Objective: To investigate the inhibitory effects and mechanisms of triterpenoids from Ganoderma lucidum (G. lucidum triterpenoids) on the growth and metastasis of hepatocellular carcinoma (HCC) both in vitro and in vivo. Methods: In in-vitro experiments, the inhibitory effects of G. lucidum triterpenoids on human HCC SMMC-7721 cell lines were investigated by observing the proliferation, apoptosis, migration and invasion phenotypes of the cell line and assessing the cell cycles as well as the cell apoptosis and proliferation. In in-vivo experiments, nude mouse SMMC-7721 tumor models were established and divided into control group, treatment group A (low concentration group) and treatment group B (high concentration group) according to the treatment models received. Magnetic resonance imaging (MRI) was performed 3 times on each mouse model to calculate their tumor volumes. The liver and kidney functions of the models were evaluated. Tissues harvested from their solid organs were subjected to HE staining, and the tumor tissues were subjected to HE staining and immunohistochemical staining (E-cad, Ki-67, and Tunel), respectively. Results: i. In in-vitro experiments, G. lucidum triterpenoids could inhibit the growth of human HCC SMMC-7721 cell lines via regulating their proliferation and apoptosis phenotype. ii. In in-vivo experiments, the comparison of tumor volumes of mouse models obtained from the second and third MIR scanning was found to be statistically significant between the control group and treatment group A (P<0.05); and statistically significant differences were also found in the tumor volumes from the second and third MRI scanning between the control group and treatment group B (P<0.05). iii. No significant acute injuries or adverse effects were observed in the liver or kidney of the nude mice. Conclusion: G. lucidum triterpenoids could inhibit the growth of tumor cells via blocking their proliferation, accelerating apoptosis, and inhibiting migration and invasion, without marked toxic effects on normal organs and tissues in the body.
Introduction
Hepatocellular carcinoma (HCC) is a common liver malignancy whose incidence ranks 5th among all malignancies and the 2nd in mortality . There are over 700,000 new HCC patients and about 500,000 to 1000,000 HCC deaths each year in the world, among which more than half of the deaths are from China . The morbidity and annual mortality of HCC have been significantly increasing over the past 20 years, with a high prevalence in Asian and African regions . Currently, surgical removal is the main treatment approach for HCC patients, but the low 5-year survival rate after the surgery is an issue that hasn’t been addressed yet . Ganoderma lucidum (G. lucidum) is a kind of plant that can extend peoples’ life span and promote our health with a long history of record in Traditional Chinese Medicine (TCM) . According to TCM records, G. lucidum can enhance peoples’ immunity and has already been used as an herbal medicine for thousands of years. G. lucidum bears ideal effects on inhibiting all types of malignant tumors, such as prostate cancer, lung cancer, breast cancer , colon cancer and cervical cancer. One study on HCC suggested that polysaccharide from G. lucidum could improve intestinal flora imbalance in HCC mouse models , and another study showed that the polysaccharide was able to inhibit the proliferation and migration of HCC cell lines Hep G2 by down-regulating the protein contents in the vascular endothelial growth factors .
Polysaccharide and triterpenoids are two major compositions that can be extracted from G. lucidum to treat tumors . Polysaccharide normally functions as an immunomodulator or an antioxidant to fight against carcinomas, while triterpenoids primarily play their part by inhibiting the proliferation and metastasis of cancer cells . Triterpenoids and polysaccharide, however, are not in a fixed proportion in G. lucidum extract, probably for the differences in the types, cultivation, growth area and extracting approach of the G. lucidum. Even in the same G. lucidum, its different composition has different triterpenoid levels. Usually, spores contain more triterpenoids than other compositions in G. lucidum.
G. lucidum Triterpenoids play a crucial role in the prevention and treatment of diseases in human beings. In addition to their anti-carcinogenic effect, they also play a role in anti-aging , liver protection , anti-atherosclerosis , lowering blood glucose and lipid , anti-inflammation, anti-androgen , anti-bacteria , immuno-regulation , heart protection , and improving one’s physical weakness , etc. Moreover, G. lucidum triterpenoids can prevent and treat cardio-cerebrovascular diseases via regulating patient’s blood pressure, blood lipid and glucose .
It has currently been reported that G. lucidum triterpenoid compounds kill tumor cells primarily through the following two approaches: one is to induce the apoptosis of tumor cells by damaging the completeness of cell membrane; the other is to inhibit the proliferation of tumor cells . Currently, many studies have confirmed that G. lucidum triterpenoid compounds can lead to cell cycle arrest primarily by down-regulating the expressions of proteins related to cell cycles . Latest studies have also suggested that G. lucidum triterpenoids play a role in inhibiting the metastasis of tumor cells. And many studies have also revealed the effectiveness of G. lucidum triterpenoids in the treatment of common cancers such as pulmonary cancer , liver cancer , cervical cancer , prostate cancer , breast cancer and colon cancer .
Even though studies have reported the therapeutic effects of G. lucidum on liver cancer, the mechanism of which still remains unclear. Therefore, our study carried out both in-vitro and in-vivo experiments to investigate the mechanism of G. lucidum on HCC.
Materials and methods
Reagents and instruments in the cell experiment
Reagents
All chemical reagents were purchased from business suppliers, which can be used directly without further isolation and purification. All solvents were purified prior to use.
Cells and G. lucidum triterpenoids used in the experiment
Human HCC SMMC-7721 cell lines were purchased from the cell bank of the Chinese Academy of Science, and G. lucidum triterpenoids from Prof. MENG Xiangxian’s research group at Hu’nan University.
Grouping and cell treatment
Grouping and CCK-8 treatment
Human HCC SMMC-7721 cell lines were divided into four groups: blank control group exempting from the treatment of G. lucidum triterpenoids, and low, medium and high dose groups receiving 2.5 µg/mL, 5 µg/mL, and 10 µg/mL G. lucidum triterpenoids, respectively. After the addition of G. lucidum triterpenoids, the cells were cultured for 12 h, 24 h and 48 h. Subsequently, 10 μL CCK-8 solution (Dojindo, CK04) was added to each well for incubation under 37°C for 4 h. Absorbance was determined with Microplate Reader (Bio-Rad, iMark) set at 450 nm. This process was repeated three times.
Effects of different concentrations of G. lucidum triterpenoids on HCC cell cycle
Cells that were growing logarithmically were added with different concentrations of G. lucidum triterpenoids after culture. Then, the cell culture medium was discarded to collect the cells, whose number was controlled between 1×105 and 1×106. After that, the cells were filtered with a 400-mesh screening net, with the use of Flow Cytometry (BD Biosciences, FACS Calibur) following standard procedures to be detected. Then, the results were analyzed. The process was repeated three times.
Effects of different concentrations of G. lucidum triterpenoids on HCC cell apoptosis
Cells that were growing logarithmically were selected, digested and counted. Subsequently, the selected cells were cultured overnight until the cell density reached 80-90%, added with medium (Gibco, 11965092) containing different concentrations of G. lucidum triterpenoids (0 µg/mL, 2.5 µg/mL, 5 µg/mL, 10 µg/mL), and incubated for 24 h. After that, the cells were digested and collected. The process was repeated 3 times. Flow Cytometry (BD Biosciences, FACS Calibur) was used to detect the Annexin V/PI activity, and the results were analyzed using FlowJo software.
Effects of different concentrations of G. lucidum triterpenoids on HCC cell apoptosis (Tunel)
HCC cells were smeared, cultured overnight until their density reached 80-90%, and added with medium containing different concentrations of G. lucidum triterpenoids (0 µg/mL, 2.5 µg/mL, 5 µg/mL, 10 µg/mL) for experimenting. The treated cells were placed under an optical microscope (Olympus, CX41) for observation and photographing. This process was repeated for 3 times.
Detection of cell migration ability by wound healing assay
Wound healing assay was employed to determine the migration ability of HCC cells. For starters, the HCC cells were digested, counted, made into suspension and inoculated evenly into a 6-well plate. Then the plates were added with mediums containing different concentrations of G. lucidum triterpenoids (0 µg/mL, 2.5 µg/mL, 5 µg/mL, 10 µg/mL) to be studied. Samples were taken at 0 h and 24 h and photographed. The process was repeated 3 times. The migration area of cells was measured with Image J and the results were recorded.
Detection of cell invasion ability with the transwell assay
Cells that were growing logarithmically were selected, digested and counted to determine their invasion ability. The treated cells were then centrifuged at 1000 rpm under room temperature and added with serum-free DMEM medium for re-suspension and adjustment of cell density to 2×105 cells/mL. Next, cell suspension of 100 μL was added to the upper chamber and DMEM medium containing 10% fetal bovine serum to the lower chamber. Mediums containing different concentrations of G. lucidum triterpenoids (0 µg/mL, 2.5 µg/mL, 5 µg/mL, 10 µg/mL) were subsequently added for experimenting. The process was repeated 3 times. In the end, the cells were observed carefully under an optical microscope and random visual fields were selected for photography. The cells were again counted and the results recorded.
Animal experiment methods
Experimental animals and cells
All animal experiments were performed sticking to the 3R principles in an effort to minimize the suffering of animals. A total of 45 female BALB/c nude mice, aged 8-10 weeks and weighed 20±2 g, were purchased successively from an experimental animal center. All nude mice were fed with standard forage and sufficient water. They received standard light treatment, which was light exposure for 14 h and the remaining 10 h in a dark environment. They were kept under 22°C in an environment with stable humidity. HCC SMMC-7721 cell lines for model construction were obtained from the cell bank of the Chinese Academy of Science (Shanghai, China). This study was approved by Experimental Animal Ethics Committee, the Second Xiangya Hospital, Central South University (2020496).
Establishment of nude mouse SMMC-7721 models
The prepared SMMC-7721 cells of 100 µL (approximately 2×106 cells) were injected into the right armpit of the 15 nude mice. Once the tumor was formed, it was harvested and inoculated into the rest 30 nude mice through their armpits.
The tumor size of the mice that had been inoculated with SMMC-7721 cells was assessed daily using a vernier caliper and received their first MRI one week after the inoculation to ensure the success of modeling. According to the first MRI results, 26 nude mice were observed to have significant tumors, the other 4 without. Next, 18 nude mice that had almost the same tumor volume (about 39.15 mm3) were selected out of the 26 and divided into control group, treatment groups A and B, with 6 nude mice in each group. In a preliminary experiment, we found that G. lucidum triterpenoids, which were diluted to 200 µg/mL and 400 µg/mL, had inhibitory effects on tumors. According to this result, the G. lucidum triterpenoid extract was also diluted to a low concentration (200 µg/mL) for nude mice in treatment group A and a high concentration (400 µg/mL) for those in treatment group B.
Treatment of nude mouse models with different concentrations of G. lucidum triterpenoids
In the preliminary experiment, because a gradually increased stress response was observed in some nude mice one week after the intragastric injection of 0.5 mL G. lucidum triterpenoids per day using a 1 mL syringe, the dose of 0.3 mL per day was decided in the subsequent experiments to avoid unnecessary death. Nude mice in the two treatment groups received 0.3 mL G. lucidum triterpenoids intragastrically using the 1-mL syringe with a No. 10 gavage needle at the same time every day, and those in the control group received normal saline in the exact same way. The needle was disinfected with alcohol before each injection. Once the needle was placed in the stomach of the mice, the triterpenoid extract was injected drop by drop at first to ensure no unexpected accident was observed. Such an unexpected accident could be the transfer of the extract into the lung via a hiccup, leading to an accidental death caused by pneumonia, etc.
MRI scanning for nude mouse models at different time points
On the day before gavage (day 0) and days 7 and 14 after gavage, the nude mice received MRI scanning using 3.0T magnetic resonance (uMR 790, Shanghai United Shadow Medical Technology Co., Ltd., China) and customized animal coils (Zhongzhi Medical Technology Co., Ltd., Suzhou, China) to obtain MRI images. Conventional sequences were as follows: T1-weighted fast spin-echo (FSE) T2-weighted single-shot fast spin-echo (SSFSE). Before MRI, all mice were injected intraperitoneally with 50 mg/kg pentobarbital sodium solution to get anesthetized.
During MRI scanning, unconscious movements were observed visually via a real-time closed-circuit television. All images were imported into the RadiAnt DICOM Viewer (64-bit) software for data analysis. Tumors were clearly observed on conventional T1- and T2-weighted sequence images. Two radiologists who have over 10-year’s working experience (YHL and CM) and one with more than 15 years (QLS) analyzed the images. All three radiologists didn’t have the knowledge of the histopathological results beforehand. However, they confirmed a tumor in the mice according to the MRI images.
Sacrifice, blood and tissue samplings of the mouse models
After the third MRI scanning, the nude mice were anesthetized by intraperitoneal injection of anesthetic, and sacrificed through cardiac puncture. The mice were prepared for further use after their eyeballs were removed and blood collected. The prepared mice, after the harvest of their tumors, were cut in their belly to obtain the heart, liver, spleen, lung and kidney. These organs and tumors of mice in different groups were fixed in 10% formalin solution within 24 h after being harvested, embedded in paraffin, sectioned, and then stained. Meanwhile, an immunohistochemical S-P method was used to evaluate the proliferation and metastasis abilities of these tumor cells.
Blood test
After the third MRI scanning, the nude mice were sacrificed, their eyeballs removed and blood collected. About 1 mL of blood was drawn from each nude mouse and centrifuged at 3000 rpm for 15 min to get the supernatant for the test. However, due to the limited amount of blood samples, liver and renal functions were the focus of the test. Their indicators were alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBIL), total bilirubin (TBIT), albumin (ALB), alkaline phosphatase (ALP), urea (BUN), creatinine (CR) and uric acid (UA). Data were subjected to one-way ANOVA using Graphpad Prism software (GraphPad company).
Pathological and immunohistochemical examinations
The tumors, hearts, livers, spleens, lungs and kidneys of the nude mice in the three groups were made into parafin sections and stained regularly with HE. As only the proliferation, apoptosis and migration phenotype were chosen for the determination of anti-carcinogenic effects of G. lucidum triterpenoids in the cell experiments, the very same phenotype was selected in the animal experiment for the immunohistochemical analysis of the tumor sections, which were Ki-67 for proliferation, Tunel for apoptosis, and E-cad for migration. Each tumor section underwent Ki-67, E-cad and Tunel immunohistochemical analysis. At the same time, both positive and negative control assays for the detection of primary antibodies were completed.
An optimal microscope (DP72, Olympus Corporation, Tokyo, Japan) and ImageJ software (Open Source, NIH Image, USA) were used for observation and data analysis. The results of HE, E-cad, Ki-67 and Tunel from microscopic observation under the same light exposure were all written down. Morphological changes were observed by an experienced pathologist (5-year experience), and the ratio of the positive areas of E-cad, Ki-67, and Tunel to the whole tumor section was calculated
Statistical analysis
GraphPad Prism 8.0 software was used for data analysis. Continuous variables were expressed as mean ± standard deviation (�̅ ± sd). One-way ANOVA was used to detect differences for further analysis and the Turkey method was used for between-group comparison. Enumeration data were expressed as cases/100 (n/%), and analyzed with the Pearson chi-square test, expressed as chi-square. P value <0.05 was considered statistically significant.
