Antitumor effects of two extracts from Oxytropis falcata on hepatocellular carcinoma in vitro and in vivo

Chinese Journal of Natural Medicines 2013, 11(5): 0519−0524

Chinese Journal of Natural Medicines

Antitumor effects of two extracts from Oxytropis falcata on hepatocellular carcinoma in vitro and in vivo YANG Guang-Ming 1a, YAN Ru 2a, WANG Zhao-Xian 3, ZHANG Fang-Fang 1, PAN Yang 1, 4*, CAI Bao-Chang 1* 1

Key Laboratory of State Administration of Traditional Chinese Medicine for Standardization of Chinese Medicine Processing & Jiangsu Key Laboratory of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, China； 2 University of Macau, Macau, China; 3 School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China; 4 Laboratory of Medical Fungi and Phyto-Biotech, Nanjing University of Chinese Medicine, Nanjing 210023, China

Available online 20 Sept. 2013

[ABSTRACT] AIMS: To investigate the antitumor effects of extracts from Oxytropis falcata on human hepatocellular carcinoma SMMC-7721 cells in vitro and in transplanted murine H22 tumors in vivo. METHODS: Cell proliferation, cell cycle distribution and apoptosis in SMMC-7721 cells were determined and tumor growth inhibition in H22 tumors was investigated. Cell cycle distribution was analyzed by flow cytometry with propidium iodide (PI) and Annexin V-FITC/ PI double staining. RESULTS: MTT assay revealed that essential oil and flavonoids of O. falcata (named EOOF and FOF) inhibited proliferation of SMMC-7721 cells in a dose-dependent manner. The IC50 value of EOOF and FOF were 0.115 and 0.097 mg·mL−1, respectively. Cell cycle was arrested at G1 phase, and induction of apoptosis occurred in SMMC-7721 cells when subjected to FOF. Growth inhibition in H22 solid tumors transplanted mice was significantly pronounced after being treated with FOF, and the inhibition ratio were 56.1% and 70.8% at the concentration of 30 and 60 mg·kg−1. CONCLUSION: The results suggest that FOF promotes apoptosis in SMMC-7721 cells and inhibits H22 tumor growth, resulting in a potential antitumor effect on hepatic cancer. [KEY WORDS] Antitumor; Oxytropis falcata; Apoptosis; SMMC-7721; Murine hepatoma22; Proliferation [CLC Number] R965

1

[Document code] A

[Article ID] 1672-3651(2013)05-0519-06

Introduction

Hepatocellular carcinoma is one of the most frequently occurring malignances of mankind and the third most common cause of cancer related death worldwide, with a tendency towards an increasingly occurrence of new cases and deaths annually [1-2]. Until recently, the prognosis for patients with hepatoma is still poor because there is no effective treatment of this metastatic disease, and effective chemotherapeutic agents have not been developed. In this

[Received on] 12-Dec.-2012 [Research funding] This project was supported by the National Natural Science Foundation of China (No. 30902012). [*Corresponding author] PAN Yang: Prof., Tel: 86-25-86798281, E-mail: [email protected]; CAI Bao-Chang: Prof., [email protected] a These authors contribute equally to this work. These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

regard, novel effective chemotherapeutic agents are desperately needed in the treatment of liver cancer to improve survival. Many crude drugs of Traditional Chinese Medicine (TCM) have been so far useful for cancer patients as antitumor drugs. Some effective compounds have been isolated from these medicinal plants to treat various cancers such as hepatic cancer, lung cancer etc. [3]. The research of antitumor mechanisms of TCM attracts more and more interests of researchers. Disturbance of the cancer cell cycle is one of therapeutic targets for development of new anticancer drugs [4], accumulated evidence has shown that cell cycle arrest might result in apoptosis due to the existence of cell cycle checkpoint and feedback control [5]. Activation of apoptotic pathway is a key mechanism by which cytotoxic drugs kill cancer cells. Recent research on the molecular mechanisms of hepatocellular carcinoma has revealed that together with deregulation of proliferation, insufficient apoptosis plays an important role in tumorigenesis [6-7].

YANG Guang-Ming, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 519−524

Oxytropis falcata Bunge, occurring in northwest of China, is a species of the genus Oxytropis in China. It is the most widely used Oxytropis species for folk remedies in Tibet and Inner Mongolia. The whole herb, known as E’daxia in Chinese traditional medicine, are clinically used for treatments of diseases such as pyrexia, influenza, tonsillitis, pharyngolaryngitis, acute and chronic bronchitis, hematochezia, dysentery, knife injury. In recent years, there is an increasing interest in Oxytropis due to their antiproliferative, antinociceptive, anti-inflammatory, hemostyptic and antitumor activities [8], and experiments also showed the antibacterial activity of O. falcata on nine Gram-positive and Gram-negative bacteria [9]. It was mainly expressed that antioxidant activities of rhamnocitrin, kaempferol and rhamnetin isolated from O. falcata appeared to be similar to ascorbic acid and were better than butylated hydroxytoluene [10]. In our previous study, five extracts obtained from O. falcata were evaluated for their cytotoxicities against several cell lines and essential oil (EOOF) and flavonoids (FOF) were proved to be the effective anticancer fractions in vitro [11]. Our previous results of Hoechst 33258 staining also demonstrated that cells treated with FOF showed several apoptotic appearances [12]. We have isolated sixteen flavonoid aglycones from the flavonoids of O. falcate (FOF), and they were identified as 2′, 4′-dihydroxychalcone, 2′, 4′-dihydroxydihydrochalcone, 2′-methoxy-4′-hydroxychalcone, 2′-hydroxy-4′-methoxychalcone, isoliquiritigenin, pinocembrin, etc. [13]. Among them, 2′, 4′-dihydroxychalcone had the highest percentage and was determined by RP-HPLC (up to 0.64%) [14], which could induce apoptosis of MGC-803 gastric adenocarcinoma cells[15-16]. The volatile oil (EOOF) obtained by steam distillation was characterized by heneicosane (22.2%), 6, 10, 14-trimethyl-2-pentadecanone (5.4%), 2-methylbenzyl cyanide (5.1%), 4a, 8-tetramethyl-2-naphthalenem ethanol (3.7%), whose contents were calculated by normalization method. Besides these hydrocarbons, there were still oxygenated sesquiterpenes, nitrogenous compounds, esters and aldehyde compounds in the volaile oil [17]. However, the extracts of O. falcate, EOOF and FOF have not been examined systematically with regard to antitumor efffcets in vitro and in vivo, and its antitumor mechanism still remained unclear. Therefore, we investigated the antitumor effects of these two extracts on human hepatocellular carcinoma SMMC-7721 cells in vitro by observing cell proliferation, cell cycle distribution and apoptosis, and in transplanted murine H22 tumors in vivo by calculating tumor growth inhibition.

2 2.1

Materials and Methods

Materials Annexin V-FITC Apoptosis Detection kit was purchased from KeyGen Corporation (Nanjing, China). Propidium iodide (PI) and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) were purchased from Sigma

Chemical Company (USA). Dimethyl Sulfoxide (DMSO) was purchased from Amresco (USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Invitrogen Corporation (USA). Fetal bovine serum was purchased from Invitrogen Corporation (USA). The whole herbs identified as O. falcata Bunge were obtained from Anduo autonomous county, Tibet, China. The voucher specimens were stored in Jiangsu Key laboratory of Chinese Medicine Processing, Nanjing, China. All other chemicals and reagents employed were of analytical grade. 2.2 Tumor cells and culture Human hepatocellular carcinoma cell line (SMMC-7721) was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). SMMC-7721 cells were cultured in DMEM supplemented with 10% FBS (V/V), 100 U·mL−1 of penicillin and 100 μg·mL−1 of streptomycin in a 37 °C incubator supplied with an atmosphere of 5% CO2. After 60%−80% confluency, cells were trypsinized with 0.25% trypsin (AMRESCO, dissolved in PBS, pH 7.4), counted and placed down at a needed density for treatment. 2.3 Extraction of EOOF and FOF from O. falcata A powdered portion of the air-dried sample (100 g) from the whole plant was extracted with 95% ethanol three times. After filtration, the residue was extracted with 50% ethanol again. The extracts were combined and evaporated to dryness. The residue was dissolved in 0.2% caustic soda solution, filtered, acidified with 1% hydrochloric acid solution, and sequentially, extracted with chloroform and ethyl acetate. The combined extracts were concentrated to dryness to yield the flavonoids fraction (FOF, 1.576 g) [13, 18]. Another powdered portion of air-dried sample (100 g) was extracted using a steam distillation process, which is the oldest form of essential oil extraction that won’t damage the plant [19]. The filtered liquid was deposited by ethanol at minus 20 °C. After 24 h, the filtered liquid was evaporated under reduced pressure to yield the essential oil fraction (EOOF, 1.284 g). Both fractions were dissolved in dimethylsulfoxide (DMSO), and the final concentration of DMSO never exceeded 0.5% (V/V). 2.4 Proliferation assay The inhibitory effects of EOOF and FOF on cell viability were measured by MTT colorimetric method [20]. SMMC-7721 cells in exponential growth were seeded at a density of 1×105 cells/well in a 96-well plate. On the second day, cells were treated with EOOF, FOF or DMSO (vehicle control, 0.5% DMSO) for 24 h, respectively. After drug treatments, attached cells were incubated with 20 μL of MTT (5 mg·mL−1, 4 h).The supernatant was then discarded and attached cells were subsequently solubilized in 100 μL DMSO to dissolve the formed violet formazan crystals within metabolically viable cells. The absorbance at 490 nm was then measured using an Automated Microplate Reader (Bio-Rad 680) (Bio-Rad Laboratories,

YANG Guang-Ming, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 519−524

Redmond, WA). The cell viability ratio was calculated based on the following formula [21]: OD of drug − treated sample Percent viability = × 100% OD of non − treated sample 2.5 Evaluation of in vivo antitumor activity in H22 tumor model All animal welfare and experimental procedures were strictly in accordance with the USA Guide for the Care and Use of Laboratory Animals of 1996 and the related ethics regulations of Nanjing University of Chinese Medicine. Five-week old male ICR (Institute of Cancer Research) mice (5−6 weeks, 18−22 g) were given food and water ad libitum. The mice were housed in a room maintained at (25 ± 1)°C with 55% relative humidity. Mice were acclimatized

Inhibition =

for one week before being injected with cancer cells. One week later, the mice were inoculated by subcutaneous injection of 5 × 106 H22 hepatocellular carcinoma cells in 200 μL PBS (phosphate buffered saline) into the right flank of the mice. Three days after tumor inoculation, forty mice in four groups were administered intragastrically with FOF or EOOF at a dose of 30 or 60 mg·kg−1 body weight respectively every day, while the control group was administrated PBS vehicle and the positive control group was injected i.p. with cyclophosphamide at 20 mg·kg−1 body weight every day. All mice were euthanized 10 days after treatment. The tumors were removed and weighed. The tumor growth inhibitory ratio was calculated based on the following formula:

tumor weight of control group − tumor weight of treated group × 100% tumor weight of control group

2.6 Cell cycle distributions The flow cytometric evaluation of the cell cycle status was performed according to a method described previously [22]. In brief, cells were seeded at a density of 1 × 106/mL in 6-well microplates, grown for 24 h and then treated with different concentrations of FOF for 24 h. Cells were then washed with PBS, trypsinized with 0.25% trypsin and harvested by centrifugation for 5 min at 1 000 r·min−1. The cell pellets were resuspended with 0.5 mL of PBS and fixed overnight with cold 70% ethanol (final concentration), followed by staining with PI solution containing 50 μg·mL−1 PI and 10 μg·mL−1 RNase A. After incubation at room temperature for 60 min, cells were analyzed by flow cytometry (FACS, Becton Dickinson, USA) using CellQuest software (BD Biosciences, San Jose, California, USA). The percentages of cells in different cycle phase were calculated. 2.7 Cell apoptosis Cell apoptosis was quantified by flow cytometry with AnnexinV labeling and PI exclusion staining. Cells undergoing apoptosis could be stained with Annexin V, because Annexin V could demonstrate the externalization of phosphatidylserine (PS) [23]. Cells were treated as described above. Briefly, SMMC-7721 cells were collected, washed with PBS and suspended in binding buffer. Then cells (1 × 106 cells/mL) were stained with 10 μL AnnexinV-FITC and 5 μL PI, incubated in dark at room temperature for 15 min according to manufacture’s instructions and subjected to flow cytometry. The data were analyzed with CellQuest software. 2.8 Statistical analysis Values were expressed as the mean ± SD Analysis of variance (ANOVA) followed by Scheffe posteriori comparison was used to assess the differences between control and treatments. A probability of 0.05 or less was considered significant.

3

Results

3.1 Effects of EOOF and FOF on proliferation of SMMC7721 cells To analyze the cytotoxicity of EOOF and FOF, SMMC-7721 cells were incubated with increasing doses of EOOF (0.05−0.3 mg·mL−1), FOF (0.05−0.3 mg·mL−1) or DMSO (0.5%, vehicle control) for 24 h, respectively. Cell viability was determined by a conventional tetrazolium-based (MTT) assay. Cyclophosphamide was applied as the reference drug. Results indicated that EOOF and FOF decreased the viability of SMMC-7721 cells in a dose-dependent manner. The IC50 (50% inhibitory concentration) value of EOOF and FOF were 0.115 and 0.097 mg·mL−1 at 24 h after treatment, respectively. 3.2 Effects of EOOF and FOF on solid tumor growth inhibition To confirm the in vivo antitumor activity of EOOF and FOF, H22 tumor model was used in ICR mice, since murine hepatoma22 cells grow rapidly in mice after inoculation. The animal study revealed that FOF significantly inhibited the growth of H22, decreased tumor weight at necropsy in a dose-dependent manner compared with PBS control. Nevertheless, EOOF did not show significant tumor growth inhibition of H22. Neither EOOF nor FOF showed any side effects such as weight loss or inactivity during the experiments (Table 1). 3.3 Effects of FOF on cell cycle on SMMC-7721 To further study the effect of FOF on tumor cell lines, cell cycle distribution was assessed on SMMC-7721l cells by flow cytometry, staining the DNA content with PI. As shown in Fig. 1A−E, cells treated with multiple doses of FOF (0.05, 0.075 and 0.1 mg·mL−1) induced G1 phase arrest (38.25%, 64.32% and 74.66%, respectively) as compared with the negative control DMSO (36%).

YANG Guang-Ming, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 519−524 Table 1

Effects of FOF and EOOF on the growth of H22 inoculated onto the right flank of ICR mice (mean ± SD) Animal number

Body weight/g

Groups

Treatment /(mg·kg−1)

Beginning

End

control

vehicle

10

10

CTX

20

10

10

17.8 ± 1.57

19.7 ± 2.83

0.02 ± 0.01**

84.2

30

10

9

18.6 ± 1.16

20.5 ± 2.67

0.94 ± 0.12

19.5

EOOF FOF

Beginning 17.8 ± 1.57

End 22.0 ± 2.51

Tumor weight/g

Inhibition/%

1.30 ± 0.42

0

60

10

8

18.0 ± 1.83

20.3 ± 3.09

0.84 ± 0.18

27.2

30

10

10

18.9 ± 1.09

25.8 ± 2.08

0.67 ± 0.13**

56.1

60

10

10

18.4 ± 1.54

24.9 ± 2.16

0.43 ± 0.14**

70.8

Three days after tumor inoculation, mice were given intragastrically of FOF, EOOF or PBS every day for 10 days (n = 10); * P< 0.05, ** P< 0.01 vs PBS control

3.4 Effects of FOF on apoptosis on SMMC-7721 cells To further characterize the apoptosis induced by FOF, we quantified apoptotic cells using flow cytometry analysis. As illustrated in Fig. 2B, about 22% of the cells experienced apoptosis after being treated with FOF at 0.05 mg·mL−1 for 24 h. More than 68% of SMMC-7721 cells experienced apoptosis when the concentration of FOF reached up to 0.075 mg·mL−1 (Fig. 2C). In addition, 70% of SMMC-7721 cells experienced apoptosis when treated with 0.1 mg·mL−1 dose of FOF (Fig. 2D).

4

Fig. 1 Effects of FOF on cell cycle distribution were performed by flow cytometry. SMMC-7721 cells were treated with 0.05 mg·mL−1 FOF (B), 0.075 mg·mL−1 FOF (C) and 0.1 mg·mL−1 FOF (D) for 24 h. Cells treated with PBS vehicle was used as control (A). Samples were analyzed by flow cytometry as described in ‘Materials and methods’ section. Histograms show number of cells per channel (vertical axis) vs. DNA content (horizontal axis). The values indicate the percentage of cells in the indicated phases of cell cycle. The data shown are representative of three independent experiments with similar findings (E)

Discussion

In this paper, we concluded that FOF, a naturally occurring polyphenols agent, potently decreased the cell viability of hepatocellular carcinoma cell line SMMC-7721 with IC50 value of 0.097 mg·mL−1. The effect was confirmed by solid tumor growth inhibition in H22 transplanted mice and the growth inhibition in H22 solid tumors was significantly pronounced with the inhibition ratio of 56.1% and 70.8% at the concentration of 30 mg·kg−1 and 60 mg·kg−1. While EOOF exhibited only in vitro antiproliferative effect with IC50 value of 0.115 mg·mL−1 without significant antitumor effect in vivo. Nevertheless, the underlying antitumor mechanism of FOF still remained unclear. Therefore, the cell cycle analysis and AnnexinV FITC/ PI staining were performed. Cell cycle is generally subdivided into G1, S, G2, and M phase. Five cellular proteins regulated the transition from one cell cycle phase to another. Cells arrested in G1 phase are mainly induced by p53-dependent DNA damage. p53 triggered cascade events of p21, Mdm2 and Bax, leading to induction of p21, a CKI (cyclin-dependent kinase inhibitor), eventually resulted in CDK (cyclin-dependent kinases) inhibition and cell cycle arrestment [24]. Accumulated evidences have shown that disturbance of the cancer cell cycle is one of novel therapeutic targets for development of new anticancer agent. Studies concerning the mechanism of how apoptosis is deregulated in cancer cells have shed light on induction of cancer cell apoptosis, which is recognized as an important target in cancer therapy [25]. FOF treatment induced a significant

YANG Guang-Ming, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 519−524

Fig. 2 Effects of FOF on apoptosis were measured by Annexin V/PI staining. SMMC-7721 cells were treated with 0.05 mg·mL−1 FOF (B), 0.075 mg·mL−1 FOF (C) and 0.1 mg·mL−1 FOF (D) for 24 h. Cells treated with PBS vehicle was used as control (A). Samples were analyzed by flow cytometry. Frames are divided into four quadrants: apoptotic cells that are positive for Annexin V and negative for PI (Annexin V+/PI-) are in quadrant IV; late apoptotic and early necrotic cells (Annexin V+/PI+) are in quadrant II; normal cells (Annexin V-/PI-) are in quadrant III; and cells undergoing necrosis (Annexin V-/PI+) are in quadrant I.

accumulation of G1 cell cycle arrest and increased portion of apoptotic bodies in a dose-dependent manner in SMMC-7721 cells. Thus, we postulated that there might be a close association between the antiproliferation of FOF in SMMC-7721 cells and cell cycle arrest as well as apoptosis. It is critical to understand and elucidate the molecular mechanisms before FOF can be used in the design of effective cancer drug therapeutics.

5

Conclusion

In conclusion, our study demonstrated that FOF exhibits multiple antitumor activities against human (SMMC-7721) and mouse (H22) tumor cells. FOF exerts G1 cell cycle arrest and causes apoptosis. Taken together, these in vitro and in vivo findings strongly demonstrate that FOF can be used potentially for hepatic cancer treatment as a chemopreventive agent. However, further investigations, such as screening of antitumor effects of the compounds isolated from FOF and EOOF, and research on mechanisms of action will be necessary to better understand the antitumor effects of O. falcata.

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镰形棘豆提取物抗肝癌的体内外活性研究 杨光明 1a, 燕

茹 2a, 王兆先 3，张芳芳 1, 潘

扬 1, 4*, 蔡宝昌 1*

1

南京中医药大学江苏省中药炮制重点实验室 国家中医药管理局中药炮制标准重点研究室，南京 210023；

2

澳门大学，澳门；

3

中国药科大学药学院，南京 210009；

4

南京中医药大学药用菌与中药生物技术研究所，南京 210023

【摘 要】 目的：探讨镰形棘豆提取物的体内外抗肝癌活性, 并探讨其可能的机制。方法: 采用 MTT 法检测镰形棘 豆提取物对 SMMC-7721 细胞的增殖抑制, 采用 PI 单染法和 Annexin V-FITC/PI 双染法进行流式细胞仪检测, 测定细胞周 期和凋亡率, 观察 H22 荷瘤小鼠的体内肿瘤抑制作用。结果: MTT 实验表明, 镰形棘豆提取物 EOOF 和 FOF 有效抑制肿 瘤细胞 SMMC-7721 的增殖, 并呈一定的剂量依赖性, 其 IC50 值分别为 0.115 和 0.097 mg·mL−1。TFOF 作用 SMMC-7721 细胞后可使细胞周期停滞于 G1 期, 凋亡细胞的比例升高，并且呈浓度依赖性。镰形棘豆提取物 FOF 能明显抑制 H22 荷 瘤小鼠肿瘤的生长，低、高剂量组的抑制率分别为 56.1%和 70.8% (P < 0.01)。结论: 镰形棘豆提取物 FOF 能抑制 SMMC-7721 细胞的增殖, 诱导其凋亡, 并对 H22 荷瘤小鼠有肿瘤生长抑制作用。显示镰形棘豆具有较好的发展为抗肝癌 药物的前景。 【关键词】 抗肿瘤; 镰形棘豆; 凋亡; SMMC-7721; H22 荷瘤小鼠; 增殖 【基金项目】

国家自然科学基金项目(No. 30902012)资助