Skip to main content
NCBI home page
AAPS PharmSciTech logoLink to AAPS PharmSciTech
. 2014 Nov 6;16(3):496–504. doi: 10.1208/s12249-014-0227-3

Anti-Hepatoma Activity of a Novel Compound Glaucocalyxin H In Vivo and In Vitro

Guangfan Hai 1, Chong Zhang 1, Yanlong Jia 1, Suping Bai 1,✉, Jinfen Han 2, Lanqing Guo 3, Taizhen Cui 1, Bingxuan Niu 1, Feng Huang 1, Yu Song 1
PMCID: PMC4444621  PMID: 25374342

Abstract

Glaucocalyxin H (GLH) is a new compound isolated from a traditional Chinese medical herb Isodon japonica var. glaucocalyx which has been used for folk medicine. This study was carried out for the first time to investigate the potential role of GLH in anti-hepatoma activity and underlying mechanisms in it. GLH could inhibit the growth of tumor in mice and induce HepG2 cells to death as assessed by the tumor reduction assay, toxic assay, morphological change, and survival rate assay. Many antitumor drugs originated from plants could inhibit the growth of tumor by inducing cells to apoptosis. The morphological changes of HepG2 cells treated with different concentrations of GLH under fluorescence and electron microscope and apoptotic rates were detected to verify its effect on apoptosis. As shown in the study, GLH could induce HepG2 cells to apoptosis in a dose-dependent manner. Bcl2 and Bax proteins played important roles in apoptosis and the disequilibrium between Bcl2 and Bax might result in apoptosis. The expression of Bax protein was upregulated and Bcl2 protein was downregulated in HepG2 cells treated with GLH assessed by Western blotting, and they were in a dose-dependent manner. Taken together, GLH can inhibit the growth of hepatoma cells in vivo and in vitro by inducing cell apoptosis due to the decreased Bcl2 and increased Bax proteins suggesting that GLH could be a potential candidate as an anti-hepatoma agent for the therapeutic treatment of hepatoma.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0227-3) contains supplementary material, which is available to authorized users.

KEY WORDS: apoptosis, Glaucocalyxin H, hepatoma, HepG2 cell

INTRODUCTION

Isodon japonica (Burm. f.) Hara var. glaucocalyx (Maxim.) Hara is widely distributed throughout northern China, and it has been used as an antibacterial, anti-inflammatory, stomachic, and anthelmintic agent in Chinese folk medicine (1). Its anticancer activities were proven in the early 1960s (2). Phytochemical analysis has shown that the main chemical compositions of I. japonica var. glaucocalyx are diterpenoids (3,4). Glaucocalyxin A and glaucocalyxin B are the two important diterpenoids from I. japonica var. glaucocalyx (5). Glaucocalyxin A is capable of inducing human leukemia HL-60 cells to apoptosis (6), inhibiting platelet-activating factor (PAF)-induced platelet aggregation (7), and suppressing immune activity (8). It has also been reported that glaucocalyxin A has cytotoxic activity (9). Glaucocalyxin B possesses cytotoxicity of AGZY-83A cells by blocking entry to the G2/M phase (10) and may induce Rb cells to apoptosis (11). As of yet, a total of more than 30 ent-kaurane diterpenoids have been extracted from I. japonica var. glaucocalyx found in different areas of China, including glaucocalyxin C, glaucocalyxin D, glaucocalyxin E, glaucocalyxin F, etc. (12–15). However, there are seldom studies relating to the pharmacological effects and mechanisms of these diterpenoids.

Glaucocalyxin H (GLH), a new natural compound, was isolated from the ethyl acetate (EtOAc) extract of the dried leaves of I. japonica var. glaucocalyx in our laboratory (16). The structural elucidation demonstrated that GLH is an acetal derivative of the two hydroxyl groups of glaucocalyxin A with the aldehyde group of protocatechuic aldehyde (Fig. 1). Glaucocalyxin A, glaucocalyxin B, and glaucocalyxin H are the ent-kaurane diterpenoids, containing the same pharmacophore α,β-unsaturated cyclopentanone in their structure (17). In our early studies, it was found that GLH had potent cytotoxicity on EC-1, U87, A549, MCF-7, Hela, K562, and HepG2 cells. Its antitumor activity on HepG2 cells was stronger than both glaucocalyxin A and glaucocalyxin B. Therefore, further studies were carried out regarding the effects of GLH on liver tumor both in vivo and in vitro to determine its potential clinical value.

Fig. 1.

Fig. 1

Open in a new tab

The structures of glaucocalyxin A, glaucocalyxin B, and glaucocalyxin H

MATERIALS AND METHODS

Reagents

Compound GLH (purity 95.5%) was provided by our laboratory (School of Pharmacy, Xinxiang Medical University). Cyclophosphamide injection (no. 09123121) was purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd., China. Medium RPMI-1640 was produced by Gibco, USA. Mouse monoclonal antibodies (Abs) to human Bcl-2, Bax, and rabbit-antimouse immunoglobulin G (IgG) Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fetal bovine serum was provided by Hangzhou Sijiqing Biological Engineering Materials Co., Ltd, China. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were provided by Sigma, USA. Propidium iodide (PI) and Hochest 33258 were purchased from Guangzhou Weiga Technology Co., Ltd., China. 5-Fluorouracil (5-Fu) (no. 100301) was purchased from Hualian Shanghai Pharmaceuticals, China.

The Scheme of Synthesis of Glaucocalyxin H

In a 50-mL round-bottom flask, two drops of concentrated sulfuric acid was added to the solution of glaucocalyxin A (332 mg, 1.0 mmol) and protocatechuic aldehyde (207.2 mg, 1.5 mmol) in CH2Cl2 (8 mL) and THF (0.5 mL). The solution was stirred for 1 h at room temperature and then 20 mL of CH2Cl2 (20 mL) was added. The mixture was washed with water (2 × 30 mL) and saturated sodium carbonate solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography eluting with chloroform/methanol (100/1) to afford glaucocalyxin H as white solid (271.5 mg, 60% yield). Mp. 282-284oC; [α]32.4 D −74.4° (c 0.23, MeOH); 1H-NMR (C5D5N, 400 MHz, ppm): δ 6.30 and 5.36 (each 1H, s, 2H-17), 4.82 (1H, s, H-14), 4.74, 4.70 (1H, dd, J = 12.88, 5.08Hz, H-7), 3.22 (1H, s, H-13), 1.13, 1.11 and 1.06 (each 3H, s, 3 × Me), 11.32 (2H, brs, ×2 Ar-OH), 6.27 (1H, s, H-1′), 7.68 (1H, s, H-7′), 7.25 (1H, s, H-3′, 6′). 13C-NMR (C5D5N, 100 MHz, ppm): δ 215.3 (s, C-3), 206.2 (s,C-15), 147.4 (s, C-16), 116.8 (t, C-17), 78.0 (d, C-14), 73.2 (d, C-7), 56.0 (s, C-8), 52.2 (d,C-9), 51.4 (d, C-5), 47.7 (s, C-4), 44.1 (d, C-13), 38.8 (s, C-10), 38.3 (t, C-1), 34.6 (t, C-2), 31.4 (t, C-12), 23.9 (t, C-6), 26.6 (q, C-18), 21.8 (q, C-19), 17.1 (q, C-20), 18.7 (t, C-11), 94.6 (d, C-1′), 131.8 (s, C-2′), 115.5 (d, C-3′), 148.5 (s, C-4′), 148.2 (s, C-5′), 116.3 (d, C-6′), 119.0 (d, C-7′). The scheme of synthesis of glaucocalyxin H was shown in Fig. 2.

Fig. 2.

Fig. 2

Open in a new tab

The scheme of synthesis of glaucocalyxin H

Animals

Adult Kunming mice weighed 20 ± 2 g, 50% female and 50% male, were purchased from the Henan Province Center of Laboratory Animals (Zhengzhou, China). All animals received humane care. The Animal Care Committee of Xinxiang Medical University of China approved the experimental protocol. These mice were maintained in a controlled environment at 24 ± 2°C with a 12-h light/dark cycle and received food and water ad libitum. They were acclimatized for at least 3 days prior to the experiments.

Cell Lines

H22 cells and HepG2 cells were obtained from Nanjing Kaiji Biological Engineering Co., Ltd. (Nanjing, China). HepG2 cell lines were originally obtained from the Cell Center of the Chinese Academy of Sciences Library. The cells were cultivated in an incubator at 37°C in air containing 5% CO2 with a relative humidity of 98%. The culture medium RPMI-1640 (Gibco, USA) contained 100 g/L of heat-inactivated fetal calf serum, 10 U/mL of penicillin, and 100 mg/mL of streptomycin. The cells were transferred to a new culture every 2–3 days. The hepatocytes in the exponential growth phase were used in the experiment.

Mouse Preparation of Experiment In Vivo

One milliliter (1 × 107cells) H22 cell solution was subcutaneously injected at the right oxter of the mice for 8 days to create the focal tumor models. Fifty mice of focal tumor models were randomly divided into five groups, namely, the model control group, 10 mg/kg cyclophosphamide control group, and 20, 40, and 80 mg/kg GLH groups. Each mouse in the GLH groups was injected with GLH by means of intraperitoneal injection every day at 8:00 am for 12 days. Physiological saline and cyclophosphamide were also injected for the model control group and 10 mg/kg cyclophosphamide control group at 8:00 am for 12 days, respectively.

Assay of Tumor Weight, Tumor Inhibitory Rate, and Morphological Changes of Tumor Tissue

Twelve days later, the mice were euthanized by dislocation after being weighed in balance. Then, they were sterilized in 75% alcohol and the skin of right axil was cut to retrieve the tumor with the membrane decoherence. The wet tumor was weighed in balance and the tumor inhibitory rate was calculated according to the following formula: tumor inhibitory rate = (mean tumor weight in model control group − mean tumor weight in GLH group) / mean tumor weight in model control group × 100%. At the same time, the extent of tumor diffusion and necrosis as well as adherence with muscles and skin was observed.

Assay of Thymus Gland and Spleen

The thoracic cavity and the belly cavity of mice were cut open to extract the thymus gland and spleen out after the mice were euthanized and sterilized. The wet thymus gland and spleen were weighed in balance. The thymus gland and spleen indexes were calculated, respectively, according to the formula: thymus index = weight of thymus / body weight of mice × 100%, spleen index = weight of spleen / body weight of mice × 100%.

Assay of HepG2 Survival Rates

The effect of GLH on cell viability was assessed by a colorimetric MTT assay. Briefly, 5 × 103 HepG2 cells were seeded in each well of a 96-well culture plate and allowed to adhere overnight at 37°C in a humidified 5% CO2 atmosphere. Twenty-four hours later, HepG2 cells were incubated with GLH at the concentrations of 0.625, 1.25, 2.5, 5.0, and 10 μmol/L, respectively. The negative control group received drug-free medium with 0.05% v/v DMSO. The positive control group was treated with 5-Fu with the same concentrations of GLH. Subsequently, 5 mg/mL MTT was added to each well. The medium was discarded and 150 μL DMSO was added into each well after incubation at 37°C for 4 h. The optical density was measured by a microplate reader at a reference wavelength of 630 nm and a test wavelength of 570 nm. The percentage of cell growth was calculated as follows: cell survival rates (%) =  A570sample /A570negative control × 100%

Assay of Morphology Under Fluorescence Microscope

HepG2 cells were seeded into 6-well culture plates at a density of 1 × 105cells per well and incubated with GLH at concentrations of 0.625, 1.25, 2.5, 5.0, and 10 μmol/L. The negative control group received drug-free medium with 0.05% v/v DMSO. The cells were washed with 4°C phosphate buffer solution (PBS) three times, and 500 μL 4% paraformaldehyde (PFA) at 4°C was dropped in every well to fix the cells for 10 min 24 h later. Being washed with PBS three times, 10 μg/L Hoechst33258 was placed into each well for 10 min at room temperature away from light. Then, the cells were observed under reverse fluorescence microscopy after being washed with PBS three times.

Assay of Morphology Under Electron Microscope

HepG2 cells (3 × 106) were seeded into each culture bottle and incubated with GLH at concentrations of 1.25 and 5.0 μmol/L for 24 h. The negative control group received drug-free medium with 0.05% v/v DMSO. Twenty-four hours later, the cells were collected and washed three times with 4°C regular saline and were detected with electron microscope.

Assay of Apoptotic Rates

HepG2 cells (1 × 106) were seeded into 6-well plates and exposed to different concentrations (0.625, 1.25, 2.5, 5.0, and 10 μmol/L) of GLH for 24 h. The negative control group exposed to 0.05% v/v DMSO for 24 h. The positive control group was treated with 5-Fu with the same concentrations of GLH. The cell solution was centrifugated at 1200 rpm for 5 min to harvest the cells, and the cells were washed three times with cold PBS. Then, the cells were gently suspended in 500 μL binding buffer. Five microliters PI were added and incubated with cells in the dark for 10 min. At the end of incubation, analysis was carried out to discriminate between live and apoptotic cells by FACScan flow cytometry.

Detection of Bcl2 and Bax Proteins

Protein expression of Bcl2 and Bax was tested by Western blot. In brief, HepG2 cells were washed with PBS (mM: NaCl 130, KCl 2.5, Na2HPO4 10, KH2PO4 1.5, pH 7.4) and lysed with solubilization buffer (mM: Tris-Cl 50, NaCl 150, 0.02%NaN3, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, NaVO3, 5 mg/mL leupeptin and 1 mg/mL aprotinin). After centrifugation, the supernatants were collected and equivalent protein concentrations were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The separated proteins were electrotransferred to polyvinylidene fluoride (PVDF) membranes. After blocking with PBST (PBS containing 0.05% Tween 20) containing 5% non-fat milk for 1 h, each membrane was incubated with primary antibodies 1 h at room temperature or overnight at 4°C, and then the membranes were probed with the appropriate secondary peroxidase-conjugated antibodies (HRP-linked anti-rabbit secondary antibody and HRP-linked anti-biotin antibody, 1 h at room temperature). The immunoblots were visualized by enhanced chemiluminescence.

STATISTICAL ANALYSIS

The results are expressed in terms of mean ± standard deviation (SD). Statistical analysis was performed using the SPSS software package (version 16.0, SPSS Company, USA). Paired t tests, Student’s t test, and one-way ANOVA, followed by the Student–Newman–Keuls test, were used to determine statistical significance. The difference between means was considered to be statistically significant when P < 0.05.

RESULTS

Effects of GLH on Tumor Weight in Mice

The tumor weight showed a clear decrease in the GLH groups and cyclophosphamide group compared to the model control group. The tumor weights in the 20 and 40 mg/kg GLH groups increased compared to the cyclophosphamide group, while the weight in the 80 mg/kg GLH group was similar to that in the cyclophosphamide group. The data were shown in Fig. 3. Tumor tissues intruded extensively into the muscle of the chest wall tightly and colliquation, cellular necrosis in the center of tumors, and skin diabrosis at the right axil were obvious in some mice of the model group. There were fewer invasions and cellular necrosis in the center of the tumor in the 20 and 40 mg/kg GLH groups. Adherence and necrosis were seldom seen in the 80 mg/kg GLH group except fewer invasions and cellular necrosis compared with the model control group. The tumor inhibitory rate in the 80 mg/kg GLH group was similar to that in the cyclophosphamide group.

Fig. 3.

Fig. 3

Open in a new tab

a Effect of GLH on the size of tumor in mice. (a) The model control group, (b) the cyclophosphamide group, (c) the 20 mg/kg GLH group, (d) the 40 mg/kg GLH group, (e) and the 80 mg/kg GLH group. b Effect of different concentrations of GLH on the tumor inhibitory rate in mice

Effects of GLH on Thymus and Spleen Indexes in Mice

The effects of GLH on the thymus and spleen are shown in Fig. 4. As shown in the figure, 80 mg/kg GLH significantly decreases the thymus and spleen indexes compared to the model control group. However, there were little changes of GLH in the 20 and 40 mg/kg dosages on the thymus and spleen indexes. There was no clear difference for thymus and spleen indexes between GLH 80 mg/kg group and cyclophosphamide group. However, the thymus index was higher in the 20 and 40 mg/kg GLH groups than that in cyclophosphamide group.

Fig. 4.

Fig. 4

Open in a new tab

Effect of glaucocalyxin H (GLH) on thymus and spleen indexes in tumor-bearing mice. Each data represents mean ± SD of ten determinations (n = 10). *P < 0.05; **P < 0.01 compared with the cyclophosphamide group. # P < 0.05; ## P < 0.01 compared with the model group

Effects of GLH on HepG2 Survival Rates

As shown in Fig. 5, GLH could significantly inhibit HepG2 cells growth. Meanwhile, GLH inhibited HepG2 cell growth in a concentration-dependent manner. A significant decrease in survival rates was shown in GLH-treated cells compared with 5-Fu-treated cells under the same experimental conditions. The survival rates were 93.17, 74.56, 59.7, 44.08, and 19.25% in GLH groups, respectively, while the survival rates were 98.66, 93.27, 82.19, 68.21, and 53.30% in 5-Fu groups, respectively.

Fig. 5.

Fig. 5

Open in a new tab

Survival rates of HepG2 cells. Each data represents mean ± SD of six determinations (n = 6)

Effects of GLH on Morphological Changes of HepG2 Cells

It was shown under reverse fluorescence microscopy that HepG2 cells treated with GLH presented obvious morphological changes. In Fig. 6, there were many apoptotic cells including shrinkage, fragmentation, and irregularity of nuclei in the GLH-treated cells, especially in the 2.5, 5.0, and 10.0 μmol/L GLH groups. Meanwhile, the number of apoptotic cells increased with the increasing of GLH concentration. There was little apoptotic cells in the non-GLH-treated group.

Fig. 6.

Fig. 6

Open in a new tab

Apoptosis of HepG2 cells induced by GLH. (a HepG2 cells un-treated with GLH, b HepG2 cells incubated with 0.625 μmol/L GLH, c HepG2 cells incubated with 1.25 μmol/L GLH, d HepG2 cells incubated with 2.5 μmol/L GLH, e HepG2 cells incubated with 5.0 μmol/L GLH, f HepG2 cells incubated with 10.0 μmol/L GLH)

Effects of GLH on Microstructure of HepG2 Cells

As shown in Fig. 7, HepG2 cells were condensed with lunate and drifted nuclei in the 1.25 μmol/L GLH-treated cells compared to the no-GLH-treated HepG2 cells with round nuclei and intercalated nuclei. There were no clear nuclei and many apoptotic bodies could be found in 5.0 μmol/L GLH-treated HepG2 cells.

Fig. 7.

Fig. 7

Open in a new tab

Effects of GLH on the microstructure of HepG2 cells. (a HepG2 cells without being treated with GLH, b 1.25 μmol/L GLH, c 5.0 μmol/L)

Effects of GLH on the Apoptotic Rates of HepG2 Cells

Different GLH concentrations could induce HepG2 cells to apoptosis and the apoptotic rates were shown in Fig. 8. GLH induced HepG2 cell to apoptosis in a concentration-dependent manner, and the apoptotic rates were 7.8, 27.6, 29.9, 33.7, and 55.4% in 0.625, 1.25, 2.5, 5.0, and 10.0 μmol/L GLH-treated cells, respectively, which showed significant difference between GLH-treated cells and no-GLH-treated cells with 0.4% apoptotic rate.

Fig. 8.

Fig. 8

Open in a new tab

The apoptotic rates of HepG2 cells. Each data represents mean ± SD of six determinations (n = 6). **P < 0.01 compared with the control group

Effects of GLH on Protein Expressions of Bcl2 and Bax of HepG2 Cells

GLH was able to significantly upregulate the protein expression of Bax and downregulate that of Bcl2 with increasing dose. The results are shown in Fig. 9. The changes of Bcl2 and Bax proteins are in a concentration-dependent manner. Bcl2/Bax ratio decreased with GLH dosage increasing.

Fig. 9.

Fig. 9

Open in a new tab

Effect of GLH on Bcl2, Bax proteins, and Bcl2/Bax ratio of HepG2 cells. a Effect of GLH on Bcl2 protein of HepG2 cells (lane 1: 0 μmol/L; lane 2: 0.625 μmol/L; lane 3:1.25 μmol/L; lane 4:2.5 μmol/L; lane 5: 5 μmol/L; lane 6:10 μmol/L). b Effect of GLH on Bax protein of HepG2 cells (lane 1: 0 μmol/L; lane 2: 0.625 μmol/L; lane 3:1.25 μmol/L; lane 4:2.5 μmol/L; lane 5: 5 μmol/L; lane 6:10 μmol/L). c Effect of GLH on Bcl2/Bax ratio of HepG2 cells. Each data represents mean ± SD of three determinations (n = 3). *P < 0.05; **P < 0.01, compared with the control group

DISCUSSION

Malignant tumors have been a serious threat to human health. However, currently, there are no effective drugs which only kill tumor cells and have little damages to normal cells. Anticancer drugs originating from natural plants provide some basis for such drugs. In China, I. japonica var. glaucocalyx is a type of folk medicine which is mainly used to cure inflammation, stomach pain, and cancer as well as heat-clearing and detoxification (18). Glaucocalyxin A and glaucocalyxin B, the main components of I. japonica var. glaucocalyx, play significant roles in disease treatment and prevention. Glaucocalyxin A is able to protect the heart and liver from injuries induced by ischemia-reperfusion and carbon tetrachloride (CCl4) by decreasing the expression of c-fos genes (19,20). It also has antitumor activity for cells such as K562, HepG2, HL-60, Hela, etc. N-Acetylcysteine (NAC), a type of antioxidant, is capable of inhibiting the cytotoxicity of glaucocalyxin A on K562 cells, which is related to reactive oxygen species (ROS) (21). Further studies regarding NAC’s structure–function relationship with antitumor activity revealed that almost all diterpene components, including α-methylene cyclopentanone building block, may cause the tumor cells to die. Based on these observations, it may be concluded that the α-methylene cyclopentanone building block of diterpenes components is the pharmacophore (22). GLH, a newly discovered compound, isolated from I. japonica var. glaucocalyx, is an acetal derivative of glaucocalyxin A. It is derived by means of condensation reaction of two hydroxyl groups of glaucocalyxin A with the aldehyde group of protocatechuic aldehyde. GLH contains the same antitumor pharmacophore of cyclopentanone, conjugated with an exomethylene group in the structure as glaucocalyxin A. However, GLH exhibits higher inhibitory proliferation activities than its parent compound glaucocalyxin A, with 50% inhibitory concentration (IC50) value of 1.97 μM against HepG2 cell line in our early studies (16). This suggests that the hydroxyl groups of protocatechuic aldehyde enhance the antitumor activities of GLH. In order to evaluate the antitumor therapeutic efficacy and mechanism of glaucocalyxin H, GLH has been synthesized artificially from glaucocalyxin A and protocatechuic aldehyde.

Cyclophosphamide, an antitumor drug, may be used to treat carcinomas of the lung, breast, ovary, etc. (23–25). It is activated by cytochrome P450-mediated microsomal oxidation in the liver to produce aldophosphamide, which spontaneously decomposes to produce reactive phosphoramide mustard, which in turn may inhibit cell growth and multiplication by promoting DNA alkylation (26). In the present study, it was shown that 10 mg/kg cyclophosphamide may resist tumor growth in mice. The results showed that GLH decreased the tumor weight of mice in GLH groups (i.e., 20, 40, and 80 mg/kg) compared with the model group, and infiltration, colliquation, and celluar necrosis in the center of the tumors were improved especially in the 80 mg/kg group compared to the cyclophosphamide group and the model group which indicated that GLH played similar roles in inhibiting tumor growth to cyclophosphamide and may be related to its anti-inflammation effect (27). As shown in Fig. 4, thymus and spleen indexes decreased in the immune systems of the mice in GLH group, which was similar to cyclophosphamide which indicated that GLH had little toxicity to immune system of mice with the treatment of tumor.

The results in the experiments in vitro showed that GLH could decrease the survival rates of HepG2 cells which made clues that GLH could inhibit the growth of HepG2 cells. How did GLH inhibit its proliferation? As known to us, most of the present anticancer drugs mediate their effect via apoptosis induction in cancer cells, and apoptosis is suggested as one of the major mechanisms for the targeted therapy of various cancers including prostate cancer. To determine whether the growth inhibition induced by GLH in human liver cells was caused by apoptosis, HepG2 cell was treated with different concentrations of GLH for 24 h and the morphological changes were examined. As shown in Fig. 6 and Fig. 7, typical characteristics (shrinkage, fragmentation, irregularity, and shift of nuclei) of apoptosis could be observed, which suggested that GLH suppress the growth of HepG2 cells by inducing apoptosis. To further confirm the apoptotic effects of GLH in the cell, we also analyzed the apoptotic rates in different concentrations of GLH. The results suggested that GLH induced HepG2 cell to apoptosis in a concentration-dependent manner (Fig. 8). These results discovered that GLH suppress the growth of HepG2 cells by inducing apoptosis.

Apoptosis is known as programmed cell death (PCD) or cell suicide and is a form of cell death in which a controlled sequence of events (or program) leads to the elimination of cells, without releasing harmful substances into the surrounding area (28). The disequilibrium between Bcl2 and Bax may cause mitochondrial permeability transition and aid cytochrome c in releasing from mitochondria to cytosol, resulting in the activation of caspase cascades (29,30). It may also be concluded that the increased Bax may induce cells to apoptosis and the decreased Bcl2 protein may reduce its anti-apoptosis effect, which could cause the cells to die. Did the changes of Bcl2 and Bax protein induce HepG2 cells to apoptosis? So, the expressions of the two kinds of proteins were detected by western blot. As shown in Fig. 9, the expression of Bax increased and Bcl2 decreased with the increasing dose which showed that the apoptotic effect of GLH on HepG2 cells was related to the regulations of Bcl2 and Bax proteins. However, the reasons inducing cells to apoptosis were complex and many factors such as cytochrome C, tumor necrosis factor (TNF), etc. could induce cells to apoptosis. So, the exact mechanism of inducing HepG2 cells to apoptosis requires further study, due to the fact that in this study, other apoptotic pathways were not covered.

CONCLUSION

In summary, there is a clear inhibitory effect of GLH on hepatoma in vivo and in vitro. The mechanism of the potent inhibitory effect of GLH on the tumor is a result of the decreased Bcl2 and increased Bax, which can induce cells to apoptosis. GLH is a promising potential antitumor agent for further drug development.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1 (442.9KB, docx)

(DOCX 442 kb)

ACKNOWLEDGMENTS

This work is supported by the National Natural Science Foundation of China (no. 81072552), Science and Technology Creative Foundation for Distinguished Young Scholars of Henan Province, China (no. 104100510016), and Henan Scientific and Technological Development Plan (no. 122102310286 and no. 122102310200).

Footnotes

Guangfan Hai and Chong Zhang are contributed equally to this work.

REFERENCES

  • 1.Sun J, Guo L, Zhuang W, Tang H. Study and advancement of glaucocalyxrabdosia herb used in drugs in phytochemistry and pharmacology in China. Chin Trad Herb Drug. 2002;33:S3–4. [Google Scholar]
  • 2.Arai T, Koyama Y, Suenaga T, Morita T. Antitumor activity of the components of Isodontrichocarpus and related plants. Antibiot. 1963;16:132–8. [PubMed] [Google Scholar]
  • 3.Sun HD, Xu YL, Jiang B. Diterpenoids from Isodon Species. Beijing: Science Press; 2001. [Google Scholar]
  • 4.Takeda Y, Otsuka H. Recent advances in the chemistry of diterpenoids from Rabdosia species. Stud Nat Prod Chem. 1995;15:111–85. doi: 10.1016/S1572-5995(06)80131-6. [DOI] [Google Scholar]
  • 5.Zhang Y, Sha D, Sha M, Yuan C. Determination of glaucocalyxin A in the leaves of Plectranthus japonicus (Burm.) Koidz. var. glaucocalyx (Maxim.) Koidz. by HPLC. Chin J Materia Med. 1991;16:679–704. [PubMed] [Google Scholar]
  • 6.Gao LW, Zhang J, Yang WH, Wang B, Wang JW. Glaucocalyxin A induces apoptosis in human leukemia HL-60 cells through mitochondria-mediated death pathway. Toxicol In Vitro. 2011;25:51–63. doi: 10.1016/j.tiv.2010.09.006. [DOI] [PubMed] [Google Scholar]
  • 7.Bin Z, Kun L. Inhibition by glaucocalyxin A of aggregation of rabbit platelets induced by ADP, arachidonic acid and platelet-activating factor, and inhibition of [3H]-PAF binding. Thromb Haemost. 1992;67:458–60. [PubMed] [Google Scholar]
  • 8.Chen ZJ, Li YS, Zhou JY, Wang ZT. Effect of glaucocalyxinA on level of Th1/Th2 type cytokines in mice. Chin J Mater Med. 2006;31:1257–60. [PubMed] [Google Scholar]
  • 9.Zhang DY, Li ZY, Shi LY, Wu XM, Hua WY. Advances in research on cytotoxinic activity of ent-Kaurane diterpenoids. Chin J Org Chem. 2008;11:1911–7. [Google Scholar]
  • 10.Hai G, Li S, Cui T, Bai S. Effect of glaucocalyxin B on cell cycle and apoptotic rates of AGZY-83A cells. Chin J Patho physiol. 2010;26:1420–2. [Google Scholar]
  • 11.Cui T, Zhang H, Hai G, Niu B, Liu J, Bai S. Effect of glaucocalyxin B on cell cycle and apoptosis of AGZY cells. Chin Recent Adv Ophth. 2011;31:711–3. [Google Scholar]
  • 12.Kim DS, Chang RG, Shen XY, Chen YP, Sun HD. Diterpenoids from rabdosia-japonica. Phytochemistry. 1992;31:677–9. [Google Scholar]
  • 13.Meng X, Meng XJ, Wang QG, Chen YZ. Diterpenoids from Iso-don japonica. Phytochemistry. 1989;28:1163–6. doi: 10.1016/0031-9422(89)80201-8. [DOI] [Google Scholar]
  • 14.Chen YZ, Li YZ, Yue JM. Diterpenoids from Rabdosiagaponica var. Glaucocalyx. J Nat Prod. 1989;52:886–7. doi: 10.1021/np50064a041. [DOI] [Google Scholar]
  • 15.Xiang ZB, Xu YX, Shen Y, Jin L, Wang HP, Chen HS. Two new diterpenoids from Rabdosia japonica var. glaucocalyx. Chin Chem Lett. 2008;19:852–4. doi: 10.1016/j.cclet.2008.05.012. [DOI] [Google Scholar]
  • 16.Liang H, Zhang Y, Hai G, Bai S, Yuan Y, Ye D, et al. Isolation, structural elucidation and cytotoxicity of three new ent-karuanediterpenoids from Isodon japonica var.glaucocalyx. Planta Med. 2012;78:589–96. doi: 10.1055/s-0031-1298265. [DOI] [PubMed] [Google Scholar]
  • 17.Fujita E, Nagao Y, Node M, Kaneko K, Nakazawa S, Kuroda H. Antitumor activity of the Isodon diterpenoids: structural requirements for the activity. Experimentia. 1976;32:203–6. doi: 10.1007/BF01937766. [DOI] [PubMed] [Google Scholar]
  • 18.Ge H, Wang YF, Xu J, et al. Anti-influenza agents from traditional Chinese medicine. Nat Prod Rep. 2010;27(12):1758–80. doi: 10.1039/c0np00005a. [DOI] [PubMed] [Google Scholar]
  • 19.Yao S, Peng N, Xu G. The protective effect of glaucocalyxinaonliver injury induced by CCL4. Hum JTCM Univ. 1988;8:46–8. [Google Scholar]
  • 20.Liu L, Ye L, Pan D, Jiang Y, Zhang T, Yang H. Effects of varglaucocalyx on c-fosgene expression during global myocardial ischemia-reperfusion in rats. J Chin Mater Med. 2003;28:358–61. [PubMed] [Google Scholar]
  • 21.Yang W, Zhang J, Gao L. Preliminary investigation on glaucocalyxin A induced cytotoxicity in K562 Cells. Chin J Exp Tradi Med. 2011;17:181–4. [Google Scholar]
  • 22.Liang Y. Anti-tumor studies of diterpenoid from Isodon japonica var. glaucocalyxon structure-function relationship and molecule mechanism .A cad Military Med Sci, 2007.
  • 23.Holzmayer TA, Hilsenbeck S, Von Hoff DD, et al. Clinical correlates of MDR1 (P-glycoprotein) gene expression in ovarian and small-cell lung carcinomas[J] J Natl Cancer Inst. 1992;84(19):1486–91. doi: 10.1093/jnci/84.19.1486. [DOI] [PubMed] [Google Scholar]
  • 24.Mackey JR, Martin M, Pienkowski T, et al. Adjuvant docetaxel, doxorubicin, and cyclophosphamide in node-positive breast cancer: 10-year follow-up of the phase 3 randomised BCIRG 001 trial. Lancet Oncol. 2013;14(1):72–80. doi: 10.1016/S1470-2045(12)70525-9. [DOI] [PubMed] [Google Scholar]
  • 25.Eichbaum M, Mayer C, Eickhoff R, et al. The PACOVAR-trial: a phase I/II study of pazopanib(GW786034) and cyclophosphamide in patients with platinum-resistant recurrent, pre-treated ovarian cancer. BMC Cancer. 2011;11(1):453. doi: 10.1186/1471-2407-11-453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Lin Y, Li S. Fundamental medical pharmacology. Changchun: Jilin Science and Technology Publishing House; 2004. [Google Scholar]
  • 27.Bai SP, Luo GS, Zhang XY, Liu W. An ent-kaurane diterpenoid from Isodon japonica var. Glaucocalyx. Acta Crystallogr Sect E Struct Rep. 2009;65:1898–906. doi: 10.1107/S1600536809027159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Kroemer G, Petit P, Zamzami N, Vayssiere JL, Mignotte B. The biochemistry of programmed cell death. FASEB J. 1995;9:1277–87. doi: 10.1096/fasebj.9.13.7557017. [DOI] [PubMed] [Google Scholar]
  • 29.Jiang XJ, Wang XD. Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-1. J Biol Chem. 2000;275:1199–203. doi: 10.1074/jbc.C000405200. [DOI] [PubMed] [Google Scholar]
  • 30.Chipuk JE, Bouchier-Hayes L, Green DR. Mitochondrial outer membrane permeabilization during apoptosis: the innocent bystander scenario. Cell Death Differ. 2006;13:1396–402. doi: 10.1038/sj.cdd.4401963. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ESM 1 (442.9KB, docx)

(DOCX 442 kb)

Articles from AAPS PharmSciTech are provided here courtesy of American Association of Pharmaceutical Scientists

RESOURCES