Chemico-Biological Interactions 220 (2014) 193–199
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
The antitumor effect of formosanin C on HepG2 cell as revealed by H-NMR based metabolic profiling
1
Yuanyuan Li a,b, Shuli Man a,b,⇑, Jing Li a,b, Hongyan Chai a,b, Wei Fan a,b, Zhen Liu c, Wenyuan Gao a,b,c,⇑ a
Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China c Tianjin Key Laboratory for Modern Drug Delivery and High Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China b
a r t i c l e
i n f o
Article history: Received 27 March 2014 Received in revised form 22 May 2014 Accepted 20 June 2014 Available online 9 July 2014 Keywords: Formosanin C S phase arrest Metabonomics Apoptosis
a b s t r a c t Formosanin C (FC) is a pure compound isolated from Rhizoma Paridis. In the past years, antitumor effects of FC have been observed in several cultural cells and animal systems. However, there was no research particular on liver cancer. In this experiment, 3-(4, 5-dimethylthiazol diphenyltetrazolium bromide (MTT) dye reduction assay was used to evaluate cell viability of HepG2 cells with FC-treatment. 40 , 6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC/PI assay and DNA fragment assay were applied to observe FC-induced apoptosis. Cell cycle analysis and NMR metabolic profiles were used to identify molecular mechanisms of FC in HepG2 cells. As a result, FC inhibited the growth of HepG2 cells through inducing apoptosis and S phase arrest. Cells cultured in the presence or absence of FC was different in metabolic profiles. The treatment decreased acetate, ethanol, choline and betaine, and increased butyrate, fatty acids, leucine and valine in HepG2 cells. In conclusion, metabolomic analysis of the exometabolome of FC-treated HepG2 cells, together with traditional methods such as apoptosis test and cell cycle analysis provided a holistic method for elucidating mechanisms of potential anti-cancer drug, FC. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is the fifth most common type of carcinoma all over the world, and accounts for about 6% of all new cancer cases diagnosed worldwide (nearly 750,000 new cases/year) [1]. It is an urgent need to develop new therapeutic agents to fight against liver cancer. Formosanin C (FC), a diosgenin glycoside with four sugars, has only recently emerged as a potential antitumor agent [2–4]. It was an effectively promoting agent for cell cycle arrest and apoptosis without deleterious effects to different normal cell types or benign neoplastic derived cells [3]. Up to now, FC appeared to be sensitive to many kinds of cancer cells including myeloid leukemia, colon cancer, liver cancer, lung cancer, cervical cancer and renal adenocarcinoma cells [5]. Abbreviations: DAPI, 40 ,6-diamidino-2-phenylindole; D2O, deuterium oxide; FC, formosanin C; HCC, hepatocellular carcinoma; MTT, 3-(4,5-dimethyl-thiagol-2yl)2,5-diplenyltertrazollium; TSP, sodium-3-(trimethylsilyl)-2,2,3,3-tetradeuteriopropionate. ⇑ Corresponding authors. Address: Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China. Tel./fax: +86 22 87401895. E-mail addresses: [email protected] (S. Man), [email protected] (W. Gao). http://dx.doi.org/10.1016/j.cbi.2014.06.023 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
The aim of the present paper was to investigate the anti-cancer effects of FC on human liver cancer cell lines HepG2 and the possible mechanisms based on metabonomics. 2. Materials and methods 2.1. Materials 3-(4,5-Dimethyl-thiagol-2yl)-2,5-diplenyltertrazollium (MTT) and 40 ,6-diamidino-2-phenylindole (DAPI) were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). Apoptotic DNA ladder isolation kit and cell cycle analysis kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Highglucose DMEM medium, fetal calf serum, penicillin–streptomycin, trypsin and EDTA were obtained from Thermo (Being, China). Sodium-3-(trimethylsilyl)-2,2,3,3-tetradeuteriopropionate (TSP) was purchased from Merck (Germany). Deuterium oxide (D2O) was purchased from J&K, China. 2.2. Isolation of FC Paris polyphylla Smith var. yunnanensis was collected from Lijiang, Yunnan province of China. The plants were air-dried,
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chipped and extracted with ethanol and partitioned with petroleum ether, EtOAc and n-BuOH, sequently. The n-BuOH soluble fraction was separated on a silica gel column with CHCl3:MeOH (100:0 ? 95:5 ? 9:1 ? 8:2 ? 7:3 ? 6:4 ? 0:10), a Sephadex LH20 column with CHCl3: MeOH (1:1) and an HPLC-ODS with MeOH ? MeOH:H2O (75:25) to obtain FC [6]. Reference standard of FC was purchased from the National Institute for the Control of Pharmaceutical and Biological Products, China. Its batch was 111,591–200,402. The purity of this monomer was determined to be more than 98% by normalization of the peak areas detected by HPLC, and was stable in methanol solution [7].
2.8. Cell cycle analysis The cell cycle distribution was analyzed by cell cycle analysis kit (Beyotime, China) according to the manufacturer’s instruction. Briefly, HepG2 cells were treated with different concentrations of FC for 24 h. Then the cells were harvested by trypsinized, washed in ice-cold phosphate buffered saline, and fixed in 70% ice cold ethanol overnight. Subsequently, the fixed cells washed with ice-cold PBS before incubation with the binding buffer containing RNase and propidium iodide for 30 min at 37 °C in the dark. Finally, the stained cells were analyzed by flow cytometry with modfit LT software (Modfit LT 4.0).
2.3. Cell culture 2.9. 1H-NMR analysis Human hepatoma cell line HepG2 was acquired from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The HepG2 cells were maintained in high-glucose DMEM supplemented with 10% heat-inactivated (56 °C, 30 min) fetal calf serum, penicillin (100 U/mL) and streptomycin (100 lg/mL). The cell culture was maintained at 37 °C in a humidified atmosphere of 5% of CO2 with passage each 3–4 days. 2.4. Cell viability assay The cell viability assay was evaluated by MTT assay [8]. HepG2 cells were seeded in 96-well microtiter plates at a density of 1 104 per well and left to adhere overnight before drugs treatment. FC was prepared in the basal medium with a final DMSO concentration of less than 0.1%. The same concentration of DMSO was used as the vehicle control in all experiments. The cells were then incubated in the presence of 0 to 50 lg/mL of FC for 24 and 48 h. MTT was added to each well at a concentration of 0.5 mg/ mL and incubated for 4 h. Absorbance was determined at 570 nm by addition of 100 lL of DMSO for each well using ELISA reader. The experiments were repeated in triplicate wells. 2.5. DAPI staining Approximately 2 105 cells/well of HepG2 cells seeded in 6-well plate and left to adhere overnight. Then different concentrations of FC (5 and 10 lg/mL) were added in the cells and incubated for 24 or 48 h. Cells were stained by DAPI (5 lg/mL) for 10 min at room temperature, and then the DAPI dye was aspirated. The DAPI staining cells were photographed by fluorescence microscope. 2.6. DNA fragmentation
Approximately 3 106 cells/well of HepG2 cells seeded in cell culture dishes and left to adhere overnight. Then different concentrations of FC were added in the cells and incubated for 24 h. Collecting cells and the change of the metabolites in the HepG2 cells was measured by 1H-NMR [9]. Each sample was mixed with 350 lL of aqueous phosphate solution (0.2 mol/L). The addition of an internal standard like TSP (100 lL, 1.5 mmol/L) was prohibited by its interaction with proteins present in the sample. Subsequently, the total volume was transferred to a 5 mm NMR tube. 1 H-NMR spectra were measured at a 1H frequency of 400 MHz using a Varian Unity INOVA 600 spectrometer, equipped with a 5-mm triple-resonance probe. Prior to Fourier transformation, the free induction decays were zero-filled to 32 K and an exponential weighing factor corresponding to a line broadening of 0.5 Hz was applied. 3. Results 3.1. FC inhibited viability and proliferation of HepG2 cells MTT assay showed that FC inhibited HepG2 cell growth in a concentration- and time-dependent manner (Fig. 1). 50% inhibitory concentration (IC50) of FC was estimated to be 13.62 ± 0.36 and 3.29 ± 0.55 lg/mL at 24 and 48 h, respectively. The IC50 of FC was the same as that in another human hepatoma cell line Bel7402 cell. 3.2. Morphological changes caused by FC It was detected by DAPI staining method that FC inducted HepG2 cells apoptosis after treatment for 24 and 48 h. As shown
Apoptosis was monitored by DNA fragmentation assay. Approximately 1 106 cells/well of HepG2 cells seeded in 6-well plate and left to adhere overnight. Then different concentrations of FC were added in the cells and incubated for 48 h. Later DNA fragmentation was extracted by Apoptotic DNA ladder isolation kit. Fragmented DNA was electrophoresed on 1.5% agarose gel to observe the appearance of DNA ladder. 2.7. Annexin V-FITC/PI staining for apoptosis evaluation Apoptosis was quantified using flow cytometry to measure the levels of detectable phosphatidylserine on the outer membrane of apoptotic cells. HepG2 cells were seeded on 6-well plates (4 105 cells/mL), and incubated with different concentrations of FC in 0.1% DMSO solutions for 48 h. Then the cells were harvested by trypsinized, washed in ice-cold phosphate buffered saline, and re-suspended in diluted binding buffer from the Annexin V-FITC kit (Bestbio Inc., Shanghai, China) based on the manufacturer’s instructions. The procedure was carried out three times.
Fig. 1. HepG2 was treated with different concentration of FC for 24 and 48 h. Cell growth was determined by MTT assay and was directly proportional to the absorbance at a wavelength of 570 nm. Data expressed as means ± S.D. from three independent experiments.
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Fig. 2. The fluorescence images of DAPI for the cells treated with different concentration of FC at 24 and 48 h (100, final magnification). Arrows in these pictures indicated morphological changes in the nucleus of cells, the red arrows indicated chromatin condensation and the white arrows indicated nucleus fragmentation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
in Fig. 2, FC induced apoptosis in a time- and concentrationdependent manner. The viable cells exhibited bright green, while the apoptotic cells caused by FC showed deep white based on DAPI staining. Changes in apoptotic cells were observed after DAPI staining in FC-treated groups including karyorrhexis and karyopyknosis. The apoptotic nuclear in the FC-treated groups were split into several nuclear apoptosis bodies as indicated by the arrows shown in Fig. 2. As incubating time increased, the nucleuses were broken into pieces at 48 h. In addition, the apoptotic area in 10 lg/mL of FC was larger than that in 5 lg/mL. 3.3. FC induces DNA damage in HepG2 cells The DNA fragmentation analysis yielded the evidence of apoptotic induction. As shown in Fig. 3, HepG2 cells were treated with different concentrations of FC for 48 h. DNA ladder which was a signal for advanced stage apoptosis and fragmentation were evidently observed on agarose gel electrophoresis, especially at the concentration of 5 lg/mL of FC for 48 h treatment. 3.4. Annexin V-FITC/PI assay of HepG2 cells apoptosis To determine whether apoptosis was induced by the compounds mentioned above, we performed flow cytometric analysis with Annexin V-FITC conjugated to propidium iodide (PI). Annexin V-FITC-positive, PI-negative (Annexin V-FITC (+) PI ()) cells were considered to be in an early apoptotic stage, while Annexin V-FITC-positive, PI-positive (Annexin V-FITC (+) PI (+)) cells were considered to be late apoptotic or necrotic. The rates of apoptosis induced by FC were significantly higher than apoptotic rate in the control group (Fig. 4).
Fig. 3. The apoptosis effect of FC on HepG2 cells detected by DNA ladder after 48 h treatment. The DNA fragmentation assay showed that apoptosis was much more apparent in cells treated with 5 lg/mL of FC than that in the non-treated one.
3.5. FC induces S-phase block in HepG2 cells An increased accumulation of subdiploid cells displayed in those cells that had been treated with FC for 24 h compared to the untreated group (Fig. 5). Furthermore, to test whether FC affected the cell cycle progression, HepG2 cells were treated with different concentration of FC for 24 h. Then they were subjected to flow cytometric analysis after DNA staining. As shown in Fig. 5, exposure of HepG2 cells to FC resulted in a significant
increase in the S phase and a decrease in the G2/M phase. After treatment with 0, 1, 3 and 5 lg/mL of FC for 24 h, the cell population in S phase increased from 43.07 ± 2.03% to 48.45 ± 3.46%, 53.51 ± 0.08% and 58.34 ± 2.44% respectively. Meanwhile, the cell population in G2/M phase decreased from 10.50 ± 0.94% to 10.06 ± 2.41%, 8.86 ± 1.96% and 2.90 ± 1.57%, respectively. These results indicated that the observed growth inhibition was due to cell cycle arrest at S phase.
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Fig. 4. The apoptosis effects of FC on HepG2 cells for 48 h were detected by Annexin V-FITC/PI assay staining. (A) 0 lg/mL (B) 3 lg/mL, (C) 5 lg/mL and (D) 10 lg/mL of FC treated with cells.
3.6. 1H-NMR analysis results Changes in the metabolite profiles of the cancer cells were measured by 1H-NMR. Different concentration of FC (0–5 lg/mL) was treated with HepG2 cells for 24 h. 1H-NMR internal standard TSP was used to estimate the absolute concentrations of metabolites. Fig. 6 showed the dose-dependent responses to FC. As a result, nine metabolites in HepG2 cells were detected by NMR. Four metabolites in FC-treated group were significantly increased, which included butyrate, fatty acids, leucine and valine. Four metabolites were significantly decreased containing ethanol, acetate, choline and betaine.
4. Discussion FC, a pure compound isolated from Rhizoma Paridis, has antitumor effect on many kinds of cancer cells, such as lung cancer [2], ovarian cancer [3], colorectal cancer [4,10] and so forth. In our previous researches, Rhizome Paridis saponins especially for FC showed anti-lung cancer and anti-hepatocarcinoma activities [2,11]. It induced apoptosis in lung cancer [8]. Other labs reported that the immune response was involved in antitumor effect of FC on MH134 mouse hepatoma [12,13]. It also activates caspase-2 and causes the dysfunction of mitochondria in human colorectal cancer HT-29 cells [10]. FC also exhibited anthelmintic activity against Dactylogyrus intermedius [14]. Its structural analog,
diosgenin, exhibits anticancer activity by attenuating lipid peroxidation via enhancing antioxidant defense system during NMUinduced breast carcinoma [15]. Through the cyclooxygenase-2 and 5- lipoxygenase pathways, diosgenin also induces apoptosis in HT-29 and HCT-116 colon cancer cells [16]. Recently, metabolic regulatory network has been used to represent drugs intervention on tumor formation and progression [9]. In this experiment, we adopted cell cycle arrest, apoptosis assay, metabonomics and network biology approaches to reconstruct the metabolic regulatory network in response to the FC intervention in hepatocarcinoma cells. This study aimed to test the possible mechanism of FC, which exerted anticancer effect on hepatoma cell HepG2. In this experiment, FC inhibited the growth of HepG2 cells with a concentrationand time-dependent manner (Fig. 1). For the early time apoptosis in 5 lg/mL of FC for 24 h, nuclei concentrated and became deep white (Fig. 2). The number of the S phase cells increased, while the G2/M phase cells decreased after the same concentration treated for 24 h (Fig. 5). With FC incubated with HepG2 cells for 48 h, the advanced stage of apoptosis appeared and the nuclei broken into pieces (Fig. 2), which indicated that FC induced HepG2 cells apoptosis in a time-dependent manner [17]. Meanwhile, DNA ladder was also a signal for the advanced stage [18]. FC-treated for 48 h with a concentration of 5 lg/mL emerged evidently DNA fragment on agarose gel electrophoresis (Fig. 3). From a metabolism point of view (Fig. 6), the observation of the elevated levels of butyrate, fatty acids, leucine and valine, and the
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Fig. 5. Cell cycle distribution after different concentration of FC (0, 1, 3, 5 lg/mL) treated with HepG2 cells for 24 h. Cells were evaluated by flow cytometry after PI staining of nuclei. (A) 0 lg/mL (B) 1 lg/mL, (C) 3 lg/mL and (D) 5 lg/mL of FC treated with cells. Data are expressed as percentages of the total cell population in each cell phase, means of three independent experiments.
Fig. 6. Dose-dependent responses to FC. (A) Typical 1H-NMR spectra of HepG2 cancer cells in the 0.7–3.4 ppm spectral range. From top to bottom, cells exposed to 0, 1, 2, 3, 4, and 5 lg/L of FC. (B) The up-regulative relative concentration of metabolites in cells. (C) The down-regulative relative concentration of metabolites in cells. Mean control value was set to 1.
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Fig. 7. The perturbed metabolic pathways in response to FC-treated HepG2 cells. Dashed line indicates a number of steps happened in this metabolic process.
descended levels of ethanol, acetate, choline and betaine (Fig. 7) were consistent with S phase arrest and apoptosis of FC-treatment in HepG2 cells. As we known, methyl group donors and intermediates of onecarbon metabolism influence DNA synthesis and DNA methylation, and thereby affect carcinogenesis. Choline, the precursor of betaine [19], and the one-carbon metabolite sarcosine have been associated with increased cancer risk [20]. Especial for high concentration of choline, it was associated with high prostate cancer risk [21], and may serve as biomarker for both diagnostic and treatment monitoring purposes for breast cancer [22–24]. Therefore, the depletion in choline and betaine indicated the inhibition of the DNA synthesis and DNA methylation in cancer cell S phase arrest. For energy production and biosynthesis of cellular components, pyruvate is metabolized to acetyl-CoA, ethanol, acetate or lactate, and via the TCA cycle, and through oxidative phosphorylation to 18 ATP molecules in normal oxygenated conditions [25]. The decreased levels of acetate and ethanol in FC-treated groups suggested that TCA cycle or energy metabolism in liver mitochondria was impaired. The decrease in acetate was also in accord with the observations that enhancing acetate incorporation into lipids and phospholipids, increased fatty acid uptake and decreased fatty acid oxidation, which was also associated with choline-decrease in HepG2 cells [26]. On the other hand, as previous report, ratio of branched-chain amino acids (leucine, valine, isoleucine) to aromatic ones (phenylalanine, tyrosine) is important for assessing liver metabolism, hepatic functional reserve and the severity of liver dysfunction [27]. Especial for valine, leucine and isoleucine, they were essential for vascular endothelial growth factor mRNA degradation in patients who were in the process of developing hepatocellular carcinoma [28]. Meanwhile, the metastatic tissue from all sites displayed a substantially decreased expression for genes involved in butyrate and propanoate metabolism and valine, leucine and isoleucine degradation [29]. Our results demonstrated that FC
up-regulated levels of butyrate, valine and leucine which involved in key cell signaling and metabolic pathways during the course of inhibiting the carcinogenesis and metastasis, which was accorded with previous report on its anti-metastasis effect [2]. In conclusion, FC inhibited the growth of HepG2 cells through inducing apoptosis and S phase arrest. Metabolomic analysis of the exometabolome of FC-treated HepG2 cells indicated that decreased choline and betaine was correlated with inhibition of the DNA synthesis and DNA methylation, down-regulated acetate and ethanol was associated with impaired TCA cycle and energy metabolism in liver mitochondria. In addition, increased butyrate, valine and leucine were involved in key cell signaling and metabolic pathways during the course of inhibiting the carcinogenesis and metastasis. Therefore, metabolomic analysis of the exometabolome of FC-treated HepG2 cells, together with traditional methods such as apoptosis test and cell cycle analysis provided a holistic method to elucidate mechanisms of FC.
Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by grants 81202952 from National Natural Science Foundation of China, Drug Creation Projects 2013ZX09103002-010 from Science and Technology in China and 12JCZDJC26200 and 13JCQNJC13400 from Tianjin Natural Science Foundation in China. References [1] J.A. Davila, Diabetes and hepatocellular carcinoma: what role does diabetes have in the presence of other known risk factors?, Am J. Gastroenterol. 105 (2010) 632–634.
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
The antitumor effect of formosanin C on HepG2 cell as revealed by H-NMR based metabolic profiling
1
Yuanyuan Li a,b, Shuli Man a,b,⇑, Jing Li a,b, Hongyan Chai a,b, Wei Fan a,b, Zhen Liu c, Wenyuan Gao a,b,c,⇑ a
Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China c Tianjin Key Laboratory for Modern Drug Delivery and High Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China b
a r t i c l e
i n f o
Article history: Received 27 March 2014 Received in revised form 22 May 2014 Accepted 20 June 2014 Available online 9 July 2014 Keywords: Formosanin C S phase arrest Metabonomics Apoptosis
a b s t r a c t Formosanin C (FC) is a pure compound isolated from Rhizoma Paridis. In the past years, antitumor effects of FC have been observed in several cultural cells and animal systems. However, there was no research particular on liver cancer. In this experiment, 3-(4, 5-dimethylthiazol diphenyltetrazolium bromide (MTT) dye reduction assay was used to evaluate cell viability of HepG2 cells with FC-treatment. 40 , 6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC/PI assay and DNA fragment assay were applied to observe FC-induced apoptosis. Cell cycle analysis and NMR metabolic profiles were used to identify molecular mechanisms of FC in HepG2 cells. As a result, FC inhibited the growth of HepG2 cells through inducing apoptosis and S phase arrest. Cells cultured in the presence or absence of FC was different in metabolic profiles. The treatment decreased acetate, ethanol, choline and betaine, and increased butyrate, fatty acids, leucine and valine in HepG2 cells. In conclusion, metabolomic analysis of the exometabolome of FC-treated HepG2 cells, together with traditional methods such as apoptosis test and cell cycle analysis provided a holistic method for elucidating mechanisms of potential anti-cancer drug, FC. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is the fifth most common type of carcinoma all over the world, and accounts for about 6% of all new cancer cases diagnosed worldwide (nearly 750,000 new cases/year) [1]. It is an urgent need to develop new therapeutic agents to fight against liver cancer. Formosanin C (FC), a diosgenin glycoside with four sugars, has only recently emerged as a potential antitumor agent [2–4]. It was an effectively promoting agent for cell cycle arrest and apoptosis without deleterious effects to different normal cell types or benign neoplastic derived cells [3]. Up to now, FC appeared to be sensitive to many kinds of cancer cells including myeloid leukemia, colon cancer, liver cancer, lung cancer, cervical cancer and renal adenocarcinoma cells [5]. Abbreviations: DAPI, 40 ,6-diamidino-2-phenylindole; D2O, deuterium oxide; FC, formosanin C; HCC, hepatocellular carcinoma; MTT, 3-(4,5-dimethyl-thiagol-2yl)2,5-diplenyltertrazollium; TSP, sodium-3-(trimethylsilyl)-2,2,3,3-tetradeuteriopropionate. ⇑ Corresponding authors. Address: Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China. Tel./fax: +86 22 87401895. E-mail addresses: [email protected] (S. Man), [email protected] (W. Gao). http://dx.doi.org/10.1016/j.cbi.2014.06.023 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
The aim of the present paper was to investigate the anti-cancer effects of FC on human liver cancer cell lines HepG2 and the possible mechanisms based on metabonomics. 2. Materials and methods 2.1. Materials 3-(4,5-Dimethyl-thiagol-2yl)-2,5-diplenyltertrazollium (MTT) and 40 ,6-diamidino-2-phenylindole (DAPI) were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). Apoptotic DNA ladder isolation kit and cell cycle analysis kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Highglucose DMEM medium, fetal calf serum, penicillin–streptomycin, trypsin and EDTA were obtained from Thermo (Being, China). Sodium-3-(trimethylsilyl)-2,2,3,3-tetradeuteriopropionate (TSP) was purchased from Merck (Germany). Deuterium oxide (D2O) was purchased from J&K, China. 2.2. Isolation of FC Paris polyphylla Smith var. yunnanensis was collected from Lijiang, Yunnan province of China. The plants were air-dried,
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chipped and extracted with ethanol and partitioned with petroleum ether, EtOAc and n-BuOH, sequently. The n-BuOH soluble fraction was separated on a silica gel column with CHCl3:MeOH (100:0 ? 95:5 ? 9:1 ? 8:2 ? 7:3 ? 6:4 ? 0:10), a Sephadex LH20 column with CHCl3: MeOH (1:1) and an HPLC-ODS with MeOH ? MeOH:H2O (75:25) to obtain FC [6]. Reference standard of FC was purchased from the National Institute for the Control of Pharmaceutical and Biological Products, China. Its batch was 111,591–200,402. The purity of this monomer was determined to be more than 98% by normalization of the peak areas detected by HPLC, and was stable in methanol solution [7].
2.8. Cell cycle analysis The cell cycle distribution was analyzed by cell cycle analysis kit (Beyotime, China) according to the manufacturer’s instruction. Briefly, HepG2 cells were treated with different concentrations of FC for 24 h. Then the cells were harvested by trypsinized, washed in ice-cold phosphate buffered saline, and fixed in 70% ice cold ethanol overnight. Subsequently, the fixed cells washed with ice-cold PBS before incubation with the binding buffer containing RNase and propidium iodide for 30 min at 37 °C in the dark. Finally, the stained cells were analyzed by flow cytometry with modfit LT software (Modfit LT 4.0).
2.3. Cell culture 2.9. 1H-NMR analysis Human hepatoma cell line HepG2 was acquired from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The HepG2 cells were maintained in high-glucose DMEM supplemented with 10% heat-inactivated (56 °C, 30 min) fetal calf serum, penicillin (100 U/mL) and streptomycin (100 lg/mL). The cell culture was maintained at 37 °C in a humidified atmosphere of 5% of CO2 with passage each 3–4 days. 2.4. Cell viability assay The cell viability assay was evaluated by MTT assay [8]. HepG2 cells were seeded in 96-well microtiter plates at a density of 1 104 per well and left to adhere overnight before drugs treatment. FC was prepared in the basal medium with a final DMSO concentration of less than 0.1%. The same concentration of DMSO was used as the vehicle control in all experiments. The cells were then incubated in the presence of 0 to 50 lg/mL of FC for 24 and 48 h. MTT was added to each well at a concentration of 0.5 mg/ mL and incubated for 4 h. Absorbance was determined at 570 nm by addition of 100 lL of DMSO for each well using ELISA reader. The experiments were repeated in triplicate wells. 2.5. DAPI staining Approximately 2 105 cells/well of HepG2 cells seeded in 6-well plate and left to adhere overnight. Then different concentrations of FC (5 and 10 lg/mL) were added in the cells and incubated for 24 or 48 h. Cells were stained by DAPI (5 lg/mL) for 10 min at room temperature, and then the DAPI dye was aspirated. The DAPI staining cells were photographed by fluorescence microscope. 2.6. DNA fragmentation
Approximately 3 106 cells/well of HepG2 cells seeded in cell culture dishes and left to adhere overnight. Then different concentrations of FC were added in the cells and incubated for 24 h. Collecting cells and the change of the metabolites in the HepG2 cells was measured by 1H-NMR [9]. Each sample was mixed with 350 lL of aqueous phosphate solution (0.2 mol/L). The addition of an internal standard like TSP (100 lL, 1.5 mmol/L) was prohibited by its interaction with proteins present in the sample. Subsequently, the total volume was transferred to a 5 mm NMR tube. 1 H-NMR spectra were measured at a 1H frequency of 400 MHz using a Varian Unity INOVA 600 spectrometer, equipped with a 5-mm triple-resonance probe. Prior to Fourier transformation, the free induction decays were zero-filled to 32 K and an exponential weighing factor corresponding to a line broadening of 0.5 Hz was applied. 3. Results 3.1. FC inhibited viability and proliferation of HepG2 cells MTT assay showed that FC inhibited HepG2 cell growth in a concentration- and time-dependent manner (Fig. 1). 50% inhibitory concentration (IC50) of FC was estimated to be 13.62 ± 0.36 and 3.29 ± 0.55 lg/mL at 24 and 48 h, respectively. The IC50 of FC was the same as that in another human hepatoma cell line Bel7402 cell. 3.2. Morphological changes caused by FC It was detected by DAPI staining method that FC inducted HepG2 cells apoptosis after treatment for 24 and 48 h. As shown
Apoptosis was monitored by DNA fragmentation assay. Approximately 1 106 cells/well of HepG2 cells seeded in 6-well plate and left to adhere overnight. Then different concentrations of FC were added in the cells and incubated for 48 h. Later DNA fragmentation was extracted by Apoptotic DNA ladder isolation kit. Fragmented DNA was electrophoresed on 1.5% agarose gel to observe the appearance of DNA ladder. 2.7. Annexin V-FITC/PI staining for apoptosis evaluation Apoptosis was quantified using flow cytometry to measure the levels of detectable phosphatidylserine on the outer membrane of apoptotic cells. HepG2 cells were seeded on 6-well plates (4 105 cells/mL), and incubated with different concentrations of FC in 0.1% DMSO solutions for 48 h. Then the cells were harvested by trypsinized, washed in ice-cold phosphate buffered saline, and re-suspended in diluted binding buffer from the Annexin V-FITC kit (Bestbio Inc., Shanghai, China) based on the manufacturer’s instructions. The procedure was carried out three times.
Fig. 1. HepG2 was treated with different concentration of FC for 24 and 48 h. Cell growth was determined by MTT assay and was directly proportional to the absorbance at a wavelength of 570 nm. Data expressed as means ± S.D. from three independent experiments.
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Fig. 2. The fluorescence images of DAPI for the cells treated with different concentration of FC at 24 and 48 h (100, final magnification). Arrows in these pictures indicated morphological changes in the nucleus of cells, the red arrows indicated chromatin condensation and the white arrows indicated nucleus fragmentation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
in Fig. 2, FC induced apoptosis in a time- and concentrationdependent manner. The viable cells exhibited bright green, while the apoptotic cells caused by FC showed deep white based on DAPI staining. Changes in apoptotic cells were observed after DAPI staining in FC-treated groups including karyorrhexis and karyopyknosis. The apoptotic nuclear in the FC-treated groups were split into several nuclear apoptosis bodies as indicated by the arrows shown in Fig. 2. As incubating time increased, the nucleuses were broken into pieces at 48 h. In addition, the apoptotic area in 10 lg/mL of FC was larger than that in 5 lg/mL. 3.3. FC induces DNA damage in HepG2 cells The DNA fragmentation analysis yielded the evidence of apoptotic induction. As shown in Fig. 3, HepG2 cells were treated with different concentrations of FC for 48 h. DNA ladder which was a signal for advanced stage apoptosis and fragmentation were evidently observed on agarose gel electrophoresis, especially at the concentration of 5 lg/mL of FC for 48 h treatment. 3.4. Annexin V-FITC/PI assay of HepG2 cells apoptosis To determine whether apoptosis was induced by the compounds mentioned above, we performed flow cytometric analysis with Annexin V-FITC conjugated to propidium iodide (PI). Annexin V-FITC-positive, PI-negative (Annexin V-FITC (+) PI ()) cells were considered to be in an early apoptotic stage, while Annexin V-FITC-positive, PI-positive (Annexin V-FITC (+) PI (+)) cells were considered to be late apoptotic or necrotic. The rates of apoptosis induced by FC were significantly higher than apoptotic rate in the control group (Fig. 4).
Fig. 3. The apoptosis effect of FC on HepG2 cells detected by DNA ladder after 48 h treatment. The DNA fragmentation assay showed that apoptosis was much more apparent in cells treated with 5 lg/mL of FC than that in the non-treated one.
3.5. FC induces S-phase block in HepG2 cells An increased accumulation of subdiploid cells displayed in those cells that had been treated with FC for 24 h compared to the untreated group (Fig. 5). Furthermore, to test whether FC affected the cell cycle progression, HepG2 cells were treated with different concentration of FC for 24 h. Then they were subjected to flow cytometric analysis after DNA staining. As shown in Fig. 5, exposure of HepG2 cells to FC resulted in a significant
increase in the S phase and a decrease in the G2/M phase. After treatment with 0, 1, 3 and 5 lg/mL of FC for 24 h, the cell population in S phase increased from 43.07 ± 2.03% to 48.45 ± 3.46%, 53.51 ± 0.08% and 58.34 ± 2.44% respectively. Meanwhile, the cell population in G2/M phase decreased from 10.50 ± 0.94% to 10.06 ± 2.41%, 8.86 ± 1.96% and 2.90 ± 1.57%, respectively. These results indicated that the observed growth inhibition was due to cell cycle arrest at S phase.
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Fig. 4. The apoptosis effects of FC on HepG2 cells for 48 h were detected by Annexin V-FITC/PI assay staining. (A) 0 lg/mL (B) 3 lg/mL, (C) 5 lg/mL and (D) 10 lg/mL of FC treated with cells.
3.6. 1H-NMR analysis results Changes in the metabolite profiles of the cancer cells were measured by 1H-NMR. Different concentration of FC (0–5 lg/mL) was treated with HepG2 cells for 24 h. 1H-NMR internal standard TSP was used to estimate the absolute concentrations of metabolites. Fig. 6 showed the dose-dependent responses to FC. As a result, nine metabolites in HepG2 cells were detected by NMR. Four metabolites in FC-treated group were significantly increased, which included butyrate, fatty acids, leucine and valine. Four metabolites were significantly decreased containing ethanol, acetate, choline and betaine.
4. Discussion FC, a pure compound isolated from Rhizoma Paridis, has antitumor effect on many kinds of cancer cells, such as lung cancer [2], ovarian cancer [3], colorectal cancer [4,10] and so forth. In our previous researches, Rhizome Paridis saponins especially for FC showed anti-lung cancer and anti-hepatocarcinoma activities [2,11]. It induced apoptosis in lung cancer [8]. Other labs reported that the immune response was involved in antitumor effect of FC on MH134 mouse hepatoma [12,13]. It also activates caspase-2 and causes the dysfunction of mitochondria in human colorectal cancer HT-29 cells [10]. FC also exhibited anthelmintic activity against Dactylogyrus intermedius [14]. Its structural analog,
diosgenin, exhibits anticancer activity by attenuating lipid peroxidation via enhancing antioxidant defense system during NMUinduced breast carcinoma [15]. Through the cyclooxygenase-2 and 5- lipoxygenase pathways, diosgenin also induces apoptosis in HT-29 and HCT-116 colon cancer cells [16]. Recently, metabolic regulatory network has been used to represent drugs intervention on tumor formation and progression [9]. In this experiment, we adopted cell cycle arrest, apoptosis assay, metabonomics and network biology approaches to reconstruct the metabolic regulatory network in response to the FC intervention in hepatocarcinoma cells. This study aimed to test the possible mechanism of FC, which exerted anticancer effect on hepatoma cell HepG2. In this experiment, FC inhibited the growth of HepG2 cells with a concentrationand time-dependent manner (Fig. 1). For the early time apoptosis in 5 lg/mL of FC for 24 h, nuclei concentrated and became deep white (Fig. 2). The number of the S phase cells increased, while the G2/M phase cells decreased after the same concentration treated for 24 h (Fig. 5). With FC incubated with HepG2 cells for 48 h, the advanced stage of apoptosis appeared and the nuclei broken into pieces (Fig. 2), which indicated that FC induced HepG2 cells apoptosis in a time-dependent manner [17]. Meanwhile, DNA ladder was also a signal for the advanced stage [18]. FC-treated for 48 h with a concentration of 5 lg/mL emerged evidently DNA fragment on agarose gel electrophoresis (Fig. 3). From a metabolism point of view (Fig. 6), the observation of the elevated levels of butyrate, fatty acids, leucine and valine, and the
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Fig. 5. Cell cycle distribution after different concentration of FC (0, 1, 3, 5 lg/mL) treated with HepG2 cells for 24 h. Cells were evaluated by flow cytometry after PI staining of nuclei. (A) 0 lg/mL (B) 1 lg/mL, (C) 3 lg/mL and (D) 5 lg/mL of FC treated with cells. Data are expressed as percentages of the total cell population in each cell phase, means of three independent experiments.
Fig. 6. Dose-dependent responses to FC. (A) Typical 1H-NMR spectra of HepG2 cancer cells in the 0.7–3.4 ppm spectral range. From top to bottom, cells exposed to 0, 1, 2, 3, 4, and 5 lg/L of FC. (B) The up-regulative relative concentration of metabolites in cells. (C) The down-regulative relative concentration of metabolites in cells. Mean control value was set to 1.
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Fig. 7. The perturbed metabolic pathways in response to FC-treated HepG2 cells. Dashed line indicates a number of steps happened in this metabolic process.
descended levels of ethanol, acetate, choline and betaine (Fig. 7) were consistent with S phase arrest and apoptosis of FC-treatment in HepG2 cells. As we known, methyl group donors and intermediates of onecarbon metabolism influence DNA synthesis and DNA methylation, and thereby affect carcinogenesis. Choline, the precursor of betaine [19], and the one-carbon metabolite sarcosine have been associated with increased cancer risk [20]. Especial for high concentration of choline, it was associated with high prostate cancer risk [21], and may serve as biomarker for both diagnostic and treatment monitoring purposes for breast cancer [22–24]. Therefore, the depletion in choline and betaine indicated the inhibition of the DNA synthesis and DNA methylation in cancer cell S phase arrest. For energy production and biosynthesis of cellular components, pyruvate is metabolized to acetyl-CoA, ethanol, acetate or lactate, and via the TCA cycle, and through oxidative phosphorylation to 18 ATP molecules in normal oxygenated conditions [25]. The decreased levels of acetate and ethanol in FC-treated groups suggested that TCA cycle or energy metabolism in liver mitochondria was impaired. The decrease in acetate was also in accord with the observations that enhancing acetate incorporation into lipids and phospholipids, increased fatty acid uptake and decreased fatty acid oxidation, which was also associated with choline-decrease in HepG2 cells [26]. On the other hand, as previous report, ratio of branched-chain amino acids (leucine, valine, isoleucine) to aromatic ones (phenylalanine, tyrosine) is important for assessing liver metabolism, hepatic functional reserve and the severity of liver dysfunction [27]. Especial for valine, leucine and isoleucine, they were essential for vascular endothelial growth factor mRNA degradation in patients who were in the process of developing hepatocellular carcinoma [28]. Meanwhile, the metastatic tissue from all sites displayed a substantially decreased expression for genes involved in butyrate and propanoate metabolism and valine, leucine and isoleucine degradation [29]. Our results demonstrated that FC
up-regulated levels of butyrate, valine and leucine which involved in key cell signaling and metabolic pathways during the course of inhibiting the carcinogenesis and metastasis, which was accorded with previous report on its anti-metastasis effect [2]. In conclusion, FC inhibited the growth of HepG2 cells through inducing apoptosis and S phase arrest. Metabolomic analysis of the exometabolome of FC-treated HepG2 cells indicated that decreased choline and betaine was correlated with inhibition of the DNA synthesis and DNA methylation, down-regulated acetate and ethanol was associated with impaired TCA cycle and energy metabolism in liver mitochondria. In addition, increased butyrate, valine and leucine were involved in key cell signaling and metabolic pathways during the course of inhibiting the carcinogenesis and metastasis. Therefore, metabolomic analysis of the exometabolome of FC-treated HepG2 cells, together with traditional methods such as apoptosis test and cell cycle analysis provided a holistic method to elucidate mechanisms of FC.
Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by grants 81202952 from National Natural Science Foundation of China, Drug Creation Projects 2013ZX09103002-010 from Science and Technology in China and 12JCZDJC26200 and 13JCQNJC13400 from Tianjin Natural Science Foundation in China. References [1] J.A. Davila, Diabetes and hepatocellular carcinoma: what role does diabetes have in the presence of other known risk factors?, Am J. Gastroenterol. 105 (2010) 632–634.
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