Effects of Ethyl Acetate Extract of Phyllanthus reticulatus Leaves on Autophagy-related Proteins Beclin-1, ATG5 and LC3 in Nude Mice Xenograft Model of HCC

Zhipeng XU, Chenyan LIANG, Cuiliu PAN*, Yunli TANG, 2*, Jianfang FENG, Tong HE

1. Guangxi University of Chinese Medicine, Nanning 530020, China; 2. Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China; 3. Wuxuan County Maternal and Child Health Care Hospital, Laibin 545900, China

Abstract [Objectives] To explore the effects of ethyl acetate extract of Phyllanthus reticulatus leaves on autophagy-related proteins Beclin-1, ATG5 and LC3 by immunohistochemistry, and to preliminarily explore their effects on autophagy. [Methods] BEL-7404 Hepatocellular Carcinoma (HCC) nude mice model was established, and blank group (same volume of pure water), positive control group (20 mg/kg fluorouracil), high dose drug group (600 mg/kg), and medium dose drug group (300 mg/kg), and low dose drug group (150 mg/kg) were set up. After 2 weeks of intragastric administration, the nude mice were sacrificed, and the tumor tissues were taken out, processed by immunohistochemistry, and then made into paraffin sections. Photos were taken under an optical microscope (10×40), and evaluation and analysis were performed with the aid of the Image-Pro Plus 6.0 image analysis software. Differences were calculated using SPSS 20.0 software. The effects of drugs on autophagy-related proteins LC3, Beclin-1 and ATG5 were observed. [Results] Compared with the blank group, the medium and high dose groups of ethyl acetate extract of P. reticulatus leaves had the effect of promoting the increase of autophagy-related proteins LC3, Beclin-1 and ATG5 (P<0.05). However, there was no significant difference between the low dose group of ethyl acetate extract of P. reticulatus leaves and the blank group (P>0.05). [Conclusions] The ethyl acetate extract of P. reticulatus leaves has a promoting effect on autophagy-related proteins LC3, Beclin-1, and ATG5.

Key words Ethyl acetate extract of Phyllanthus reticulatus leaves, Autophagy, Hepatocellular Carcinoma (HCC), Nude mice

1 Introduction

China is a high incidence area of liver cancer or Hepatocellular Carcinoma (HCC), which has seriously affected the life, health and life of the Chinese people. At present, surgery and chemotherapy drugs are the most commonly used treatment methods, but most of the chemotherapy drugs have large toxic and side effects. Therefore, it is a research hotspot to find drugs resisting HCC with high efficiency and low toxicity. Modern medicine has shown that autophagy plays an important role in the immune activity of the human body. It can catabolize some pathogenic cells,etc., thereby reducing the occurrence of diseases. Extensive studies have shown that many Chinese herbal medicines can inhibit the proliferation of tumor cells by promoting the increase of autophagy.

PhyllanthusreticulatusPoir.var.glaber Muell.Arg belongs to Euphorbiaceae plants[1]. Extensive studies have shown thatP.reticulatushas the effects of expelling wind, dampness, and promoting blood circulation, and has a good effect on the treatment of hepatitis, rheumatism and joint pain, nephritis, enteritis, dysentery, and bruises,etc.Using MTT method to conductinvitroexperiments, Tang Yunlietal.[2]found that the ethyl acetate extract ofP.reticulatushas a good inhibitory effect on human liver cancer cell line BEL-7404. Based on the previous research, we established a BEL-7404 xenograft model, to explore the possible action mechanism of ethyl acetate extract ofP.reticulatusin inhibiting the human liver cancer cell line BEL-7404, and provide a further scientific basis for the follow-up in-depth research and clinical application of ethyl acetate extract ofP.reticulatus.

2 Materials and methods

2.1 Materials

2.1.1Cell lines. The human liver cancer cell line BEL-7404 was purchased from Nanjing KeyGen Biotech Co., Ltd.

2.1.2Drugs. Zhuang medicineP.reticulatuswas collected in Gaofeng Forest Farm and identified by deputy professor Guo Min from Medicinal Plant Teaching and Research Office, College of Pharmacy of Guangxi University of Chinese Medicine as leaves ofPhyllanthusreticulatusPoir.var.glaber Muell.Arg.

2.1.3Animals. BALB/C nude mice, half male and half female, weighing 18-22 g, were provided by the Laboratory Animal Center of Guangxi Medical University, the laboratory animal production license No.: SCXK Gui 2014-0002, and the laboratory animal use license No.: SYXK Gui 2014-0003.

2.1.4Main reagents and instruments. The main reagents were listed in Table 1 and main instruments were listed in Table 2.

Table 1 Main reagents

Table 2 Main instruments

2.2 Methods

2.2.1Extraction of ethyl acetate fromP.reticulatusleaves.P.reticulatusleaves were collected for cleaning, drying, and extracted with 80% ethanol after drying. A large rotary evaporator and a water bath were used to dry the liquid into a dry extract, took 50.00 g of the dry extract, and dissolved it in a 300 mL volumetric flask. Used ethyl acetate to extract until the liquid got transparent and colorless, combined all the extracts and then dried under reduced pressure to obtain the ethyl acetate extract ofP.reticulatusleaves. Dissolved with distilled water to prepare high, medium and low dose solutions, and stored them in a 4 ℃ refrigerator for later use.

2.2.2Primary culture of liver cancer BEL-7404 cells. After the nude mice were weighed, 2×107liver cancer BEL-7404 cells were inoculated into the axilla of the right forelimb of the nude mice using a syringe, and the tumor tissue was removed when the volume was greater than 500 mm3. Cut the tumor tissue into about 1-2 mm, and placed in a tissue grinder. Added normal saline, grinded to homogenate, added 10 mL of normal saline for washing, collected the cell suspension, filtered through a 200-mesh sieve, centrifuged, and then washed with normal saline three times. After centrifugation, added the culture medium, and cultured the cells in a cell incubator with the constant temperature of 37 ℃ and 5% CO2.

2.2.3Establishment of nude mice model of liver cancer BEL-7404 cells. Took the cells in the logarithmic growth phase in the clean bench, digested with trypsin, collected the cells in a 15 mL centrifuge tube, and centrifuged at 1 000 rpm/min for 5 min. Discarded the supernatant, added RPMI Medium 1640 basic complete culture medium, gently pipetted evenly to form a single-cell suspension, and counted on a hemocytometer. Stained with trypan blue and adjusted the cell number to 2×107cells/mL with normal saline. Used a syringe to take 0.2 mL of immune tumor cell suspension and inoculated it in the axilla of the right forelimb of nude mice to wait for tumor growth. When the tumor tissues successfully grew to 500 mm3, randomly divided them into five groups: blank group, positive control group, and drug low, medium and high dose groups.

2.2.4Expression of auto-related proteins Beclin-1, ATG5 and LC3 in the transplanted tumor tissue of human liver cancer BEL-7404 nude mice was measured by immunohistochemical method. Weighed and made a record of the body weight of each nude mouse and started drug administration. At regular intervals every day, the blank group (same volume of pure water), high (600 mg/kg), medium (300 mg/kg) and low (150 mg/kg) doses of the drug were intragastrically administered, and the positive control group was injected with 20 mg/kg fluorouracil. After administration for 2 weeks, nude mice were sacrificed, and the tumors were removed and cleaned. Prepared 10% formaldehyde for tumor fixation, and embedded with conventional paraffin. Tumors were sectioned with a tissue microtome, dewaxed and hydrated with gradient concentrations of xylene and ethanol, and washed 2-3 times with tap water. Added 0.01 mol/L (pH=6) citrate buffer and put it into the autoclave for high pressure repair for about 20 min, took out the sections and cooled in a gradient manner. After cooling to room temperature, washed 3 times with 5 min each time. Added serum blocking solution dropwise for about 10 min, shook off excess liquid. Added the corresponding antibodies (Beclin-1, ATG5, LC3 antibodies) dropwise, incubated at 4 ℃ overnight, and placed them at room temperature for 30 min when taking them out. Added Biotin-labeled secondary antibody (goat anti-rabbit IgG) dropwise and incubated at room temperature for 10 min, washed 3 times with PBS. Added tertiary antibody (Horseradish Peroxidase) and incubated for 10 min, wash 3 times with PBS, developed color with DAB for 5 min, and stained with hematoxylin after washing. Used hydrochloric acid alcohol to differentiate, rinse three times with water, and then used neutral gum to mount the slides for microscopic examination.

2.2.5Detection of autophagy-related proteins Beclin-1, ATG5, and LC3. The positive expression of proteins Beclin-1, ATG5, and LC3 in animal models of liver cancer after immunohistochemistry was yellow-brown, and the positive expression is in the cytoplasm. The tumor was sectioned to make paraffin sections. Took photos by an optical microscope (cytoplasmic staining at 10×40 times), and 5 images of different fields of view were taken for each section. Used Image-Pro Plus 6.0 image processing software to process, and calculated the average optical density and the mean value. Calculated thePvalue with the aid of SPSS 20.0 software to evaluate the effect of ethyl acetate extract ofP.reticulatusleaves on expression of Beclin-1, ATG5, and LC3 proteins.

Average Optical Density=IOD/Area

3 Results and analysis

3.1 Effects of ethyl acetate extract ofP.reticulatusleaves on expression of Beclin-1 proteinAfter immunohistochemistry, the positive expression of Beclin-1 protein was yellow-brown, and it was located in the cytoplasm. As shown in Fig.1A, there was no significant difference between the low dose drug group and the blank group, and the yellow immunohistochemical substances in the medium and high dose drug groups were significantly increased compared with the blank group. There was no significant difference in the average optical density between the low dose drug group and the blank group (P>0.05), and the average optical density between the medium and high dose drug groups was statistically different from the blank group (P<0.01), as shown in Table 3, indicating that the medium and high dose drugs can promote the increase of Beclin-1 protein.

Table 3 Effects of ethyl acetate extract of Phyllanthus reticulatus Leaves on expression of Beclin-1 protein (n=6,

3.2 Effects of ethyl acetate extract ofP.reticulatusleaves on expression of ATG5 proteinAfter immunohistochemistry, the positive expression of ATG5 protein was yellow-brown and was located in the cytoplasm. As shown in Fig.1B, there was no significant difference between the low dose drug group and the blank group, and the yellow immunohistochemical substances in the medium and high dose drug groups were significantly increased compared with the blank group. There was no significant difference in the average optical density between the low dose drug group and the blank group (P>0.05). The average optical density of the medium and high dose drug groups was significantly different from that of the blank group (medium dose group:P<0.05; high dose group:P<0.01), as shown in Table 3, indicating that the medium and high dose drugs can promote the increase of ATG5 protein.

3.3 Effects of ethyl acetate extract ofP.reticulatusleaves on expression of LC3 proteinAfter immunohistochemistry, the positive expression of LC3 protein was yellow-brown and was located in the cytoplasm. As shown in Fig.1C, there was no significant difference between the low dose drug group and the blank group, and the yellow immunohistochemical substances in the medium and high dose drug groups were significantly increased compared with the blank group. There was no significant difference in the average optical density between the low dose drug group and the blank group (P>0.05). The average optical density of the medium and high dose drug groups was significantly different from that of the blank group (P<0.01), as shown in Table 3, indicating that the medium and high dose drugs can promote the increase of LC3 protein.

Note: A. Beclin-1 protein, B. ATG5 protein, C. LC3 protein.Fig.1 Immunohistochemical staining of autophagy-related proteins Beclin-1, ATG5, and LC3

4 Discussion

Liver cancer is the sixth most common cancer worldwide, with an extremely high incidence rate[3]. Primary liver cancer can be divided into four types, hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), hepatoblastoma and combined hepatocellular-cholangiocarcinoma (CHC)[4]. The common one is HCC, which is also a major problem in current clinical treatment, and its incidence rate and mortality rate rank among the top three of all cancers[5-7]. China is a high-incidence and frequent-incidence area of liver cancer. The incidence of liver cancer in China accounts for 43.7% of the global total. At present, hepatectomy and chemotherapeutic drug therapy are the main methods for the treatment of liver cancer, but the effect is not significant, and the recurrence rate is as high as 80%. In addition, the toxic and side effects of chemotherapeutic drugs cause great harm to the human body. Therefore, it is of great significance to find drugs that can effectively treat liver cancer and have less toxic and side effects.

The theory of traditional Chinese medicine (TCM) has a long history. Chinese herbal medicine has a good curative effect in the treatment of intractable diseases and cancer. It is a treasure of the Chinese nation and has an important influence in the medical field. According to the current research, some Chinese herbal medicines have good anti-cancer effects and few adverse reactions[8], so they are often used in clinical treatment. In some areas, TCM physicians often choose some Chinese herbal medicines to differentiate and treat tumor patients. For example, the Chinese Pharmacy of Chang Gung Memorial Hospital often uses Angelicae Sinensis Radix and Astragali Radix decoction to treat lung adenocarcinoma. There are also examples of using Chinese herbal medicines to treat tumors in the folk. For example, residents in Yunnan use thirteen kinds of folk Chinese herbal medicines to treat cancer. The experimental results also show that the drugs in these examples have strong antitumor effects. In recent years, scientists have discovered and successfully extracted many tumor suppressor substances from plants such asTaxuswallichianavar. chinensis,Camptothecaacuminata, andRabdosiarubescens, and also found that the mechanism of action of these substances is related to the related proteins in autophagy. Studies about LC3, Beclin-1, and ATG5 proteins in autophagy cells are flourishing.

Autophagy is one of the ways of programmed cell death. The occurrence of autophagy can be roughly summarized into the following stages: the occurrence of autophagic vacuoles, the formation of autophagosomes, the transport and fusion of autophagy, the fusion of autophagosomes and lysosomes, and the degradation by autophagy. Autophagy can catabolize and phagocytose some foreign pathogenic cells to maintain normal and stable cells in the human body and achieve an inhibitory effect. LC3 protein is involved in the formation of autophagosome membrane, and its expression intensity is closely related to autophagy activity. LC3 protein is a marker of autophagy, so it is often used as one of the commonly used indicators to detect autophagy[9]. Levine discovered the Beclin-1 gene from autophagy, located on human chromosome 17q21, which is involved in autophagy and vacuolar protein sorting. By establishing a mouse model of Beclin-1 heterozygosity disruption, Cianfanelli Vetal.[10]found that not only an increase in the number of tumor cells but also a decrease in the number of autophagic cells was observed in mice, indicating that Beclin-1 protein in autophagy cells plays an extremely important role in the process of suppressing tumors. The current study also found that ATG5 is a key protein involved in phagocytic membrane extension in autophagic vesicles. ATG5 promotes the generation of autophagic cells by participating in the formation of autophagic vacuoles in autophagic cells, and indirectly inhibits the growth of tumor cells. Kim MSetal.[11]also found that ATG5 protein is related to autophagy in prostate cancer, confirming that the formation of autophagy is inseparable from ATG5.

PhyllanthusreticulatusPoir.var.glaber Muell.Arg belongs to Euphorbiaceae plants[1]. It is a common plant in Guangdong, Guangxi, Hainan, and Yunnan. It was included in theGuangxiTraditionalChineseMedicineStandardsin the second edition of 1990, also called Lantiebo, Shanbingdou, and Longyanjing[12]. Folks often use this medicine to treat rheumatoid arthritis pain, bruises,etc., and its effect is remarkable. Both domestic and foreign studies have found that Zhuang medicineP.reticulatusleaves have the effect of inhibiting tumor. Its pharmacological activity shows that the ethanol extract ofP.reticulatusleaves has a certain curative effect in acute liver injury caused by carbon tetrachloride (CCl4)[13]. Wu Huaienetal.[14]found that some extracts ofP.reticulatusleaves have antibacterial effects, including water extracts and alcohol extracts, and the antibacterial effect of water extracts is significantly lower than that of ethanol extracts. Huang Binbinetal.[15]found thatP.reticulatushas analgesic and anti-inflammatory effects, the mechanism may come from the triterpenoids inP.reticulatus. Tang Yunlietal.[2]conductedinvitroexperiment using the MTT method and found that its ethyl acetate extract had a good effect on inhibiting the HCC. They also isolated the active substance monomer from the effective part and observed its effect on the liver cancer BEL-7404 cells. Based on the above related studies, it has been shown that Zhuang medicineP.reticulatushas a good inhibitory effect on cancer, but its action mechanism has not been studied, and its dose-effect relationship has not been clearly introduced.

In this experiment, we established a nude mouse liver cancer BEL-7404 cell model, and used the blank control method to study the ethyl acetate extracted fromP.reticulatusleaves. Besides, we used immunohistochemistry to process and analyze autophagy-related proteins LC3, Beclin-1, and ATG5, to explore their possible mechanisms. Preliminary experiments have confirmed thatP.reticulatushas an inhibitory effect on the liver cancer cells. On this basis, our study found that both the blank group and theP.reticulatusleaves low-dose ethyl acetate group showed low expression; the medium and high dose groups ofP.reticulatusleaves ethyl acetate extract and the positive drug group (fluorouracil) showed high expression (P<0.01, ATG5 group:P<0.05). The results showed that the medium and high dose groups of ethyl acetate extract ofP.reticulatusleaves had the effect of promoting the increase of LC3, Beclin-1 and ATG5 proteins. The ethyl acetate extract ofP.reticulatusleaves may promote the increase of autophagy cells by promoting the increase of LC3, Beclin-1, and ATG5 proteins to inhibit tumor. These findings indicate that this drug has a good effect in inhibiting tumors and it is of great research significance. It is hoped that this drug will be further developed, researched and utilized in the future. This experimental result provided a scientific basis for our further study on the mechanism of ethyl acetate extract ofP.reticulatusleaves inhibiting human liver cancer BEL-7404 cells.

In summary, we detected LC3, Beclin-1, and ATG5 proteins in liver cancer cells by immunohistochemistry, and used the average optical density to evaluate them. The experimental results showed that the ethyl acetate extract ofP.reticulatusleaves promoted the increase of LC3, Beclin-1 and ATG5 proteins. The possible action mechanism is that it promotes the increase of LC3, Beclin-1, and ATG5 proteins to increase autophagy cells, to achieve the effect of inhibiting liver cancer BEL-7404 cells. This experimental result provided a basis for our further study on the effect ofP.reticulatusleaves on inhibiting the liver cancer BEL-7404 cells.