Cell Biochem Biophys DOI 10.1007/s12013-014-0025-2
ORIGINAL PAPER
Induction of Apoptosis in Human Hepatocarcinoma SMMC-7721 Cells In Vitro by Psoralen from Psoralea corylifolia Zhongyong Jiang • Jie Xiong
Ó Springer Science+Business Media New York 2014
Abstract Psoralen is a major active component of Psoralea corylifolia. In the present study, we analyzed psoralen-induced changes in human hepatocarcinoma cell viability and apoptosis, and investigated the underlying mechanisms of the proapoptotic effect of the compound on SMMC-7721 cells. We measured human hepatocarcinoma cell viability by MTT assay and Annexin V-FITC/PI double staining, and evaluated the activity of caspase 3 and the expression of p53, Bax, and Bcl-2 proteins, involved in regulating cell apoptosis. Psoralen was able to inhibit the growth of SMMC-7721 cells in a dose- and time-dependent manner and had a strong proapoptotic effect on these cells. We show a dose-dependent increase in caspase-3 activity, and elevated levels of p53 and Bax proteins in psoralentreated cells, that coincided with dose-dependent decrease in Bcl-2 expression. These results suggest that psoralen induces apoptosis in cancer cells via mechanisms that involve caspase-3, p53, Bax, and Bcl-2 pathway. Our results may provide a molecular basis for the further development of natural compounds as novel anticancer agents for human hepatomas. Keywords Psoralen SMMC-7721 cell Apoptosis Caspase-3 p53 Bax Bcl-2
Zhongyong Jiang and Jie Xiong contributed equally in this article. Z. Jiang (&) J. Xiong Department of Clinical Laboratory, Chengdu Military General Hospital, Chengdu Military District, Chengdu 610083, People’s Republic of China e-mail: [email protected]
Introduction Hepatocellular carcinoma is one of the most common malignant tumors worldwide, which is responsible for approximately one million deaths annually. Since surgical and pharmacological therapeutic approaches, available today, are rarely effective in treating the advanced disease, the outcomes of HCC patients remain poor [1]. Hepatocellular carcinoma is usually insensitive to chemotherapeutic drugs currently used in clinical setting, and is well known for its expression of multidrug resistance (MDR) gene, as well as the poor response to chemotherapeutic agents [2]. There is, therefore, a great need for alternative more efficient therapeutic approaches with less side effects. Previous studies suggested that bioactive natural compounds found in medicinal plants could in theory serve as alternatives to chemically designed anticancer agents [3]. Plant-derived natural products play a very important role in the area of cancer therapy research as a source of numerous currently used anticancer agents including vincristine, vinblastine, paclitaxel, camptothecin derivatives, epipodophyllotoxin, and many others [4, 5]. Therefore, there is a great need for continuous search for new antineoplastic agents from plant sources. Psoralea corylifolia is a leguminous plant that is widely used in traditional Chinese medicine (TCM) for treating pollakiuria, nephritis, osteoporosis, hypertension, and cardiovascular diseases [6–8], and is officially listed in the Pharmacopoeia of the Peoples’ Republic of China (Edit 2010 Vol II). In vitro and in vivo studies have demonstrated that the active fractions from the seeds and roots of Psoralea corylifolia have distinct antimicrobial, antioxidative, immunomodulatory, and anticancer properties [6, 9]. Previous reports show that P. corylifolia extract contains coumarins, flavonoids, and merotepenes, such as
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psoralen, isopsoralen, neobavaislfoavone, bovachin, bavaislfoavone, bavachromene, psoralidin, corylifolinin, bavachinin, and bavachalcone [4]. Psoralen, a tricyclic furocoumarin, is the major and the most active furocoumarin present in P. corylifolia, and is widely used as an antineoplastic agent in treatment of leukemia and other cancers [6, 9–11]. In the present study, we are describing for the first time the proapoptotic effect of psoralen on human hepatoma SMMC-7721 cells. Our results suggest that the potent anticancer activity of psoralen correlates with the up-regulation of expression of caspase-3, p53, Bax, and Bcl-2, the components of the intrinsic apoptosis pathway.
Methods Regents and Materials Psoralen was purchased from the National Institute of Control of Pharmaceutical and Biological Products. Cell culture reagents were obtained from GIBCO Life Technology (Grand Island, NY, USA). Antibodies specific for the Bcl-2, Bax, p53, and antirabbit IgG, HRP-linked antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). b-Actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-3 activity kit was obtained from Beyotime Institute of Biotechnology (Haimen, China), and Annexin V-FITC Apoptosis Detection kit was purchased from Pharmingen (Becton–Dickinson Company, San Jose, CA, USA). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) and other reagents were purchased from Sigma (St. Louis, MO, USA). Preparation of Psoralen Psoralen was dissolved in dimethyl sulfoxide (DMSO), and aliquots of 10 mg/mL stock solution were stored at -20 °C. For all the experiments, psoralen was further diluted in DMSO to the final concentrations of 10, 30, 50, or 100 lg/mL. Carrier solvent (0.1 % DMSO) was used as control. Cell Culture Human hepatocarcinoma cell line (SMMC-7721) was purchased from Jiangsu Institute of Cancer Research (JICR. Jiangsu, China). Cells were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum, 100 U/mL penicillin G, and 100 lg/mL streptomycin in a humidified atmosphere of 5 % CO2 at 37 °C. The culture medium was changed for every 2 days.
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Cell Viability Assay Inhibition of cell proliferation by psoralen was measured by MTT assay. SMMC-7721 cells were cultured in 96-well plates at a cell density of 2 9 104 cells per well in 100 lL of RPMI 1640 for 24 h. The medium was then removed and replaced by fresh medium containing different concentrations of psoralen (10, 30, 50, and 100 lg/mL) for 24, 48, 72, and 96 h. Positive control group was treated with 5-fluorouracil (5-FU) solution at the final concentration of 250 lg/mL. Cells in the negative control group were incubated with sterilized saline solution. Cells were then incubated with 10 lL of 5 mg/mL MTT solution in 100 lL of medium for 3 h at 37 °C. MTT solution was then replaced by 150 lL DMSO and incubated for another 10 min with agitation. Absorbance was measured by automatic microplate reader (EL800, BIO-TEK instruments, Inc.) at 550 nm. Growth inhibition rate (GIR) was calculated as follows: GIR ð%Þ ¼ ðODnegative group ODtest group Þ=ODnegative group 100 %: The results were presented as mean ± SD. The morphological changes of SMMC-7721 cells were observed with an inverted microscope at 2009 magnification. Determination of Cell Apoptosis by Annexin V and PI Double Staining Measurement of phosphatidylserine redistribution in a plasma membrane of SMMC-7721 cells was conducted according to the protocol outlined by the manufacturer of the Annexin V-FITC Apoptosis Detection kit (Becton–Dickinson Company, San Jose, CA, USA). Briefly, cells were seeded in 6-well tissue culture plates to the final density of 1 9 106 cells/well, and incubated for 24 h at 37 °C. Psoralen (10, 30, 50, and 100 lg/mL) was directly added to the 6-well plates and incubated with cells for an additional 48 h. Positive control group was treated with 250 lg/mL 5-FU solution, and sterilized saline was used for the negative control group. Cells were then harvested and resuspended in PBS. Apoptotic cells were identified by FITC-conjugated Annexin-V/PI double staining, according to the manufacturer’s protocol, and evaluated by FACSCalibur flow cytometry (Becton–Dickinson, San Jose, CA). Caspase-3 Activity Assay Activity of caspase-3 was determined using the caspase-3 activity kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s protocol. Briefly, following 48 h incubation with different concentrations of psoralen (0, 10, 30, 50, and 100 lg/mL), SMMC-7721 cells
Cell Biochem Biophys Table 1 Inhibition rates of hepatoma SMMC-7721 cells following treatment with increasing concentrations of psoralen for 24–96 h Groups
Inhibition rate (%) 24 h
P 48 h
72 h
96 h
10 lg/mL
18.84 ± 1.75
36.96 ± 1.21
54.28 ± 0.92
51.20 ± 2.13
0.000
30 lg/mL
40.33 ± 1.91
61.86 ± 1.26
71.40 ± 1.21
65.95 ± 2.48
0.000
50 lg/mL
47.43 ± 1.61
82.25 ± 1.35
86.61 ± 1.31
78.35 ± 3.04
0.000
100 lg/mL
53.17 ± 1.08
82.00 ± 1.16
86.39 ± 1.51
81.15 ± 1.14
0.000
Positive
48.67 ± 1.16
75.84 ± 1.31
86.40 ± 2.06
80.34 ± 1.57
0.000
P
0.000
0.000
0.000
0.000
Cells treated with 250 lg/mL of 5-FU were used as positive control; Values represent mean ± standard deviation of triplicate measurements
Fig. 1 Inhibition of SMMC-7721 cell proliferation by psoralen in a dose- and time-dependent manner. Cells were treated with different concentrations of psoralen. Absorbance was measured at 24, 48, 72, and 96 h by MTT assay. Cells treated with 250 lg/mL of 5-FU were used as positive control. P \ 0.05
were lysed. Caspase-3 activity assays were carried out in 96-well microtitre plates by incubating 10 lL of cell lysate in 80 lL reaction buffer (1 % NP-40, 20 mM Tris–HCl (pH = 7.5), 137 mM Nad and 10 % glycerol) containing 10 lL caspase-3 substrate (Ac-DEVD-pNA) (2 mM). Samples were incubated at 37 °C for 4 h and absorbance was measured at 405 nm.
Proteins were separated on 10 % SDS–polyacrylamide gel (SDS-PAGE) and transferred onto the PVDF membranes (Millipore, Billerica, MA) blocked with. Immune bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, Mo) and probed with rabbit antiBax, rabbit antiBcl-2, and mouse antiactin antibodies (1:500, Santa Cruz, CA) overnight at 4 °C. Blots were washed and incubated for 1 h with IRDyeTM800-conjugated antimouse and antirabbit secondary antibodies (1:15,000, Santa Cruz, CA), washed with 0.1 % Tween-20 in TBS, and the proteins were visualized by chemiluminescence using the Amersham ECL Plus Western Blotting Detection kit (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) according to the manufacturer’s instructions. b-Actin was used as a loading control. Statistical Analysis All data were expressed as mean ± SD, and analyzed by one-way analysis of variance followed by Least Significance Difference multiple comparison or Dunnett’s multiple comparison tests using SPSS 13.0 software (SPSS, Chicago, IL, USA). Multiple comparison tests were used when appropriate. A P value of 0.05 was considered statistically significant.
Western Blot Analysis
Results
SMMC-7721 cells were incubated with psoralen (0, 10, 30, 50, and 100 lg/mL) for 48 h, and total cell proteins were extracted by lysing the cells in buffer, containing 100 mM Tris–Cl, pH 6.8, 4 % (m/v) SDS, 20 % (v/v) glycerol, 200 mM b-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 g/mL aprotinin. Protein concentrations were determined by BCA assay using Varioskan spectrofluorometer and spectrophotometer (Thermo, Waltham, MA) at 562 nm.
Effect of Psoralen on the Growth of Human Hepatoma SMMC-7721 Cells Traditional antineoplastic therapy is based on the use of chemotherapeutic compounds which have a cytotoxic effect on proliferating cells and promote the destruction of sensitive tumors. We first researched possible antiproliferative activity of psoralen by evaluating its in vitro cytotoxic effect on human hepatoma SMMC-7721 cells
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Cell Biochem Biophys Fig. 2 Morphological changes in hepatoma SMMC-7721 cells after treatment with psoralen for 48 h. a Untreated cells (negative control); b cells treated with 10 lg/mL of psoralen; c cells treated with 50 lg/mL of psoralen; d cells treated with 250 lg/mL of 5-FU (positive control). Cells were visualized using inverted microscope at 2009 magnification
using MTT assay. Cells, treated with 10 lg/mL of psoralen for 24 h exhibited only slightly reduced growth inhibition. Increasing psoralen concentration or the length of the treatment led to further increase in proliferation inhibition rate of hepatoma SMMC-7721 (Table 1; Fig. 1). The highest concentration of psoralen (50 and 100 lg/mL) resulted in GIRs similar to 5-FU-treated positive control cells. These results suggest that psoralen could inhibit growth of hepatoma SMMC-7721 cells in a time- and dosedependence manner. Analysis of Psoralen-Induced Changes in SMMC-7721 Cell Morphology
with the increased concentration of psoralen for 48 h, and the rate of cell apoptosis was determined by Annexin V-FITC/PI staining, followed by the flow cytometric analysis (FACS) (Fig. 3). Even at the lowest concentration (10 lg/mL), psoralen treatment of the hepatoma cells resulted in increased number of double-stained Annexin V-FITC-positive and PI-negative cells, indicative of early apoptosis. As summarized in Table 2, cells, treated with 30 lg/mL of psoralen exhibited significantly higher rate of apoptosis (23.32 ± 1.91 %) as compared with the negative control group (2.57 ± 1.23 %, P \ 0.01). Effect of Psoralen on the Activation of Caspase-3
To evaluate psoralen-induced morphological changes in human hepatoma cells, SMMC-7721 cells were treated with increased concentrations of psoralen for 48 h and analyzed using inverted microscope at 2009 magnification. Number of SMMC-7721 cells was significantly decreased after treatment with psoralen when compared to untreated cells (negative control) in a dose-dependent manner (Fig. 2), and cells exhibited progressive morphological changes with increased duration of exposure to psoralen (data not shown).
Caspase-3 plays a central role in mediating various cellular apoptotic responses [12]. We measured caspase-3 protease activity in human hepatoma cells treated with the various concentrations of psoralen. Psoralen treatment caused increased caspase-3 protease activity in a dose-dependent manner: over threefold increase at the concentration of 30 lg/mL, and almost fivefold increase (569.94 ± 7.74 %) at the maximal concentration of 100 lg/mL as compared to untreated cells (Fig. 4).
Flow Cytometric Analysis of Psoralen-Induced Apoptosis
Effects of Psoralen on Levels of p53, Bax, and Bcl-2 Proteins in SMMC-7721 Cells
We next tested the proapoptotic effect of psoralen on the human hepatoma cells. SMMC-7721 cells were treated
We next evaluated the effect of psoralen on the expression of p53, Bax, and Bcl-2, important components of the
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Cell Biochem Biophys Fig. 3 Proapoptotic effect of psoralen. SMMC-7721 cells were treated with psoralen (10, 30, 50, and 100 lg/mL) for 48 h, incubated with Annexin V and PI for 30 min at 37 °C and then subjected to FACS analysis. a Untreated cells represent negative control group; b cells, treated with 5-FU were used as a positive control; c-1 to c-4 SMMC-7721 cells treated with 10, 30, 50, and 100 lg/mL of psoralen, respectively. The viable cell population is presented in the lower left quadrant (Annexin V-PI-). The cells at the early stages of apoptosis are in the lower right quadrant (Annexin V ? PI-)
intrinsic apoptotic pathway, in SMMC-7721 cells. After 48 h incubation with the increasing concentrations of psoralen (0, 10, 30, 50, and 100 lg/mL), the expression of p53, Bax, and Bcl-2 proteins was analyzed by western blotting. At higher concentrations (30, 50, and 100 lg/mL) psoralen led to a significant increase in protein levels of p53 and Bax in human hepatoma. In contrast, the level of Bcl-2 protein significantly decreased in a dose-dependent manner after psoralen treatment (Fig. 5).
Discussion HCC is a rapidly fatal disease, with a life expectancy of about 6 months from the time of the diagnosis. Therapeutic strategies employed to date have significantly improved the prognosis for patients with unresectable HCC [13]. This emphasizes the need for investigating the molecular mechanisms responsible for HCC development and seeking effective and noncytotoxic chemical agents for
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Cell Biochem Biophys Table 2 Apoptosis rates of SMMC-7721 cells treated with psoralen for 48 h Groups Negative control Positive control Psoralen 30 lg/mL
Apoptosis rate (%) 2.57 ± 1.23 1.48 ± 0.36 23.32 ± 1.91ab
Apoptosis rate of negative group, positive group, and 30 lg/mL of psoralen-treated cells were determined by Annexin V-FITC and PI staining and FACS. The values expressed were mean ± standard deviation of triplicate measurements a
P \ 0.01 vs. negative control group
b
P \ 0.01 vs. positive control group
Fig. 5 Effect of psoralen on the Expression of p53, Bax, and Bcl-2 in SMMC-7721 cells. Cells were treated with psoralen (0, 10, 30, 50, and 100 lg/mL) for 48 h, and the expression of p53, Bax, and Bcl-2 proteins was detected by western blotting. b-Actin is used as a loading control
Fig. 4 Effects of psoralen on caspase-3 activity in SMMC-7721 cells. Cells were incubated with 0, 10, 30, 50, and 100 lg/mL psoralen for 48 h at 37 °C. Data represent mean ± SD of three independent experiments. P \ 0.05 and P \ 0.01 from Dunnett’s test comparing to untreated cells (control group). Each value is expressed as the ratio of caspase-3 activation level to control level, which was assigned a value of 100
chemoprevention and treatment. Since only few synthetic antineoplastic compounds are effective for the treatment of this disease, the focus of more and more studies has shifted recently to researching natural active agents for cancer chemoprevention and treatment [14]. The pharmacological mechanism of the anticancer activity of P. corylifolia L. extract, psoralen is still not fully understood. Previous reports show that psoralen decreases the energy supply of cancer cells and inhibits nucleic acid and protein synthesis by destroying cell mitochondria [15]. Other studies report that psoralen and isopsoralen induce cancer cell apoptosis, and the differences in apoptotic response of cells to psoralen and isopsoralen may provide some insight on whether the proapoptotic effect of P. corylifolia L. extract is dependent on p53 [9].
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In the present study, we demonstrate that psoralen has an anticancer activity on SMMC-7721 cells. Psoralen has a significant growth inhibitory effect on human hepatoma cells, probably through the cytotoxic mechanism that involves the induction of apoptosis. The latter was identified by typical characteristic features of apoptosis and dose-dependent increase in apoptotic rate. Proapoptotic effect of psoralen correlated with the dose-dependent elevated activity of caspase-3 in psoralen-treated SMCC-7721 cells. Caspases, a family of cysteine proteases, have long been recognized as critical components of the apoptotic pathway, and caspase-3 is required for a majority of the nuclear changes associated with apoptosis [12]. We demonstrated that psoralen induced dose-dependent increase in the levels of p53 and Bax proteins and decrease in the levels of Bcl-2 protein in treated hepatoma cells. Numerous studies demonstrated that p53, a tumor suppressor gene, plays an important role in tumor growth inhibition and induction of apoptosis, and exerts its control on apoptosis by interacting with other important apoptotic molecules, including the members of the Bcl-2 family [16–18]. This protein family regulates the intrinsic or mitochondrial pathway of apoptosis, and is involved in positive (Bax, Bak, Blk, Bad, and Bid) and negative (Bcl-2, Bcl-XL, Bclw, Bfl-1, Bag-1, Mcl-1, and A1) regulation of apoptotic cell death [19]. Antiapoptotic Bcl-2 and proapoptotic Bax are two of the major members of the Bcl-2 family [20]. Bcl-2 is known as an upstream effector of the apoptotic pathway and is identified as a potent suppressor of apoptosis [21]. Studies also show that Bcl-2 promotes cell survival by preserving the integrity of the external mitochondrial membrane, thus preventing the release of
Cell Biochem Biophys
cytochrome c from mitochondria and subsequent cell death [22]. Interestingly, the increased apoptotic cell death correlates with an elevated intracellular Bax/Bcl-2 ratio [23], and the Bcl-2/Bax ratio rather than the levels of bcl-2 alone, plays an important role in regulating the effects of the apoptosis induction [24]. Our recent results suggest that psoralen would increase Bax/Bcl-2 ratio in human hepatoma cells, given the fact that the treatment results in upregulation of Bax expression that coincides with the dosedependent decrease in Bcl-2 protein levels. In conclusion, we have demonstrated that psoralen inhibits the growth of SMMC-7721 human hepatoma cells by inducing cell apoptosis through mechanism that involves regulation of caspase-3 activity, and p53 and Bcl2/Bax protein expression. These results may provide further insight on the mechanisms involved in psoraleninduced cytotoxic effects on cancer cells.
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References 1. Kern, M. A., Breuhahn, K., & Schirmacher, P. (2002). Molecular pathogenesis of human hepatocellular carcinoma. Advances in Cancer Research, 86, 67–112. 2. Geng, C. X., Zeng, Z. C., & Wang, J. Y. (2003). Docetaxel inhibits SMMC-7721 human hepatocellular carcinoma cells growth and induces apoptosis. World Journal of Gastroenterology, 9(4), 696–700. 3. Ramirez-Mares, M. V., Chandra, S., & de Mejia, E. G. (2004). In vitro chemopreventive activity of Camellia sinensis, Ilex paraguariensis and Ardisia compressa tea extracts and selected polyphenols. Mutation Research, 554(1–2), 53–65. 4. Cragg, M. G., & Newman, D. J. (2005). Plants as a source of anticancer agents. Journal of Ethnopharmacology, 100(1–2), 72–79. 5. Cragg, M. G., Kingston, D. G. I., & Newman, D. J. (2005). Anticancer agents from natural products (Chap. 7, pp. 123–136). Boca Raton, FL: Brunner-Routledge Psychology Press, Taylor & Francis Group. 6. Latha, P. G., Evans, D. A., Panikkar, K., & Jayavardhanan, K. K. (2000). Immunomodulatory and antitumor properties of Psoralea corylifolia seeds. Fitoterapia, 71, 223–231. 7. Zhao, L., Wu, M., & Xiang, B. (2005). Analysis of Psoralea corylifolia L. fruits in different regions. Chemical & Pharmaceutical Bulletin, 53(8), 1054–1057. 8. Zhao, L. H., Huang, C. Y., Shan, Z., Xiang, B. G., & Mei, L. H. (2005). Fingerprint analysis of Psoralea corylifolia by HLPC and LC-MS. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 821(1), 67–74. 9. Wang, Y., Hong, C., Zhou, C., Xu, D., Qu, H., & Cheng, Y. (2009). Screening antitumor compounds psoralen and isopsoralen
16.
17. 18. 19.
20.
21.
22.
23.
24.
from Psoralea corylifolia L. seeds. Evidence-Based Complementary and Alternative Medicine, 8, 1–7. Oliveira, A. M., Raposo, M. M., Oliveira-Campos, A. M., Machado, A. E., Puapairoj, P., Pedro, M., et al. (2006). Psoralen analogues: Synthesis, inhibitory activity of growth of human tumor cell lines and computational studies. European Journal of Medicinal Chemistry, 41(3), 367–372. Xin, D., Wang, H., Yang, J., Su, Y. F., Fan, G. W., Wang, Y. F., et al. (2010). Phytoestrogens from Psoralea corylifolia reveal estrogen receptor-subtype selectivity. Phytomed, 17(2), 126–131. Lee, W. R., Shen, S. C., Lin, H. Y., Hou, W. C., Yang, L. L., & Chen, Y. C. (2002). Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by decrease of reactive oxygen species, and activation of caspase-3 and Ca (2?)dependent endonuclease. Biochemical Pharmacology, 63(2), 225–236. Farazi, P. A., & DePinho, R. A. (2006). Hepatocellular carcinoma pathogenesis: From genes to environment. Nature Reviews Cancer, 6(9), 674–687. Middleton, E., Kandaswami, C., & Theoharides, T. C. (2000). The effects of plant flavonoids on mammalian cells: Implications for inflammation, heart disease, and cancer. Pharmacological Reviews, 52(4), 673–751. Shi, C., Wu, C. Q., Cao, A. M., Sheng, H. Z., Yan, X. Z., & Liao, M. Y. (2007). NMR-spectroscopy-based metabonomic approach to the analysis of Bay41-4109, a novel anti-HBV compound, induced hepatotoxicity in rats. Toxicology Letters, 173(3), 161–167. Agarwal, M., Taylor, W. R., Chernov, M. V., Chernova, O. B., & Stark, G. R. (1998). The p53 network. Journal of Biological Chemistry, 273, 1–4. Pucci, B., Kasten, M., & Giordano, A. (2000). Cell cycle and apoptosis. Neoplasia, 2, 291–299. Vidal, A., & Koff, A. (2000). Cell-cycle inhibitors: Three families united by a common cause. Gene, 247(1–2), 1–15. Vander Heiden, M. G., & Thompson, C. B. (1999). Bcl-2 proteins: Regulators of apoptosis or of mitochondrial homeostasis. Nature Cell Biology, 1(18), 209–216. Wolter, K. G., Hsu, Y. T., Smith, C. L., Nechushtan, A., Xi, X. G., & Youle, R. J. (1997). Movement of Bax from the cytosol to mitochondria during apoptosis. Journal of Cell Biology, 139(5), 1281–1292. Hockenbery, D. M., Oltvai, Z. N., Yin, X. M., Milliman, C. L., & Korsmeyer, S. J. (1993). Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell, 75(2), 241–251. Scorrano, L., & Koresmeyer, S. J. (2003). Mechanisms of cytochrome c release by proapoptotic Bcl-2 family members. Biochemical and Biophysical Research Communications, 304(3), 437–444. Zha, H., & Reed, J. C. (1997). Heterodimerization-independent functions of cell death regulatory proteins Bax and Bcl-2 in yeast and mammalian cells. Journal of Biological Chemistry, 272(50), 31482–31488. Pettersson, F., Dalgleish, A. G., Bissonnette, R. P., & Colston, K. W. (2002). Retinoids cause apoptosis in pancreatic cancer cells via activation of RAR-gamma and altered expression of Bcl-2/ Bax. British Journal of Cancer, 87(5), 555–561.
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ORIGINAL PAPER
Induction of Apoptosis in Human Hepatocarcinoma SMMC-7721 Cells In Vitro by Psoralen from Psoralea corylifolia Zhongyong Jiang • Jie Xiong
Ó Springer Science+Business Media New York 2014
Abstract Psoralen is a major active component of Psoralea corylifolia. In the present study, we analyzed psoralen-induced changes in human hepatocarcinoma cell viability and apoptosis, and investigated the underlying mechanisms of the proapoptotic effect of the compound on SMMC-7721 cells. We measured human hepatocarcinoma cell viability by MTT assay and Annexin V-FITC/PI double staining, and evaluated the activity of caspase 3 and the expression of p53, Bax, and Bcl-2 proteins, involved in regulating cell apoptosis. Psoralen was able to inhibit the growth of SMMC-7721 cells in a dose- and time-dependent manner and had a strong proapoptotic effect on these cells. We show a dose-dependent increase in caspase-3 activity, and elevated levels of p53 and Bax proteins in psoralentreated cells, that coincided with dose-dependent decrease in Bcl-2 expression. These results suggest that psoralen induces apoptosis in cancer cells via mechanisms that involve caspase-3, p53, Bax, and Bcl-2 pathway. Our results may provide a molecular basis for the further development of natural compounds as novel anticancer agents for human hepatomas. Keywords Psoralen SMMC-7721 cell Apoptosis Caspase-3 p53 Bax Bcl-2
Zhongyong Jiang and Jie Xiong contributed equally in this article. Z. Jiang (&) J. Xiong Department of Clinical Laboratory, Chengdu Military General Hospital, Chengdu Military District, Chengdu 610083, People’s Republic of China e-mail: [email protected]
Introduction Hepatocellular carcinoma is one of the most common malignant tumors worldwide, which is responsible for approximately one million deaths annually. Since surgical and pharmacological therapeutic approaches, available today, are rarely effective in treating the advanced disease, the outcomes of HCC patients remain poor [1]. Hepatocellular carcinoma is usually insensitive to chemotherapeutic drugs currently used in clinical setting, and is well known for its expression of multidrug resistance (MDR) gene, as well as the poor response to chemotherapeutic agents [2]. There is, therefore, a great need for alternative more efficient therapeutic approaches with less side effects. Previous studies suggested that bioactive natural compounds found in medicinal plants could in theory serve as alternatives to chemically designed anticancer agents [3]. Plant-derived natural products play a very important role in the area of cancer therapy research as a source of numerous currently used anticancer agents including vincristine, vinblastine, paclitaxel, camptothecin derivatives, epipodophyllotoxin, and many others [4, 5]. Therefore, there is a great need for continuous search for new antineoplastic agents from plant sources. Psoralea corylifolia is a leguminous plant that is widely used in traditional Chinese medicine (TCM) for treating pollakiuria, nephritis, osteoporosis, hypertension, and cardiovascular diseases [6–8], and is officially listed in the Pharmacopoeia of the Peoples’ Republic of China (Edit 2010 Vol II). In vitro and in vivo studies have demonstrated that the active fractions from the seeds and roots of Psoralea corylifolia have distinct antimicrobial, antioxidative, immunomodulatory, and anticancer properties [6, 9]. Previous reports show that P. corylifolia extract contains coumarins, flavonoids, and merotepenes, such as
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Cell Biochem Biophys
psoralen, isopsoralen, neobavaislfoavone, bovachin, bavaislfoavone, bavachromene, psoralidin, corylifolinin, bavachinin, and bavachalcone [4]. Psoralen, a tricyclic furocoumarin, is the major and the most active furocoumarin present in P. corylifolia, and is widely used as an antineoplastic agent in treatment of leukemia and other cancers [6, 9–11]. In the present study, we are describing for the first time the proapoptotic effect of psoralen on human hepatoma SMMC-7721 cells. Our results suggest that the potent anticancer activity of psoralen correlates with the up-regulation of expression of caspase-3, p53, Bax, and Bcl-2, the components of the intrinsic apoptosis pathway.
Methods Regents and Materials Psoralen was purchased from the National Institute of Control of Pharmaceutical and Biological Products. Cell culture reagents were obtained from GIBCO Life Technology (Grand Island, NY, USA). Antibodies specific for the Bcl-2, Bax, p53, and antirabbit IgG, HRP-linked antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). b-Actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-3 activity kit was obtained from Beyotime Institute of Biotechnology (Haimen, China), and Annexin V-FITC Apoptosis Detection kit was purchased from Pharmingen (Becton–Dickinson Company, San Jose, CA, USA). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) and other reagents were purchased from Sigma (St. Louis, MO, USA). Preparation of Psoralen Psoralen was dissolved in dimethyl sulfoxide (DMSO), and aliquots of 10 mg/mL stock solution were stored at -20 °C. For all the experiments, psoralen was further diluted in DMSO to the final concentrations of 10, 30, 50, or 100 lg/mL. Carrier solvent (0.1 % DMSO) was used as control. Cell Culture Human hepatocarcinoma cell line (SMMC-7721) was purchased from Jiangsu Institute of Cancer Research (JICR. Jiangsu, China). Cells were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum, 100 U/mL penicillin G, and 100 lg/mL streptomycin in a humidified atmosphere of 5 % CO2 at 37 °C. The culture medium was changed for every 2 days.
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Cell Viability Assay Inhibition of cell proliferation by psoralen was measured by MTT assay. SMMC-7721 cells were cultured in 96-well plates at a cell density of 2 9 104 cells per well in 100 lL of RPMI 1640 for 24 h. The medium was then removed and replaced by fresh medium containing different concentrations of psoralen (10, 30, 50, and 100 lg/mL) for 24, 48, 72, and 96 h. Positive control group was treated with 5-fluorouracil (5-FU) solution at the final concentration of 250 lg/mL. Cells in the negative control group were incubated with sterilized saline solution. Cells were then incubated with 10 lL of 5 mg/mL MTT solution in 100 lL of medium for 3 h at 37 °C. MTT solution was then replaced by 150 lL DMSO and incubated for another 10 min with agitation. Absorbance was measured by automatic microplate reader (EL800, BIO-TEK instruments, Inc.) at 550 nm. Growth inhibition rate (GIR) was calculated as follows: GIR ð%Þ ¼ ðODnegative group ODtest group Þ=ODnegative group 100 %: The results were presented as mean ± SD. The morphological changes of SMMC-7721 cells were observed with an inverted microscope at 2009 magnification. Determination of Cell Apoptosis by Annexin V and PI Double Staining Measurement of phosphatidylserine redistribution in a plasma membrane of SMMC-7721 cells was conducted according to the protocol outlined by the manufacturer of the Annexin V-FITC Apoptosis Detection kit (Becton–Dickinson Company, San Jose, CA, USA). Briefly, cells were seeded in 6-well tissue culture plates to the final density of 1 9 106 cells/well, and incubated for 24 h at 37 °C. Psoralen (10, 30, 50, and 100 lg/mL) was directly added to the 6-well plates and incubated with cells for an additional 48 h. Positive control group was treated with 250 lg/mL 5-FU solution, and sterilized saline was used for the negative control group. Cells were then harvested and resuspended in PBS. Apoptotic cells were identified by FITC-conjugated Annexin-V/PI double staining, according to the manufacturer’s protocol, and evaluated by FACSCalibur flow cytometry (Becton–Dickinson, San Jose, CA). Caspase-3 Activity Assay Activity of caspase-3 was determined using the caspase-3 activity kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s protocol. Briefly, following 48 h incubation with different concentrations of psoralen (0, 10, 30, 50, and 100 lg/mL), SMMC-7721 cells
Cell Biochem Biophys Table 1 Inhibition rates of hepatoma SMMC-7721 cells following treatment with increasing concentrations of psoralen for 24–96 h Groups
Inhibition rate (%) 24 h
P 48 h
72 h
96 h
10 lg/mL
18.84 ± 1.75
36.96 ± 1.21
54.28 ± 0.92
51.20 ± 2.13
0.000
30 lg/mL
40.33 ± 1.91
61.86 ± 1.26
71.40 ± 1.21
65.95 ± 2.48
0.000
50 lg/mL
47.43 ± 1.61
82.25 ± 1.35
86.61 ± 1.31
78.35 ± 3.04
0.000
100 lg/mL
53.17 ± 1.08
82.00 ± 1.16
86.39 ± 1.51
81.15 ± 1.14
0.000
Positive
48.67 ± 1.16
75.84 ± 1.31
86.40 ± 2.06
80.34 ± 1.57
0.000
P
0.000
0.000
0.000
0.000
Cells treated with 250 lg/mL of 5-FU were used as positive control; Values represent mean ± standard deviation of triplicate measurements
Fig. 1 Inhibition of SMMC-7721 cell proliferation by psoralen in a dose- and time-dependent manner. Cells were treated with different concentrations of psoralen. Absorbance was measured at 24, 48, 72, and 96 h by MTT assay. Cells treated with 250 lg/mL of 5-FU were used as positive control. P \ 0.05
were lysed. Caspase-3 activity assays were carried out in 96-well microtitre plates by incubating 10 lL of cell lysate in 80 lL reaction buffer (1 % NP-40, 20 mM Tris–HCl (pH = 7.5), 137 mM Nad and 10 % glycerol) containing 10 lL caspase-3 substrate (Ac-DEVD-pNA) (2 mM). Samples were incubated at 37 °C for 4 h and absorbance was measured at 405 nm.
Proteins were separated on 10 % SDS–polyacrylamide gel (SDS-PAGE) and transferred onto the PVDF membranes (Millipore, Billerica, MA) blocked with. Immune bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, Mo) and probed with rabbit antiBax, rabbit antiBcl-2, and mouse antiactin antibodies (1:500, Santa Cruz, CA) overnight at 4 °C. Blots were washed and incubated for 1 h with IRDyeTM800-conjugated antimouse and antirabbit secondary antibodies (1:15,000, Santa Cruz, CA), washed with 0.1 % Tween-20 in TBS, and the proteins were visualized by chemiluminescence using the Amersham ECL Plus Western Blotting Detection kit (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) according to the manufacturer’s instructions. b-Actin was used as a loading control. Statistical Analysis All data were expressed as mean ± SD, and analyzed by one-way analysis of variance followed by Least Significance Difference multiple comparison or Dunnett’s multiple comparison tests using SPSS 13.0 software (SPSS, Chicago, IL, USA). Multiple comparison tests were used when appropriate. A P value of 0.05 was considered statistically significant.
Western Blot Analysis
Results
SMMC-7721 cells were incubated with psoralen (0, 10, 30, 50, and 100 lg/mL) for 48 h, and total cell proteins were extracted by lysing the cells in buffer, containing 100 mM Tris–Cl, pH 6.8, 4 % (m/v) SDS, 20 % (v/v) glycerol, 200 mM b-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 g/mL aprotinin. Protein concentrations were determined by BCA assay using Varioskan spectrofluorometer and spectrophotometer (Thermo, Waltham, MA) at 562 nm.
Effect of Psoralen on the Growth of Human Hepatoma SMMC-7721 Cells Traditional antineoplastic therapy is based on the use of chemotherapeutic compounds which have a cytotoxic effect on proliferating cells and promote the destruction of sensitive tumors. We first researched possible antiproliferative activity of psoralen by evaluating its in vitro cytotoxic effect on human hepatoma SMMC-7721 cells
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Cell Biochem Biophys Fig. 2 Morphological changes in hepatoma SMMC-7721 cells after treatment with psoralen for 48 h. a Untreated cells (negative control); b cells treated with 10 lg/mL of psoralen; c cells treated with 50 lg/mL of psoralen; d cells treated with 250 lg/mL of 5-FU (positive control). Cells were visualized using inverted microscope at 2009 magnification
using MTT assay. Cells, treated with 10 lg/mL of psoralen for 24 h exhibited only slightly reduced growth inhibition. Increasing psoralen concentration or the length of the treatment led to further increase in proliferation inhibition rate of hepatoma SMMC-7721 (Table 1; Fig. 1). The highest concentration of psoralen (50 and 100 lg/mL) resulted in GIRs similar to 5-FU-treated positive control cells. These results suggest that psoralen could inhibit growth of hepatoma SMMC-7721 cells in a time- and dosedependence manner. Analysis of Psoralen-Induced Changes in SMMC-7721 Cell Morphology
with the increased concentration of psoralen for 48 h, and the rate of cell apoptosis was determined by Annexin V-FITC/PI staining, followed by the flow cytometric analysis (FACS) (Fig. 3). Even at the lowest concentration (10 lg/mL), psoralen treatment of the hepatoma cells resulted in increased number of double-stained Annexin V-FITC-positive and PI-negative cells, indicative of early apoptosis. As summarized in Table 2, cells, treated with 30 lg/mL of psoralen exhibited significantly higher rate of apoptosis (23.32 ± 1.91 %) as compared with the negative control group (2.57 ± 1.23 %, P \ 0.01). Effect of Psoralen on the Activation of Caspase-3
To evaluate psoralen-induced morphological changes in human hepatoma cells, SMMC-7721 cells were treated with increased concentrations of psoralen for 48 h and analyzed using inverted microscope at 2009 magnification. Number of SMMC-7721 cells was significantly decreased after treatment with psoralen when compared to untreated cells (negative control) in a dose-dependent manner (Fig. 2), and cells exhibited progressive morphological changes with increased duration of exposure to psoralen (data not shown).
Caspase-3 plays a central role in mediating various cellular apoptotic responses [12]. We measured caspase-3 protease activity in human hepatoma cells treated with the various concentrations of psoralen. Psoralen treatment caused increased caspase-3 protease activity in a dose-dependent manner: over threefold increase at the concentration of 30 lg/mL, and almost fivefold increase (569.94 ± 7.74 %) at the maximal concentration of 100 lg/mL as compared to untreated cells (Fig. 4).
Flow Cytometric Analysis of Psoralen-Induced Apoptosis
Effects of Psoralen on Levels of p53, Bax, and Bcl-2 Proteins in SMMC-7721 Cells
We next tested the proapoptotic effect of psoralen on the human hepatoma cells. SMMC-7721 cells were treated
We next evaluated the effect of psoralen on the expression of p53, Bax, and Bcl-2, important components of the
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Cell Biochem Biophys Fig. 3 Proapoptotic effect of psoralen. SMMC-7721 cells were treated with psoralen (10, 30, 50, and 100 lg/mL) for 48 h, incubated with Annexin V and PI for 30 min at 37 °C and then subjected to FACS analysis. a Untreated cells represent negative control group; b cells, treated with 5-FU were used as a positive control; c-1 to c-4 SMMC-7721 cells treated with 10, 30, 50, and 100 lg/mL of psoralen, respectively. The viable cell population is presented in the lower left quadrant (Annexin V-PI-). The cells at the early stages of apoptosis are in the lower right quadrant (Annexin V ? PI-)
intrinsic apoptotic pathway, in SMMC-7721 cells. After 48 h incubation with the increasing concentrations of psoralen (0, 10, 30, 50, and 100 lg/mL), the expression of p53, Bax, and Bcl-2 proteins was analyzed by western blotting. At higher concentrations (30, 50, and 100 lg/mL) psoralen led to a significant increase in protein levels of p53 and Bax in human hepatoma. In contrast, the level of Bcl-2 protein significantly decreased in a dose-dependent manner after psoralen treatment (Fig. 5).
Discussion HCC is a rapidly fatal disease, with a life expectancy of about 6 months from the time of the diagnosis. Therapeutic strategies employed to date have significantly improved the prognosis for patients with unresectable HCC [13]. This emphasizes the need for investigating the molecular mechanisms responsible for HCC development and seeking effective and noncytotoxic chemical agents for
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Cell Biochem Biophys Table 2 Apoptosis rates of SMMC-7721 cells treated with psoralen for 48 h Groups Negative control Positive control Psoralen 30 lg/mL
Apoptosis rate (%) 2.57 ± 1.23 1.48 ± 0.36 23.32 ± 1.91ab
Apoptosis rate of negative group, positive group, and 30 lg/mL of psoralen-treated cells were determined by Annexin V-FITC and PI staining and FACS. The values expressed were mean ± standard deviation of triplicate measurements a
P \ 0.01 vs. negative control group
b
P \ 0.01 vs. positive control group
Fig. 5 Effect of psoralen on the Expression of p53, Bax, and Bcl-2 in SMMC-7721 cells. Cells were treated with psoralen (0, 10, 30, 50, and 100 lg/mL) for 48 h, and the expression of p53, Bax, and Bcl-2 proteins was detected by western blotting. b-Actin is used as a loading control
Fig. 4 Effects of psoralen on caspase-3 activity in SMMC-7721 cells. Cells were incubated with 0, 10, 30, 50, and 100 lg/mL psoralen for 48 h at 37 °C. Data represent mean ± SD of three independent experiments. P \ 0.05 and P \ 0.01 from Dunnett’s test comparing to untreated cells (control group). Each value is expressed as the ratio of caspase-3 activation level to control level, which was assigned a value of 100
chemoprevention and treatment. Since only few synthetic antineoplastic compounds are effective for the treatment of this disease, the focus of more and more studies has shifted recently to researching natural active agents for cancer chemoprevention and treatment [14]. The pharmacological mechanism of the anticancer activity of P. corylifolia L. extract, psoralen is still not fully understood. Previous reports show that psoralen decreases the energy supply of cancer cells and inhibits nucleic acid and protein synthesis by destroying cell mitochondria [15]. Other studies report that psoralen and isopsoralen induce cancer cell apoptosis, and the differences in apoptotic response of cells to psoralen and isopsoralen may provide some insight on whether the proapoptotic effect of P. corylifolia L. extract is dependent on p53 [9].
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In the present study, we demonstrate that psoralen has an anticancer activity on SMMC-7721 cells. Psoralen has a significant growth inhibitory effect on human hepatoma cells, probably through the cytotoxic mechanism that involves the induction of apoptosis. The latter was identified by typical characteristic features of apoptosis and dose-dependent increase in apoptotic rate. Proapoptotic effect of psoralen correlated with the dose-dependent elevated activity of caspase-3 in psoralen-treated SMCC-7721 cells. Caspases, a family of cysteine proteases, have long been recognized as critical components of the apoptotic pathway, and caspase-3 is required for a majority of the nuclear changes associated with apoptosis [12]. We demonstrated that psoralen induced dose-dependent increase in the levels of p53 and Bax proteins and decrease in the levels of Bcl-2 protein in treated hepatoma cells. Numerous studies demonstrated that p53, a tumor suppressor gene, plays an important role in tumor growth inhibition and induction of apoptosis, and exerts its control on apoptosis by interacting with other important apoptotic molecules, including the members of the Bcl-2 family [16–18]. This protein family regulates the intrinsic or mitochondrial pathway of apoptosis, and is involved in positive (Bax, Bak, Blk, Bad, and Bid) and negative (Bcl-2, Bcl-XL, Bclw, Bfl-1, Bag-1, Mcl-1, and A1) regulation of apoptotic cell death [19]. Antiapoptotic Bcl-2 and proapoptotic Bax are two of the major members of the Bcl-2 family [20]. Bcl-2 is known as an upstream effector of the apoptotic pathway and is identified as a potent suppressor of apoptosis [21]. Studies also show that Bcl-2 promotes cell survival by preserving the integrity of the external mitochondrial membrane, thus preventing the release of
Cell Biochem Biophys
cytochrome c from mitochondria and subsequent cell death [22]. Interestingly, the increased apoptotic cell death correlates with an elevated intracellular Bax/Bcl-2 ratio [23], and the Bcl-2/Bax ratio rather than the levels of bcl-2 alone, plays an important role in regulating the effects of the apoptosis induction [24]. Our recent results suggest that psoralen would increase Bax/Bcl-2 ratio in human hepatoma cells, given the fact that the treatment results in upregulation of Bax expression that coincides with the dosedependent decrease in Bcl-2 protein levels. In conclusion, we have demonstrated that psoralen inhibits the growth of SMMC-7721 human hepatoma cells by inducing cell apoptosis through mechanism that involves regulation of caspase-3 activity, and p53 and Bcl2/Bax protein expression. These results may provide further insight on the mechanisms involved in psoraleninduced cytotoxic effects on cancer cells.
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References 1. Kern, M. A., Breuhahn, K., & Schirmacher, P. (2002). Molecular pathogenesis of human hepatocellular carcinoma. Advances in Cancer Research, 86, 67–112. 2. Geng, C. X., Zeng, Z. C., & Wang, J. Y. (2003). Docetaxel inhibits SMMC-7721 human hepatocellular carcinoma cells growth and induces apoptosis. World Journal of Gastroenterology, 9(4), 696–700. 3. Ramirez-Mares, M. V., Chandra, S., & de Mejia, E. G. (2004). In vitro chemopreventive activity of Camellia sinensis, Ilex paraguariensis and Ardisia compressa tea extracts and selected polyphenols. Mutation Research, 554(1–2), 53–65. 4. Cragg, M. G., & Newman, D. J. (2005). Plants as a source of anticancer agents. Journal of Ethnopharmacology, 100(1–2), 72–79. 5. Cragg, M. G., Kingston, D. G. I., & Newman, D. J. (2005). Anticancer agents from natural products (Chap. 7, pp. 123–136). Boca Raton, FL: Brunner-Routledge Psychology Press, Taylor & Francis Group. 6. Latha, P. G., Evans, D. A., Panikkar, K., & Jayavardhanan, K. K. (2000). Immunomodulatory and antitumor properties of Psoralea corylifolia seeds. Fitoterapia, 71, 223–231. 7. Zhao, L., Wu, M., & Xiang, B. (2005). Analysis of Psoralea corylifolia L. fruits in different regions. Chemical & Pharmaceutical Bulletin, 53(8), 1054–1057. 8. Zhao, L. H., Huang, C. Y., Shan, Z., Xiang, B. G., & Mei, L. H. (2005). Fingerprint analysis of Psoralea corylifolia by HLPC and LC-MS. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 821(1), 67–74. 9. Wang, Y., Hong, C., Zhou, C., Xu, D., Qu, H., & Cheng, Y. (2009). Screening antitumor compounds psoralen and isopsoralen
16.
17. 18. 19.
20.
21.
22.
23.
24.
from Psoralea corylifolia L. seeds. Evidence-Based Complementary and Alternative Medicine, 8, 1–7. Oliveira, A. M., Raposo, M. M., Oliveira-Campos, A. M., Machado, A. E., Puapairoj, P., Pedro, M., et al. (2006). Psoralen analogues: Synthesis, inhibitory activity of growth of human tumor cell lines and computational studies. European Journal of Medicinal Chemistry, 41(3), 367–372. Xin, D., Wang, H., Yang, J., Su, Y. F., Fan, G. W., Wang, Y. F., et al. (2010). Phytoestrogens from Psoralea corylifolia reveal estrogen receptor-subtype selectivity. Phytomed, 17(2), 126–131. Lee, W. R., Shen, S. C., Lin, H. Y., Hou, W. C., Yang, L. L., & Chen, Y. C. (2002). Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by decrease of reactive oxygen species, and activation of caspase-3 and Ca (2?)dependent endonuclease. Biochemical Pharmacology, 63(2), 225–236. Farazi, P. A., & DePinho, R. A. (2006). Hepatocellular carcinoma pathogenesis: From genes to environment. Nature Reviews Cancer, 6(9), 674–687. Middleton, E., Kandaswami, C., & Theoharides, T. C. (2000). The effects of plant flavonoids on mammalian cells: Implications for inflammation, heart disease, and cancer. Pharmacological Reviews, 52(4), 673–751. Shi, C., Wu, C. Q., Cao, A. M., Sheng, H. Z., Yan, X. Z., & Liao, M. Y. (2007). NMR-spectroscopy-based metabonomic approach to the analysis of Bay41-4109, a novel anti-HBV compound, induced hepatotoxicity in rats. Toxicology Letters, 173(3), 161–167. Agarwal, M., Taylor, W. R., Chernov, M. V., Chernova, O. B., & Stark, G. R. (1998). The p53 network. Journal of Biological Chemistry, 273, 1–4. Pucci, B., Kasten, M., & Giordano, A. (2000). Cell cycle and apoptosis. Neoplasia, 2, 291–299. Vidal, A., & Koff, A. (2000). Cell-cycle inhibitors: Three families united by a common cause. Gene, 247(1–2), 1–15. Vander Heiden, M. G., & Thompson, C. B. (1999). Bcl-2 proteins: Regulators of apoptosis or of mitochondrial homeostasis. Nature Cell Biology, 1(18), 209–216. Wolter, K. G., Hsu, Y. T., Smith, C. L., Nechushtan, A., Xi, X. G., & Youle, R. J. (1997). Movement of Bax from the cytosol to mitochondria during apoptosis. Journal of Cell Biology, 139(5), 1281–1292. Hockenbery, D. M., Oltvai, Z. N., Yin, X. M., Milliman, C. L., & Korsmeyer, S. J. (1993). Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell, 75(2), 241–251. Scorrano, L., & Koresmeyer, S. J. (2003). Mechanisms of cytochrome c release by proapoptotic Bcl-2 family members. Biochemical and Biophysical Research Communications, 304(3), 437–444. Zha, H., & Reed, J. C. (1997). Heterodimerization-independent functions of cell death regulatory proteins Bax and Bcl-2 in yeast and mammalian cells. Journal of Biological Chemistry, 272(50), 31482–31488. Pettersson, F., Dalgleish, A. G., Bissonnette, R. P., & Colston, K. W. (2002). Retinoids cause apoptosis in pancreatic cancer cells via activation of RAR-gamma and altered expression of Bcl-2/ Bax. British Journal of Cancer, 87(5), 555–561.
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