Anti-tumour Effect and Pharmacokinetics of an Active Ingredient Isolated from Inonotus hispidus

Abstract

Inonotus hispidus is an anti-tumour drug used in folk medicine. (4S,5S)-4-Hydroxy-3,5-dimethoxycyclohex-2-enone (HDE) is a compound isolated from Inonotus hispidus for the first time. However, the mechanisms underlying its therapeutic effects have not been elucidated. In this study, the in vitro screening, in vivo anti-tumour effects, mechanism of action, pharmacokinetics, and tissue distribution of HDE were investigated. HDE could inhibit the proliferation of HepG2 cells. Additionally, its half-maximal inhibitory concentration was 7.9 µg/mL. Increasing HDE concentrations significantly increased apoptosis rate in a dose-dependent manner. Furthermore, HDE was rapidly absorbed into mouse plasma, reaching a maximum concentration at 30 min. The area under the plasma HDE concentration–time curves for the studied organs were as follows: spleen > liver > lung > kidney > muscle > thymus > heart > brain. HDE also inhibited tumour growth up to 69%. The weights of organs harvested from HDE-treated mice were not significantly different from those harvested from control mice. Furthermore, HDE upregulated Fas expression and downregulated FasL expression in HepG2 cells. HDE significantly increased caspase-3 and caspase-8 activity. The anti-tumour effect of HDE might be realized by activating the Fas-mediated apoptotic pathway. We also found that HDE undergoes enterohepatic circulation or is quickly absorbed by the body, and the drug is released back into systemic circulation. In conclusion, HDE significantly inhibited H22 hepatocarcinoma cells (H22)tumour growth in mice without damaging organs; therefore, it may be suitable for treating liver cancer.

INTRODUCTION

Inonotus hispidus is an annual parasitic fungus that attaches itself to Basidiomycota, Agaricomycetes, Hymenochaetales, Hymenochaetaceae, and Inonotus species.¹⁾ It is also the main parasite of deciduous trees such as Fraxinus mandshurica, Ulmus macrocarpa var. mongolica, Populus, Morus alba, Juglans mandshurica, Malus pumila, and Sophora japonica. It is mainly distributed in the regions of Jilin, Liaoning, Heilongjiang, Hebei, Inner Mongolia, Beijing, Shandong, Shanxi, Ningxia, and Xinjiang in China,^2,3) and often used to treat indigestion in the north-eastern region of China.⁴⁾ It has been reported that I. hispidus has antioxidant and antiviral effects.⁵⁾ Additionally, it is effective for treating diabetes, cancer, inflammation, oral ulcers, arthritis, and gout.^6,7)

Given that there are no reported side effects of I. hispidus, and its known anti-tumour effect, we aimed to find if (4S,5S)-4-hydroxy-3,5-dimethoxycyclohex-2-enone (HDE) can kill tumour cells or inhibit their proliferation without damaging normal cells. HDE was isolated from the fruiting body of I. hispidus, and its pharmacokinetics, in vitro and in vivo anti-tumour effects, internal distribution, and mechanism of action were investigated.

MATERIALS AND METHODS

Fungus Sample

HDE was isolated from the fruiting body of I. hispidus taken from F. mandshurica at Northeast Forestry University (Heilongjiang, China). Prof. Tolgor from the Mycological Institute of Jilin Agricultural University (Jilin, China) identified the fungus. The specimen of I. hispidus was deposited in the Herbarium of traditional Chinese medicine in Changchun Institute for Food and Drug Control (HTCM1203), Jilin, China.

Chemicals and Reagents

The following reagents were purchased for this study: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation and cytotoxicity assay kit/radio immunoprecipitation assay (RIPA) lysate solution/bicinchoninic acid (BCA) protein assay kit/Western sealing fluid/Caspase 3 activity assay kit/Caspase 8 activity assay kit (Beyotime Biotechnology Company, Jiangsu, China); 1×phosphate-buffered saline (PBS, pH 7.4; Shanghai Biological Technology Co., Ltd., Shanghai, China); Annexin V-fluorescein Isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Mbchem, Shanghai, China); ethylenediaminetetraacetic acid (EDTA)/diethypyrocarbonate (DEPC)-treated water/methanol/Fas/FasL/β-actin/anti-Fas/anti-FasL/anti-β-actin/rabbit anti-rat secondary antibody (Thermo Fisher Scientific Co., Ltd., Suzhou, China); trypsin–EDTA solution (Beyotime Biotechnology Company); chloroform (Sigma-Aldrich Trading Company Ltd., Shanghai, China); isopropyl alcohol (Shanghai Haoran Biological Technology Co., Ltd., Shanghai, China); ethanol (Sigma-Aldrich Trading Company Ltd.); TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.); reverse transcription kit (Bao Biological Engineering Co., Ltd., Dalian, China); reverse-phase C18 (Silicycle Inc., Quebec, Canada); heparin sodium salt (Nanjing Chemical Reagent Co., Ltd., Nanjing, China); Hank’s balanced salt solution (Shanghai Haoran Biological Technology Co., Ltd.); haematoxylin and eosin staining solution (Beijing leagene Biotechnology Co., Ltd., Beijing, China); optimum cutting temperature compound (Shanghai Solarbio Bioscience & Technology Co., Ltd., Shanghai, China); and 5-fluoro-2,4-(1H,3H)-pyrimidinedione (5-FU; Nantong Essence Pharmaceutical Co., Ltd., Jiangsu, China).

Instruments

The following instruments were used in the study: Multiskan GO Microplate Reader (Thermo Fisher Scientific Co., Ltd., Waltham, MA, U.S.A.); CO₂ incubator (Memmert Co., Ltd., Shanghai, China); inverted microscope (CKX31-A12PHP; Olympus Optical Co., Ltd., Nagano, Japan); FACS Calibur Flow Cytometer (BD Biosciences, San Jose, CA, U.S.A.); 7300 StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, U.S.A.); UV-visible spectrophotometer (UV-2550; Shimadzu, Kyoto, Japan); centrifuge (ROTINA 380R; Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany); HPLC system (1260 Infinity; Agilent Technologies Co., Ltd., Santa Clara, CA, U.S.A.); freezing microtome (CM1950; Leica Microsystems GmbH, Wetzlar, Germany); and optical microscope (BX51; OLYMPUS Co., Ltd., Okaya, Nagano, Japan).

Animals and Cell Culture

ICR mice (23 ± 0.5 g) were purchased from the Laboratory Animal Center of Jilin University (Jilin, China) for the study. The mice were shut individually in cages in a constant greenhouse room under a 12/12-h light/dark cycle. The mice were acclimatised for one week and fed food and water before use in the study. The protocols for all animal experiments were approved by the Animal and Ethics Review Committee of the Faculty of Jilin Agricultural University. Human cancer cells (HepG2, MCF-7, HeLa, and A549), and mouse H22 hepatocarcinoma cells (H22) were provided by Jilin Province Institute of Tumour Research (Jilin, China) for this study. H22 cells were administered intraperitoneally as a suspension in Hank’s balanced salt solution (2 × 10⁶ cells per 0.1 mL) to each mouse. H22 cells were removed from the abdominal cavity of a mouse within 9 to 11 d after inoculation.⁸⁾ In the experiment to assess the anti-tumour effect of HDE, H22 cells (1 × 10⁶) were prepared by Hank’s balanced salt solution and inoculated subcutaneously (0.1 mL) into the left axillary region of each mouse. When the tumour size reached to 4 to 6 mm, the experiment was initiated.⁹⁾

Extraction and Separation of HDE

The fruiting body of I. hispidus was crushed, and the powder was sifted through a 0.90-mm sieve. The powder and methanol were mixed at a ratio of 1 : 20 each time and extracted by ultrasound for 3 times (each time for 1 h). The extracted solution was concentrated at 50°C by reducing pressure. Then, the extract was suspended with water and centrifuged (3404 × g, 4°C, 10 min). The liquid in the middle layer was subjected to reverse phase C18 column chromatography. Gradient elution was performed with methanol–water.

MTT Assay for Assessing Cell Proliferation

First, HepG2, HeLa, McF-7, A549, and H22 tumour cells were cured with HDE or 5-FU. A blank test was performed simultaneously. Each cell suspension was adjusted with logarithmic phase cells to obtain a cell density of 5000 cells/well. The cells were added to six wells and treated with HDE. The final concentration was 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, or 100 µg/mL. The cells were cultured for 48 h in an incubator (5% CO₂, 37°C), until a monolayer of cells covered the bottom of the plate. Next, 10 µL of MTT solution (5 mg/mL) was added to each well, and the cells were cultured for 4 h. Thereafter, 100 µL of formazan-dissolving solution from the MTT cell proliferation and cytotoxicity assay kit was added to each well. The plates were incubated until total dissolution of formazan was observed, which was monitored using an inverted microscope. Absorbance at 570 nm was detected by using a Multiskan GO Microplate Reader.

Apoptosis Detection by Flow Cytometry

HepG2 cells were treated with HDE (3.13, 12.5, and 50 µg/mL) for 24 h. At the same time, HepG2 cells were pretreated with FasL neutralizing antibody (10 µg/mL, 37°C, 1 h) were disposed with HDE (3.13, 12.5, and 50 µg/mL) for 24 h. A blank test was performed simultaneously. The culture medium was removed and placed in a suitable centrifuge tube, after which the cells were rinsed with PBS. The tube was filled with the appropriate digestive juice containing pancreatic enzymes and EDTA. Adherent cells were incubated at 23°C, followed by removal of pancreatic juice. Next, the cells were collected and suspended in PBS. Approximately 1 × 10⁵ heavy suspended cells were centrifuged for 5 min, and the supernatant was then removed. The cells were lightly suspended in 195 µL annexin V-FITC binding solution, to which 5 µL annexin V-FITC and 10 µL iodide dye were added. The suspension was mixed gently, kept at 25°C for 15 min, then placed in an ice-bath. The tube containing the suspension was covered with aluminium foil to avoid exposure to light. The sample was then detected using a FACS Calibur Flow Cytometer.

Quantitative (q)RT-PCR

HDE was dissolved in PBS for the experiment. The solution was filtered using a pinhole filter and set aside. HepG2 cells were cultured in a T25 cell culture bottle until around 80% confluence was reached. The cells were digested using trypsin–EDTA solution, centrifuged, resuspended in PBS, added to 24-well plates, and then cultivated followed by treatment with HDE (3.13, 12.5, or 50 µg/mL). Next, each well was added to 1 mL TRIzol reagent and 24-well plates were placed on ice for 5 min. The liquid was collected into a 1.5-mL Ribonuclease (RNase)-free centrifuge tube, and 0.2 mL chloroform was added to it. The centrifuge tube was violently shaken for 15 s, and allowed to stand for 5 min, after which it was centrifuged (12000 × g, 4°C, 15 min). The supernatant was transferred to another RNase-free centrifuge tube and a similar amount of isopropyl alcohol was added. The suspension was placed for 5 min and then centrifuged (12000 × g, 4°C, 15 min). The supernatant was discarded, and the precipitate was suspended in 75% ethanol (1 mL). The suspension was vortex-mixed for 30 s and centrifuged (8000 × g, 4°C, 5 min). The supernatant was discarded and the above steps were repeated. The precipitate was placed on an ultra-clean table for 5–10 min and 20 µL DEPC–water was added. Total RNA was identified by agarose gel electrophoresis and quantified using an UV-visible spectrophotometer. The reverse transcription kit was used for reverse transcription. The primer sequences used were as follows: Fas, 5′-TTC TGC CAT AA GCC CTG TC-3′ (forward) and 5′-TTG GTG TTG CTG GTG AG T-3′ (reverse); FasL, 5′-TTC AGC TCT TCC ACC TAC AG-3′ (forward) and 5′-ACA TTC TCG GTG CCT GTA AC-3′ (reverse); and β-actin, 5′-GGT GCT GAG TAT GTC GTG GAG-3′ (forward) and 5′-ATG CAG GGA TGA TGT TCT AGG-3′ (reverse). PCR was performed simultaneously. The amplification conditions were as follows: 95°C, pre-denaturation for 3 min; 95°C, denaturation for 30 s; 62°C, renaturation for 30 s; and 72°C, extension for 30 s, repeated 40 cycles. Data were analysed using 7300 system sodium dodecyl sulfate (SDS) software installed on the 7300 StepOnePlus Real-Time PCR system. The level of Fas, and FasL relative mRNA expression to β-actin was calculated by the gene expression’s C (T) difference method.¹⁰⁾

Western Blot Analysis

HepG2 cells were treated with different concentrations of HDE (3.13, 12.5, and 50 µg/mL). The cells were cleaved through RIPA lysate solution. The protein concentration was determined using the BCA protein assay kit, and the same amount of protein (30 µg) was added to SDS polyacrylamide gel electrophoresis gel. The proteins were then electrophoretically transferred to polyvinylidene fluoride membranes. The membranes were sealed by Western sealing fluid that was incubated with anti-Fas (1 : 1000), anti-FasL (1 : 1000), and anti-β-actin (1 : 1000) antibodies at 4°C overnight respectively. They were then incubated with horseradish peroxidase-conjugated rabbit anti-rat secondary antibody. The results were analysed by Image Lab 3.0 software.

Detection of Caspase-3 and Caspase-8 Activities

HepG2 cells were treated with HDE (3.13, 12.5, and 50 µg/mL) for 24 h. Caspase-3 and caspase-8 activities in the cells were determined according to the instructions of Caspase-3 activity assay kit and Caspase-8 activity assay kit. Samples were measured at 405 nm by Multiskan GO Microplate Reader. The absorbance of peptide nucleic acids from every sample was compared with the control group.

Pharmacokinetics of HDE in Mice

The pharmacokinetic study was conducted in ICR mice, which were fasted for 12 h before the experiment. The mice were divided into two groups (each group of 66 mice, half male and half female) and orally administered with HDE (9.44 and 18.88 mg/kg, respectively). Approximately 0.5 mL blood samples were collected into 1.5-mL heparinised tubes (using heparin sodium salt) by eye puncture at 5, 15, 30, 45, 60, 90, 120, 180, 240, 360, and 720 min after dosing. Six mice were euthanised by dislocation of the cervical spine immediately after the last blood sample was collected, and plasma was collected by centrifugation of blood (4863 × g, 15 min). For each sample, 0.2 mL of plasma was added to 1.5-mL centrifuge tube and vortex-mixed with 1 mL methanol for 3 min. The mixture was then centrifuged (3404 × g, 4°C, 10 min). The supernatant was collected into a 1.5-mL centrifuge tube and dried at 40°C using nitrogen. Subsequently, each sample and blank biological sample was dissolved in 0.5 mL mobile phase and analysed by HPLC. Chromatographic separation was completed with a C18 column (250 × 4.6 mm, 5 µm) under the following conditions: temperature, 30°C; detector wavelength, 254 nm; mobile phase, methanol : water (10 : 90, v/v); flow rate, 1 mL/min; and run time, 15 min. The plasma concentration of HDE was analysed using PKSolver 2.0.

In Vivo Tissue Distribution of HDE in Mice

Three groups (n = 6 per group) of ICR mice were used for this study, and they were orally administered 18.88 mg/kg HDE. The mice were euthanised at 30, 60, or 120 min after dosing, and their liver, lung, kidney, spleen, thymus, muscle, heart, and brain were harvested, cleaned, and washed using cold saline. The water was removed using a filter paper and weighed. Each organ was homogenised in water, and the resulting homogenate was concentrated to 0.2 mL at 40°C using nitrogen. Finally, the amount of HDE was analysed using HPLC.

In Vivo Anti-tumour Effect of HDE

The anti-tumour effect of HDE was evaluated by tumour growth inhibition rate (TGI). H22 tumour-bearing mice were randomly assigned to the following five groups (half male and half female): saline (10 mL/kg); HDE1 (4.72 mg/kg); HDE2 (9.44 mg/kg); HDE3 (18.88 mg/kg); and 5-FU. The 5-FU group was injected intraperitoneally using 50 mg/kg 5-FU every other day (four times in total), whereas the other groups were orally administered the respective treatments every day. The mice were euthanised one day after the administration of the last dose. The tumours were taken and weighed. The heart, liver, spleen, thymus, lung, kidney, and brain of each animal were also harvested and weighed. The formula of TGI is as follows:

  

The tumours and organs were frozen in optimum cutting temperature compound and sectioned using a freezing microtome. Slices of tumours and organs were dyed by haematoxylin and eosin staining solution, and observed by an optical microscope.

Statistical Analysis

Results are expressed as mean ± standard deviation (S.D.; in vitro experiments, n = 3; animal study, n = 6). Statistical significance was determined using ANOVA and Dunnett’s post hoc test. p-Values less than 0.05 were considered statistically significant.

RESULTS

Identification of HDE

HDE was obtained from I. hispidus for the first time. The structure of HDE (Fig. 1) was identified and confirmed by LC-MS/MS and NMR spectroscopy. Additionally, the [M + H]⁺ ion of HDE was detected at m/z 173.1 by LC-MS/MS (electrospray ionization). The ¹H-NMR and ¹³C-NMR data for HDE are listed in Table 1. Furthermore, two-dimensional NMR (2D-NMR) data for HDE are as follows: ¹H-detected heteronuclear multiple quantum coherence; ¹H–¹H correlation spectroscopy; and heteronuclear multiple bond connectivity. These findings are similar to the results of Boyd et al.¹²⁾ and Xie et al.¹³⁾

Fig. 1. Chemical Structure of HDE

Table 1. ¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) Data for HDE in DMSO-d₆

Carbon	δC	δH	Carbon	δC	δH
1	195.4		5	79.0	3.63 (1H, br s)
2	101.6	5.25 (1H, s)	6	37.7	2.65 (1H, d, J = 16.0 Hz) 2.34 (1H, d, J = 16.0 Hz)
3	174.9		3-OCH3	55.9	3.67 (3H, s)
4	67.2	4.08 (1H, br s)	5-OCH3	56.5	3.27 (3H, s)

d, doublet; J, coupling constant; s, singlet; br s, broad single.

In Vitro Anti-tumour Screening and Apoptosis

In this study, the anti-tumour effect of HDE and 5-FU at different concentrations was evaluated by MTT. At a concentration of 100 µg/mL, the proliferation inhibitory rate of HDE on HepG2, McF-7, HeLa, A549, and H22 tumour cells was 86, 88, 85, 88, and 85%, respectively (Fig. 2a). The half-maximal inhibitory concentration values of HDE were 7.9, 10.7, 14.0, 10.7, and 9.5 µg/mL, respectively. At a concentration of 12.5 µg/mL, the proliferation inhibitory rate of 5-FU on HepG2, McF-7, HeLa, A549, and H22 tumour cells was 85, 83, 79, 81, and 83%, respectively (Fig. 2b)

Fig. 2. Effects of (a) HDE and (b) 5-FU on the Proliferation of HepG2, MCF-7, HeLa, A549, and H22 Cells

(Color figure can be accessed in the online version.)

The apoptosis rates of HepG2 cells and HepG2 cells treated with FasL neutralizing antibody at HDE concentrations of 3.13, 12.5, and 50 µg/mL were 18.21, 45.47, and 77.43% and 7.94, 26.47, and 49.78%, respectively (Fig. 3).

Fig. 3. Apoptosis Rates of HepG2 Cells and HepG2 Cells Treated with FasL Neutralizing Antibody Induced by HDE

This assay was performed in triplicate. * indicates p < 0.05 and ** indicates p < 0.01 when compared to the control group; ♯ indicates p < 0.01 when compared to the HDE group at the same concentration. (Color figure can be accessed in the online version.)

Protein and mRNA Expression of Fas and FasL

The relative mRNA expression levels of Fas and FasL after treatment of HepG2 cells with HDE are shown in Fig. 4. When the concentrations of HDE was 50 µg/mL, the relative mRNA expression levels of Fas and FasL were found to be 176.4 ± 2.6% and 39.2 ± 3.5% after 24 h of treatment, respectively. After treating HepG2 cells with HDE, the relative protein expression levels of Fas and FasL were shown in Fig. 5. When the concentrations of HDE was 50 µg/mL, the relative protein expression levels of Fas and FasL were found to be 165.1 ± 6.3% and 51.3 ± 5.5% after 24 h of treatment, respectively.

Fig. 4. Effect of HDE on Fas and FasL mRNA Expression in HepG2 Cells

HepG2 Cells were treated with various concentrations of HDE (3.13, 12.5, and 50 µg/mL) for 24 h, after which Fas and FasL mRNA expression were determined by qRT-PCR. ** indicates p < 0.01 when compared to the control group.

Fig. 5. Effect of HDE on Fas and FasL Protein Expression in HepG2 Cells

HepG2 Cells were treated with various concentrations of HDE (3.13, 12.5, and 50 µg/mL) for 24 h, after which Fas and FasL protein expression were determined by Western blot analysis. * indicates p < 0.05 and ** indicates p < 0.01 when compared to the control group.

Activities of Caspase-3 and Caspase-8

When the concentrations of HDE was 3.13, 12.5, and 50 µg/mL, the relative Caspase-3 activities were 1.25 ± 0.15, 1.46 ± 0.16, and 1.87 ± 0.15, and the relative Caspase-8 activities were 1.32 ± 0.12, 1.51 ± 0.17, and 1.93 ± 0.19. Increasing HDE concentrations significantly increased relative Caspase-3 and Caspase-8 activities in a dose-dependent manner (Figs. 6a, 6b.).

Fig. 6. Relative Activities of Caspase-3 (a) and Caspase-8 (b)

* indicates p < 0.05 and ** indicates p < 0.01 when compared to the control group.

Pharmacokinetic Study and Tissue Distribution of HDE in Mice

The plots of plasma HDE concentration versus time obtained after oral administration of HDE (9.44 or 18.88 mg/kg) in mice are shown in Fig. 7a. The pharmacokinetic parameters obtained from the plots are listed in Table 2. The results show that HDE was eliminated quickly and was not detectable at 720 min. Furthermore, it was quickly absorbed and reached the maximum concentration in blood at 30 min. The HDE concentration in the muscle and different organs at 30, 60, and 120 min after dosing are shown in Fig. 7b, whereas HDE distribution in the mice is shown in Fig. 8. The area under the plasma concentration–time curves (AUC) of HDE in mice were of the following order: spleen > liver > lung > kidney > muscle > thymus > heart > brain. The AUC values indicate HDE distribution and concentration in mice.

Fig. 7. (a) Plasma Concentration–Time Curve for HDE after Oral Administration to Mice; (b) Distribution of HDE in the Mouse Heart, Liver, Thymus, Spleen, Lung, Kidney, Brain, and Muscle after Oral Administration

(Color figure can be accessed in the online version.)

Fig. 8. AUC of HDE for the Heart, Liver, Thymus, Spleen, Lung, Kidney, Brain, and Muscle after Oral Administration to Mice

AUC, area under the curve.

Table 2. Pharmacokinetic Parameters of HDE after Oral Administration to Mice (n = 6)

Parameter	Unit	HDE dose (mg/kg)
		9.44	18.88
t1/2	min	114.9 ± 17.6	118.9 ± 15.3
Tmax	min	30	30
Cmax	mg/mL	0.0149 ± 0.0011	0.0361 ± 0.0029
AUC(0–t)	mg*min/mL	1.944 ± 0.199	4.597 ± 0.406
AUC(0–∞)	mg*min/mL	1.965 ± 0.175	4.652 ± 0.476
CL/F	mL/min/kg	4.803 ± 0.457	4.058 ± 0.0423

AUC, area under the curve; CL/F, clearance; C_max, maximum plasma concentration; T_max, time to reach C_max.

In Vivo Anti-tumour Effect of HDE

The anti-tumour effect of HDE acting on H22 tumour-bearing mice is summarised in Fig. 9a. After treatment for 8 d, the average tumour weight in the 5-FU, HDE1, HDE2, and HDE3 groups significantly decreased (p < 0.01). Furthermore, thymus and spleen weights were significantly higher in control group than in 5-FU-treated group (p < 0.01), which may have been due to the immunosuppressive effect of 5-FU. However, spleen and thymus weights in the control group were not obviously different compared to those in the HDE-treated groups. Additionally, the other organ weights were not significantly different between the HDE-treated, 5-FU, and control groups (Fig. 9b). Pathological examination of tumour tissues showed that tumour cells in the control group grew vigorously. However, HDE and 5-FU were shown to cause necrosis and dissolution of tumour cells. Histological images of the tumour are shown in Fig. 10.

Fig. 9. Effects of HDE on (a) Tumour Weight, TGI, and (b) Organ Weight

(Color figure can be accessed in the online version.)

Fig. 10. Histological Images of the Tumour

(Color figure can be accessed in the online version.)

DISCUSSION

Studying the pharmacokinetics and tissue distribution of drugs is important for understanding traditional medicine.^14–16) One or more active ingredients in a herbal medicine can be studied when evaluating the pharmacokinetics and tissue distribution of the herb.^17,18)

The MTT assay method is a simple, sensitive, and reproducible method that is widely used to screen anti-tumour drugs.¹⁹⁾ Our study found that the proliferation of HepG2, McF-7, HeLa, A549, and H22 cells was inhibited by HDE; however, this effect was more pronounced in HepG2 cells. The apoptosis rate increased significantly in proportion to HDE concentration increase, the apoptosis rate decreased with the addition of FasL neutralizing antibody in HepG2 cells, relative activities of Caspase-3 and Caspase-8 increased when HepG2 cells were treated with HDE, suggesting that HDE may exert anti-tumour effects by inducing apoptosis. Many factors induce apoptosis; nevertheless, in general, exogenous and endogenous apoptosis pathways are the two main pathways involved.²⁰⁾ During the development of human malignant tumours, FasL expression is usually significantly increased and Fas expression on the tumour cell surface is decreased. Additionally, there may be loss of Fas function. Therefore, inducing or enhancing Fas expression and reducing FasL expression could promote apoptosis of tumour cells.²¹⁾ In the present study, Fas expression increased or FasL expression reduced in proportion to the HDE concentration administered to HepG2 cells (3.13, 12.5, and 50 µg/mL) for 24 h. However, Fas mRNA expression was slightly higher when the treatment duration was 24 h. Fas mRNA expression was reduced when the cells were treated for 72 h with increasing concentrations of HDE. This could have been due to an overabundance of dead cells.

The plasma concentration-time curve of HDE displayed significant bimodal characteristics after the mice were orally administered with the drug. This could be due to different absorption rates in each parts of the body, enterohepatic circulation, or quick absorption by the body and release of the drug back into systemic circulation.^22,23) HDE concentration was higher in the spleen and liver than in other organs and muscles (Fig. 7b). Furthermore, HDE was not found in the brain, which indicates that it is unable to pass through the blood–brain barrier.

The results of the in vivo study on H22 tumour-bearing mice showed that HDE (4.72, 9.44, and 18.88 mg/kg) significantly reduced tumour weight. TGI was slightly higher in 5-FU group than in HDE-treated groups, and found to be 49.6, 57.2, 69.0, and 77.4% in HDE1, HDE2, HDE3, and 5-FU groups, respectively. Moreover, the immune organs of the HDE-treated mice remained undamaged, which was demonstrated by the weights of the spleen and thymus.

Hepatocellular carcinoma is a common type of liver malignancy.²⁴⁾ Currently, chemotherapy is an important treatment method for hepatocellular carcinoma; however, its effective application is restricted by side effects and high toxicity.²⁵⁾ Importantly, the findings of this study show that HDE has a definite anti-tumour effect, and thus provides insight into the mechanisms underlying its use as herbal medicine.

CONCLUSION

This study showed that HDE upregulated Fas expression and downregulated FasL expression, increased Caspase-3 and Caspase-8 activities, activated the Fas-mediated apoptosis pathway, and induced apoptosis in HepG2 cells. Additionally, the pharmacokinetics and tissue distribution of HDE was evaluated by an HPLC method that we developed based on detection of HDE in mouse plasma. Our findings indicate that HDE is quickly absorbed by body and released back into systemic circulation or undergoes enterohepatic circulation. Furthermore, HDE significantly inhibited tumour growth in mice and did not have a negative effect on organs. Therefore, HDE may be an effective anti-tumour drug.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (NSFC Nos. 31270088).

Conflict of Interest

The authors declare no conflict of interest.

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