Kang-Lai-Te (KLT) is extracted from the

Original Article / Liver Experimental study on the effect of Kang-Lai-Te induced apoptosis of human hepatoma carcinoma cell HepG2 Yun Lu, Li-Qun Wu, Qian Dong and Chang-Sheng Li Qingdao, China BACKGROUND: Kang-Lai-Te (KLT) is extracted from the traditional Chinese herbal medicine Semen Coicis, which has been used in China as an effective clinical drug for over a thousand years. It contains numerous ingredients with anti-tumor effects. In our previous studies on transplanted hepatomas in rats, KLT could stop the cells in the G2+M stage of cell cycle and then reduce the number of cells entering the stage G0 and G1, but the mechanism of the anti-proliferative effect was unknown. In this experiment, we examined whether KLT inhibits HepG2 cell growth, if so, tried to explore its mechanism. METHODS: KLT at different concentrations was used for the treatment of hepatocellular carcinoma cells in vitro, respectively. The proliferation inhibitory rate was evaluated by MTT assay, induction of cell apoptosis rate and the protein levels of Fas and Fas ligand (FasL) were determined by flow cytometry (FCM), and the expression of Fas and FasL mrna was detected by real-time fluorescent quantitative RT-PCR. RESULTS: KLT produced an obvious time and dosedependent inhibitory effect on HepG2 cells, and marked apoptosis was detected by FCM. The protein of Fas increased by 11.01%, 18.71%, 28.71% and 37.15%; the protein of FasL increased by 1.49%, 1.91%, 3.27% and 3.38% in comparison with the control (P<0.05). Real-time fluorescent quantitative RT-PCR showed that treating HepG2 cells with KLT caused the upregulation of Fas and FasL mrna. Author Affiliations: Department of Hepatobiliary Surgery (Lu Y and Wu LQ), Department of Pediatric Surgery (Dong Q), and Center of Cell and Molecular Pathology (Li CS), Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, China Corresponding Author: Yun Lu, MD, Department of Hepatobiliary Surgery, Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, China (Tel: 86-532-82911369; Fax: 86-532-82911999; Email: cloudylucn@126.com) 2009, Hepatobiliary Pancreat Dis Int. All rights reserved. CONCLUSION: KLT inhibits HepG2 growth by inducing apoptosis, which may be mediated through activation of the Fas/FasL pathway. (Hepatobiliary Pancreat Dis Int 2009; 8: 267-272) KEY WORDS: Kang-Lai-Te; drugs, Chinese herbal; hepatocellular carcinoma; HepG2; Fas/FasL Introduction Kang-Lai-Te (KLT) is extracted from the traditional Chinese herbal medicine Semen Coicis, which has been used in China as an effective clinical drug for over a thousand years and contains numerous ingredients with anti-tumor effects. [1] KLT injections can increase the sensitivity of some tumors to chemotherapeutic drugs and reduce the toxicity of chemotherapy. [2, 3] As an emulsion, it is thought to provide vital energy for the human body in some Chinese philosophical sense and could also be used to reduce cachexia in patients with end-stage diseases. [4] As reported in Chinese Medical News (2001), KLT injection was approved by the American Food and Drug Administration as one of chemotherapeutic agents, while clinical trials in the United States had been premised since 2001. Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, especially in several Asian countries. [5] Both clinical operation and chemotherapy could lead to the death of some tumor cells. However, some tumor cells are survival and could result in HCC metastasis, then lead to poor prognosis. In our previous research involving transplanted hepatomas in rats, [3] KLT could stop the cells in the G2+M stage of cell cycle and then reduce the number of cells entering the stages G0 and G1. As Hepatobiliary Pancreat Dis Int,Vol 8,No 3 June 15,2009 www.hbpdint.com 267

Hepatobiliary & Pancreatic Diseases International a result, the percentage of cells in the S period and karyokinesis was reduced, preventing hyperplasia and causing apoptosis. However, the mechanism of this anti-proliferative effect is unknown. The present experiment was to examine whether KLT inhibits HepG2 cells growth and, if so, to explore its mechanism. KLT induced apoptosis in HepG2 cells, suggesting that KLT induces apoptosis through the activation of the Fas/FasL pathway. Methods Cell culture and KLT treatment Hepatoma carcinoma cells line HepG2 was provided by Nanjing Kaiji Biotechnology Ltd., China. Tumor cells were cultured in RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco BRL) at 37 in a humidified atmosphere containing 5% CO 2. After 60%-80% confluency, the cells were trypsinized with 0.25% trypsin (AMRESCO, dissolved in PBS, ph 7.4), counted and placed down at a needed density for treatment. 10% KLT injection was purchased from Kang-Lai-Te Pharmaceutical of Zhejiang province, China (specifications, 100 ml/ bottle; registration, 060706 1-2). Growth inhibition assay To examine the effect of KLT, we plated the cells in 96-well plates at 1 10 5 cells/ml per well with RPMI 1640 containing 10% fetal bovine serum (FBS). Prior to the KLT treatment, the cell monolayers were rinsed and serum-deprived for 24 hours with RPMI 1640 supplemented with 1% FBS (serum-deprivation medium). After serum deprivation, we replaced the medium with fresh serum-deprivation medium with or without various concentrations of KLT. After treatments, 100 μl MTT (Sigma Chemical Co., America) was added, and the cells were further incubated at 37 for 4 hours. The supernatant was then discarded, and 200 μl DMSO (Gibco BRL) was added. The 96-well plate was vibrated on a micro-vibrator for additional 10 minutes, and the optical density of each well was measured with enzymeimmunoassay instrument (Bio-Rad2550, America) at 490 nm. The cell inhibition rate was calculated with the following formula: Cell inhibition rate (%)=(1 average A 490 nm of treated group/average A 490 nm of control group) 100%. Flow cytometry (FCM) assay of apoptosis To estimate early apoptotic cell number, cells were plated in 24-well plates and incubated in the absence (10 mmol HEPES/NaOH, 140 mmol NaCl, 2.5 mmol CaCl 2 ) or presence of various concentrations of KLT. After 48 hours, the cells were collected and washed with ice-cold PBS and binding buffer. They were then incubated with GFP-tagged Annexin V (Jingmei Company, Beijing, China) on ice for additional 15 minutes. PI (Jingmei Company, Beijing, China) was added immediately before analysis by FCM (FACScan Becton Dickinson, America). 10 000 cells were counted, per sample. The data were analyzed with CellQuest software (Becton Dickinson). FCM assay of the Fas/FasL protein HepG2 cells were cultured in 24-well plates for 24 hours and incubated in the absence or presence of various concentrations of KLT. After 48 hours, the adherent and nonadherent cells were collected and washed with PBS. Stop buffer was added. After 10 minutes, the 20 μl fluorescent antibodies (Santa Cruz Biotechnology, America) of Fas and FasL were added to the treating cells with KLT, and 20 μl mouse IgG1/IgG1 (Jingmei Company, Beijing) was added to the cells without KLT. Then the cells were centrifugated for 10 minutes and were suspended by paraformaldehyde. The result was detected by a flow cytometer. RNA extraction and real-time fluorescent quantitative RT-PCR Total RNA was isolated using a Trizol reagent (TaKaRa Biotechnology Co, Ltd., Dalian) according to the manufacturer's recommendations. cdna was synthesized from 2 μg of total RNA and reverse transcriptase with random hexanucleotide according to the manufacturer's instruction. The reaction mixture was subjected to PCR to amplify sequences to the desired primers (Table 1). Then the fluorescent probes (Table 2) were added and amplification was performed with a 7500 RT-PCR meter (Applied Biosystemswith) on cycles of denaturation at 95 for 10 seconds, annealing at 95 for 5 seconds, and Table 1. Gene-specific primers for RT-PCR Name Orientation Sequence of primer Fas Sense 5'-TGG AAG GCC TGC ATC ATG A-3' Anti-sense 5'-TGT CTG TGT ACT CCT TCC CTT CTT G-3' FasL Sense 5'-ACA GCA TCA TCT TTG GAG AAG CA-3' Anti-sense 5'-ACA ATT CCA TAG GTG TCT TCC CAT T-3 GAPDH Sense 5'-CAT GGG TGT GAA CCA TGA GAA GTA T-3' Anti-sense 5'-GAC TGT GGT CAT GAG TCC TTC CA-3' GAPDH: glyceraldehyde-3-phosphate dehydrogenase. 268 Hepatobiliary Pancreat Dis Int,Vol 8,No 3 June 15,2009 www.hbpdint.com

Kang-Lai-Te induced apoptosis on HepG2 cells extension at 60 for 34 seconds. Finally Ct value and quantitation were calculated with the following (Ct normal group Ct formula: corresponding quantitation=2 medication administration (Ct normal group Ct medication group)(destination gene)/2 administration group) (GAPDH). Statistical analysis Biostatistical analyses were done using the SPSS 11.5 software package. All experiments were repeated at least three times. Results of multiple experiments are expressed as mean±sd. The statistical analysis involving two groups was made by Student's t test, whereas analysis of variance (ANOVA) followed by Dunnett's multiple comparison test was used to compare more than two groups. A P value less than 0.05 was accepted as statistically significant. Results Growth inhibition by KLT treatment Table 2. Taqman probe Name Sequence of primer Fas 5'-TGC CAT AAG CCC TGT CCT CCA GGT G-3'; FasL 5'-CAC CCC AGT CCA CCC CCT GAA AAA-3'; GAPDH 5'-AAG ATC ATC AGC AAT GCC TCC TGC-3' GAPDH: glyceraldehyde-3-phosphate dehydrogenase. To investigate whether KLT has anti-proliferative effects against HepG2 cells, then we did growth inhibition assay in cells treated with KLT using the MTT method. KLT treatment resulted in a time- and dose-dependent growth inhibition of HepG2 cells (Fig. 1). Treatment with 5, 10, 15 and 20 μl/ml KLT for 2 days significantly decreased viable cell numbers by 30.89±1.55%, 49.91±2.03%, 60.78±3.31% and 73.73± 5.71%, respectively. Induction of apoptosis by KLT treatment To test whether the decrease in cell viability observed after treatment with KLT was due to induction of apoptosis, we estimated apoptotic cell numbers using Annexin V and PI double staining followed by FCM. Apoptotic cell numbers were increased in a KLT dose-dependent manner (Figs. 2, 3). Compared to the control, a statistically significant increase in apoptotic cell numbers was observed. Induction of Fas/FasL expression by KLT treatment Since the Fas/FasL system is a key signaling transduction pathway of apoptosis, the effects of KLT on their mrna and protein levels in HepG2 Table 3. Effects of KLT on levels of Fas and FasL protein Group Concentration (μl/ml) Fas FasL Control 0 1.05±0.14 0.37±0.34 KLT 5 12.06±2.08 # 1.86±0.24 # 10 19.76±3.78 # 2.28±0.62 # 15 29.76±2.63 # 3.64±0.81 # 20 38.20±2.78 # 3.75±0.55 # One-way ANOVA method was used. F(Fas)=239.09, P<0.05; F(FasL)=32.07, P<0.05. Dunnett was adjusted for comparisons between groups, and compared to the control, #: P<0.01. Fig. 1. The inhibition of KLT on HepG2 cells at different times after treatment. Table 4. Effects of KLT on levels of Fas and FasL mrna Group Concentration (μl/ml) Fas mrna FasL mrna Control 0 0.54±0.24 0.012±0.08 KLT 5 0.75±0.07 # 0.027±0.15 # 10 0.99±0.16 # 0.038±0.21 # 15 1.06±0.59 # 0.053±0.14 # 20 1.37±0.22 # 0.061±0.04 # One-way ANOVA method was used. F(Fas mrna)=3.18, P<0.05; F(FasL mrna)=32.63, P<0.05. Dunnett was adjusted for comparisons between groups, and compared to the control, #: P<0.01. Fig. 2. KLT induces apoptosis of HepG2 cells. KLT induces apoptosis of HepG2 cells. Each bar represents the mean± SD. Comparisons between groups that yielded significant differences (P<0.05) are indicated by different concentration above each bar. Hepatobiliary Pancreat Dis Int,Vol 8,No 3 June 15,2009 www.hbpdint.com 269

Hepatobiliary & Pancreatic Diseases International Fig. 3. The scatterplot of apoptosis induced by KLT. The apoptosis ratio of HepG2 cells (B-E) following exposure to KLT (5, 10, 15 and 20 μl/ml) determined by FCM analysis. Cells treated with no KLT (A) as controls for 48 hours. After treatments, the cells stained with double-stained with GFP-tagged annexin V (1 2000) and PI (1 μl/ml) in the presence of binding buffer without fixation. 10000 cells were counted per sample. The data analyzed with CellQuest software. Each experiment performed in triplicate. The numbers in panels 1 indicate the percentage of cells at the early phase of apoptosis. Fig. 4. The results of Fas and FasL protein expression by KLT treatment. a-e: the expression of Fas protein; a: control group; b: 5 μl/ml KLT; c:10 μl/ml KLT; d: 15 μl/ml KLT ; e: 20 μl/ml KLT. A-E: the expression of FasL protein; A: control group; B: 5 μl/ml KLT; C: 10 μl/ml KLT; D: 15 μl/ml KLT; E: 20 μl/ml KLT. Quantitation 1.5 1.0 0.5 Fas mrna FasL mrna 0 0 5 10 15 20 KLT ( μl/ml) Fig. 5. Induction of Fas/FasL mrna by KLT treatment in HepG2 cells. After 48 hours treatment with KLT, total RNAs were isolated, and real-time fluorescent quantitative RT-PCR analyses were performed using the indicated primers and the fluorescent probes. Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as a housekeeping control gene. cells using FCM assay and real-time fluorescent quantitative RT-PCR analysis were examined. KLT treatment significantly increased the expression of Fas/FasL mrna as well as protein levels (Figs. 4, 5). Fas/FasL mrna and protein levels were markedly increased after KLT treatment in a concentrationdependent fashion (Tables 3, 4). Discussion Many studies confirm that malignancy involves inhibition of apoptosis. [6-8] This has led to keen interest over recent years in induced tumor cell apoptosis. Chemotherapeutic agents such as 5-fluorouracil, methotrexate and doxorubicin can inhibit the proliferation of tumor cells and even kill [9, 10] tumor cells, but their concomitant destruction of normal cells leads to major problems with adverse reactions. KLT is an effective, natural substance extracted from the traditional Chinese medicine Coicis Semen. Some studies have show that KLT can block tumor cell mitosis, inhibit tumor cell proliferation, and induce [1, 2] tumor cells apoptosis. Some studies found that 270 Hepatobiliary Pancreat Dis Int,Vol 8,No 3 June 15,2009 www.hbpdint.com

Kang-Lai-Te induced apoptosis on HepG2 cells KLT could impact the oncogene expression, inhibit tumor angiogenesis and regulate cytokine level. [2, 4] In our previous research involving KLT and transplanted hepatoma rats, [3] we found KLT could inhibit the cells in the G2+M stage of cell cycle and then reduce the number of cells entering the stage G0 and G1. The rate of tumor restriction on the 3th day after injection of KLT ranged from 49.4% to 52.7%, and on the 8th day from 49.4% to 68.2%. On the 3rd day after injection, the increase of cell nuclear antigen levels in transplant hepatoma rats varied from 32.4% to 40.6%, on the 8th day from 39.8% to 49.2%. As a result, the percentage of cells in the S period and karyokinesis are reduced, preventing hyperplasia and causing apoptosis. The present study demonstrated that KLT induces apoptosis in HepG2 cells, which appears to account for its antiproliferative activity (Fig. 2). The Fas/FasL system is a key signaling transduction pathway of apoptosis in cells and tissues. Many observations have highlighted the role of the Fas/FasL system in chemotherapy-induced apoptosis of tumors with upregulation of Fas/APO-1 or its ligand, FasL. [11] Fas is a glycosylated cell surface protein, which localizes on the plasma membrane as well as in the cytosol, in particular in the Golgi complex. [12] Ligation of Fas by agonistic antibody or its mature ligand induces receptor oligomerization and formation of deathinducing signaling complex, followed by activation of caspase-8, and then further activation of a series of caspase cascades resulting in cell apoptotic death. [11] As a tumor necrosis factor-related type Ⅱ membrane protein, FasL can be proteolytically cleaved by a signal, generating a soluble form that has apoptosisinducing ability. Recruitment and accumulation of pro-caspase-8 result in spontaneous activation of caspase-8 via autoproteolytic cleavage of FasL and initiation of the apoptosis signal. [13-15] Therefore, the expression levels of Fas and FasL in KLT-treated cells were examined by FCM assay and real-time fluorescent quantitative RT-PCR in order to determine if KLT induces HepG2 cells death through a change in the expression of Fas and FasL. [16-18] As shown in Fig. 5, KLT markedly induced the levels of Fas and FasL expression, and the upregulation of both Fas and FasL by KLT occurred in a concentration-dependent manner similar to that observed with the inhibition of cell viability and induction of apoptosis. [19-22] Thus, we suggest that the Fas death receptor pathway may at least in part be involved in KLT-mediated anti-proliferation and apoptosis in HepG2 cells. We concluded that KLT potently suppresses proliferation of HepG2 cells by induction of apoptosis, and that KLT-induced apoptosis induction is initiated by the Fas/FasL pathway, which is associated with a significant increase in the level of Fas/FasL protein expression and mrna expression. Although further studies are needed, these findings may contribute to the understanding of the anticancer activity of KLT. Funding: This study was supported by grants from the Distinguished Young Scholar Foundation of Shandong province, China (No. 2006BS03039); and the Science & Technology Key Project of Shandong province, China (No. 2007G30002014). Ethical approval: Not needed. Contributors: LY proposed the study and wrote the first draft. LY and WLQ analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. LY is the guarantor. 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