induction of apoptosis is known to be an efficient strategy for cancer therapy. Recently, extracts prepared from a variety of plants were demonstrated to possess the ability in triggering the apoptotic pathway (Liu et al., 2000; Liu et al., 2001). In Taiwan, P. angulata is commonly used for treating hepatitis. The present study demonstrated that EEPP was an effective inducer of apoptosis in Hep G2 cells, suggesting the presence of bioactive compounds in the extracts. Previous studies have isolated a number of physalins, which are believed to be the bioactive compounds of the genus Physalis (Chiang et al., 1992a, 1992b; Ismail and Alam, 2001). Physalin B and physalin F, but not physalin D, inhibit the growth of human leukemia cells in vitro. Physalin F also exhibited a strong activity against human hepatoma cell lines. Our results also showed that all tested extracts did not produce cytotoxic effect towards normal cells, suggesting that the anticancer activity of EEPP might be specific to many other types of tumor cells such as leukemia, hepatoma, cervix uteri etc (Chiang et al., 1992a, 1992b; Ismail and Alam, 2001). Thus, much effort was devoted to the study on the influence of EEPP on mitochondria dysfunction and the understanding of their mechanism of action in cultured Hep G2 cells. Hydrogen peroxide is an important member of ROS and is generated predominantly by mitochondria (Gosslau and Rensing, 2002). Excessive production of hydrogen peroxide in mitochondria will damage lipid, proteins, and DNA, as well as leading to reactions that can cause cells to die due to necrosis or apoptosis (Sakurai and Cederbaum, 1998; Gosslau and Rensing, 2002). Interestingly, EEPP revealed the damaging effects at a higher concentration (50 Ag/ml) as noted by an elevated intracellular ROS generation (Table 3), suggesting EEPP might acts as a prooxidant. This finding was consistent with reported results indicating that that oxidants or prooxidants can induce apoptosis (Ueda et al., 2001; Jin et al., 2002). Addition of the antioxidants such as vitamin E or EEPP pretreatment with vitamin E can inhibit ROS production in cells. ROS can be reduced by antioxidants (e.g. by vitamin C, vitamin E and glutathione) (Gosslau and Rensing, 2002; Jin et al., 2002). Our results have demonstrated that vitamin E possesses a powerful ROS-scavenging activity. Cellular glutathione is a major component of the intracellular reducing factor and a critical determinant of apoptosis (Ueda et al., 2001; Armstrong et al., 2002; Nakamura et al., 2002). GSH in mitochondria originates from the cytosolic compartment by a transport system that translocates GSH into the matrix (Kurosawa et al., 1990). In Fig. 5, the Rh-123 fluorescence intensity was effectively reduced by CCCP-treated cells. The protonophore CCCP-treated cells showed a loss of Dcm and morphological swelling (Minamikawa et al., 1999). Treatment with 10~50 Ag/ml EEPP induced a decrease in GSH contents and a loss of Dcm in dose-dependent pattern (Fig. 4 and Fig. 5). The depletion of mitochondrial GSH, the disruption of Dcm and the subsequent increase in production of ROS are believed to cause cell death. Excessive ROS can impair cells and thus become important regulators of cell cycle arrest and apoptosis (Jin et al., 2002). The results showed that EEPP exhibited a dose-dependent accumulation of
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sub-G1 peak (Fig. 1) and resulted in G0/G1 phase arrest and subsequently reduced the DNA synthesis in S phase (Table 2). Redistribution of membrane phosphatidylserine from the inner leaflet of the plasma membrane to the outer surfaces is common in many apoptotic cells (Martin et al., 1995; Wu et al., 2002). Treatment with EEPP resulted in an increase of cell population that was positive for annexin V staining (Fig. 2). The data exhibited that EEPP induced the exposure of phosphatidylserine on the surface of Hep G2 cells in a concentration-dependent manner. The apoptotic morphological changes such as chromatin condensation and DNA fragmentation were observed in the EEPP-treated cells (Fig. 3). These results indicated that the apoptosis induced by EEPP was possibly mediated through multiple pathway, suggesting many compounds, rather than single component, in P. peruviana may act to induce the apoptosis in human Hep G2 cells. In conclusion, the detailed molecular mechanism for the anticancer effect of P. peruviana and the isolation of its active components still need to be further investigated.
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