Part Ⅰ：Cistanche Tubulosa Phenylethanoid Glycosides Induce Apoptosis Of Hepatocellular Carcinoma Cells By Mitochondria-Dependent And MAPK Pathways And Enhance Antitumor Effect Through Combination With Cisplatin

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Pengfei Yuan, Changshuang Fu, Yi Yang, Aipire Adila, Fangfang Zhou, Xianxian Wei, Weilan Zhang, Jie Lv, Yijie Li, Lijie Xia, Jinyao Li

Abstract

Cistanche tubulosa is a type of Chinese herbal medicine and exerts various biological functions. Previous studies have been demonstrated that Cistanche tubulosa phenylethanoid glycosides (CTPG) exhibit antitumor effects on a variety of tumor cells. However, the antitumor effects of CTPG on HepG2 and BEL-7404 hepatocellular carcinoma (HCC) cells are still elusive. Our study showed that CTPG significantly inhibited the growth of HepG2 and BEL-7404 cells through the induction of cell cycle arrest and apoptosis, which was associated with the activation of MAPK pathways characterized by the up-regulated phosphorylation of p38, JNK, and ERK1/2 and mitochondrial-dependent pathway characterized by the reduction of mitochondrial membrane potential. The release of cytochrome c and the cleavage of caspase-3, -7, -9, and PARP were subsequently increased by CTPG treatment. Moreover, CTPG significantly suppressed the migration of HepG2 by reducing the levels of matrix metalloproteinase-2 and vascular endothelial growth factors. Interestingly, CTPG not only enhanced the proliferation of splenocytes but also reduced the apoptosis of splenocytes induced by cisplatin. In the H22 tumor mouse model, CTPG combined with cisplatin further inhibited the growth of H22 cells and reduced the side effects of cisplatin. Taken together, CTPG inhibited the growth of HCC through the direct antitumor effect and indirect immunoenhancement effect and improved the antitumor efficacy of cisplatin.

Keywords

Cistanche tubulosa phenylethanoid glycosides, apoptosis, MAPK pathway, mitochondrial-dependent pathway, cisplatin, immunoenhancement.

Introduction

Liver cancer was predicted to be the sixth most commonly diagnosed cancer and the fourth leading cause of cancer death worldwide in 2018, with about 841 000 new cases and 782000 deaths annually in South-Eastern Asia (Mongolia, Cambodia, and Vietnam).1 In China, liver cancer was the third leading cause of cancer-related death in 2015.2 More than 90% of primary liver cancers are hepatocellular carcinoma (HCC) throughout the world. Currently, hepatectomy, liver transplantation, and percutaneous ablation are the main methods for the treatment of HCC. Unfortunately, these treatments are effective for 30% of patients diagnosed with early liver cancer, while patients diagnosed with advanced liver cancer (accounting for 40%) have to rely on palliative treatment to extend their survival time.3,4 Sorafenib in combination with hepatic arterial chemoembolism is an important and common palliative therapy for most patients with advanced HCC in the Asia-Pacific region.5 Sorafenib, approved by FDA in 2006 for the treatment of advanced liver cancer, is a multiple kinase inhibitor that inhibits tumor cell proliferation and vascular production and promotes tumor cell apoptosis. Sorafenib significantly extends the patient’s survival time by 3 to 5months but has serious side effects and is closely related to the occurrence of drug resistance.6 Therefore, it is urgent to develop new drugs or strategies for HCC.

Traditional Chinese medicine (TCM) alone or in combination with other strategies has been used to treat HCC and has shown clinical benefits including extended survival time, improved life quality, reduced adverse reactions, and so on.7,8 Cistanche is a TCM containing phenylethanoid glycosides (PhGs), iridoids, lignin, and polysaccharides which has various biological functions, such as anti-oxidation, anti-inflammation, anti-aging, memory enhancement, and immune enhancement functions.9 PhGs have been considered as the major active components of Cistanche, and have multiple functions including anti-oxidation, anti-inflammation, anti-apoptosis, hepatoprotection, and neuroprotection.10-12 Our group had reported that Cistanche tubulosa phenylethanoid glycosides (CTPG) could inhibit the growth of melanoma B16-F10 cells, esophageal carcinoma Eca-109 cells, and HCC H22 cells through extrinsic or intrinsic signaling pathways in vitro or in vivo; in addition, CTPG had an immunostimulatory effect.13-15

In this study, we investigated the antitumor effect and mechanism of CTPG on HepG2 and BEL-7404 cells in vitro, analyzed the immunomodulatory function of CTPG in vitro and in vivo, and further evaluated the therapeutic effect of CTPG combined with the chemotherapy drug cisplatin on HCC H22 tumor mice in vivo. We found that CTPG could inhibit the growth of HepG2 and BEL-7404 cells through mitochondria-dependent apoptosis and the MAPK signaling pathway. CTPG significantly inhibited the migration of HepG2 cells by reducing the levels of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF). In addition, CTPG promoted the proliferation and activation of immune cells and enhanced the immunity of mice. Importantly, CTPG combined with cisplatin can further inhibit the growth of H22 cells in vivo and reduced the side effects of cisplatin.

Materials and Methods

Animals

About 6 to 8week old male BALB/c, Kunming mice, and female C57BL/6 mice were purchased from the Animal Laboratory Center, Xinjiang Medical University (Urumqi, Xinjiang, China) and housed in an animal facility of Xinjiang University with the room temperature (RT) of 25±3°C, and a 12/12 hours light-dark period.

Cell Lines and Cell Culture

The murine H22 cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, Hubei, China), and human HCC HepG2 and BEL-7404 cells were obtained from the Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Xinjiang University (Urumqi, Xinjiang, China) and cultured in RPMI 1640 medium (Gibco, USA) or Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) containing 10% heat-inactivated fetal bovine serum (MRC, China), 1% L-glutamine (100mM), 100U/mL penicillin, and 100μg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2.

High-Performance Liquid Chromatography (HPLC)

The major compounds of CTPG (Upbio Tech Co., Ltd., Shanghai, China) were qualified and quantified by HPLC as previously reported.13 A ZORBAX SB-C18 Column (250×4.6mm; 5μm) was used and the mobile phase consisted of 0.2% formic acid solution and methanol with a gradient from 23% to 31%. A total of 10μL sample was injected and detected at 330 nm. The echinacoside and acteoside standards (Yuanye, Shanghai, China) were used to analyze the components of CTPG.

Analysis of Cell Viability

The antitumor effects of CTPG on HepG2 and BEL-7404 cells were assessed using MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide). HepG2 and BEL-7404 cells were plated into 96-well plates (5 × 104 cells/well) and treated with 0, 200, 400, and 600 μg/mL CTPG for 24 and 48 hours, respectively, after 24 hours of incubation at 37°C. About 35 μg/mL cisplatin (Yuanye) was used as a positive control. Then 100 μL MTT (0.5mg/mL, diluted by medium without FBS medium) was added to each well and cultured for 3 hours at 37°C and 5% CO2. After incubation, the plates were centrifuged at 1200 rpm for 7minutes, the medium was removed and 200 μg/mL DMSO was added to each well to dissolve the formed formazan crystals. The OD490 values were measured by a 96-well microplate reader (Bio-Rad Laboratories, CA, USA). To evaluate the effects of CTPG on splenocytes, the cells were isolated from C57BL/6 mice and plated into 96-well plates at a density of 1 × 105 cells/well. Splenocytes were treated with 0, 200, 400, and 600 μg/mL for 24, 48, and 72 hours, respectively. The cell viability was calculated as the followed formula: Cell viability (%) = (ODtreated/ODuntreated) × 100%.

Detection of Ki-67

Detection of Ki-67 was done according to our previous study.16 In brief, BEL-7404 cells were treated with different concentrations (0, 200, 400, and 600μg/mL) of CTPG or cisplatin (35μg/mL). After 24 hours, cells were harvested and washed with PBS, then fixed and permeabilized with Foxp3 Staining Buffer Set (eBioscience, USA) according to the manufacturer’s instructions. Intracellular staining was performed using FITC conjugated Ki-67 antibody (BD Biosciences, San Jose, CA, USA) for 15minutes at RT. The samples were analyzed by flow cytometry (BD FACSCalibur, CA, USA).

Analysis of Cell Apoptosis and Cell Cycle

HepG2 and BEL-7404 cells were seeded at a density of 2.5×105 cells/dish and incubated at 37°C overnight. Cells were trypsinized and harvested by centrifugation after treatment with various concentrations of CTPG or pretreated with caspase inhibitor (Z-VAD-FMK) or caspase-3 inhibitor (Ac-DEVD-CHO) (Beyotime, China) for 2 hours before CTPG treatment. After 24hours, apoptosis was detected by flow cytometry. In brief, the collected cells were washed with cold PBS (Gibco) and resuspended in annexin-binding buffer with 2.5μL Annexin V-FITC and 5μL PI-PE Staining Solution (Solarbio, Beijing, China), and then cells were incubated at RT in the dark for 15minutes. For analysis of the cell cycle distribution, cells were harvested after CTPG treatment and fixed in cold 70% ethanol at 4°C for 30minutes. Cells were stained with PI (BD Biosciences) in the dark for 30minutes. Samples were analyzed by flow cytometer (BD FACSCalibur). Expression levels of cell apoptosis and cycle-related proteins were detected by Western blot.

Western Blot

Western blot was done according to our previous study.17 HCC were treated with CTPG for 24hours. After washing with ice-cold PBS twice, all adherent and floating cells were collected and lysed in RIPA Lysis Buffer (Beijing ComWin Biotech Co., Ltd) for 20minutes on ice. After centrifugation at 12000 rpm 4°C for 10minutes, protein concentration was determined using a Bicinchoninic Acid Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The same concentration of proteins was separated by 12% SDS-PAGE and transferred to PVDF membranes. After washing with PBST buffer (PBS with 0.05% Tween-20), the membranes were blocked with 5% non-fat milk at 37°C for 1 hour and then incubated with the primary antibodies (Cell Signaling Technology, MA, USA) at proper dilutions overnight at 4°C. After washing 3 times with PBST, the membrane was incubated with the corresponding HRP-conjugated secondary antibodies (eBioscience) for 2hours at 37°C. The target proteins were detected using an ECL assay kit (Beyotime). Grayscale scanning data were obtained by Image J.

Analysis of Mitochondrial Membrane Potential (Δψm)

Δψm was determined by membrane-permeable JC-1 dye (Beyotime). Briefly, HepG2 and BEL-7404 cells were treated with different concentrations of CTPG (0, 200, 400, and 600μg/mL) for 24 hours. All cells were collected and washed with JC-1 washing buffer. Cells were stained with the JC-1 fluorescent probe according to the manufacturer’s instruction for 20minutes at RT. After washing with PBS twice, all samples were analyzed by flow cytometry (BD FACSCalibur).

Hoechst 33342 Staining

Hoechst 33342 staining was done according to our previous study.16 The morphological changes of nuclei were examined using membrane-permeable DNA-binding dye Hoechst 33342 (Beyotime). Briefly, the cells were inoculated in a 6-well plate at the concentration of 1×105 cells/well in a 2mL medium. When reaching 70% to 80% confluence, the cells were treated with 200, 400, and 600μg/mL of CTPG or cisplatin for 24 hours. The cells were washed with PBS and fixed with 4% ice-cold paraformaldehyde at 4°C for 10minutes. After washing with PBS, cells were stained with Hoechst 33342 at 4°C for 10minutes. Samples were observed by fluorescence inverted microscope (Nikon Eclipse Ti-E, Japan).

Migration Assay

HCC cell migration was detected through the wound healing assay. In brief, HepG2 cells (2×104 /well) were seeded in a 24-well plate. After reaching 80% confluence, the center of each well was scratched once with a 20μL pipette tip. After washing with PBS, cells were treated with cisplatin (35μg/mL) or different concentrations (0, 200, 400, and 600μg/mL) of CTPG at 37°C. After 24hours, images of each sample were taken under a microscope (Nikon Eclipse Ti-E). The average distances of cell migration were analyzed by Image J. The percentage of wound healing was calculated by the equation: wound healing (%)=(1−scratch area at indicated time point/scratch area at 0hours)×100%. Expression levels of cell migration-related proteins MMP-2 and VEGF were detected by Western blot.

Proliferation and Apoptosis of Splenocytes

For proliferation analysis, splenocytes were isolated from 3 C57BL/6 mice and stained with CFSE (eBioscience). CFSE labeled cells were inoculated into 24-well plates at a density of 2 × 106 cells in 1 mL medium per well and treated with different concentrations of CTPG (200, 400, and 600 μg/mL) or combined with 35 μg/mL cisplatin for 72 hours. Flow cytometry analysis was performed after staining with CD3-APC and CD19-PE (BD Biosciences). For apoptosis analysis, splenocytes were inoculated into 24-well plates at a density of 2 × 106 cells in 1 mL medium per well and treated with different concentrations of CTPG (200, 400, and 600 μg/mL) or combined with cisplatin for 24 hours. Flow cytometry analysis was performed after staining with 2.5 μL Annexin V-FITC and 5 μL PI-PE Solution.

Safety Evaluation and Immunostimulatory Activities of CTPG In Vivo

For evaluating the in vivo immunostimulatory activities of CTPG, 6 to 8 weeks male Kunming mice were randomly divided into 7 groups (5 mice/group). Subcutaneous injection (s.c., 200, 400 mg/kg), intraperitoneal injection (i.p., 200, 400 mg/kg), and intragastric administration (i.g., 200, 400 mg/kg) was applied every 2 days for a total of 7 times, while the control group did not receive any treatment. The mice have weighed every other day and the status of the mice was observed every day. After CTPG treatment, the organs of the mice were separated and weighed. The organ indexes were calculated according to the formula: organ index = organ weight (mg)/body weight (g). The splenocytes were collected, counted, and stained with anti-CD3-APC, anti-CD19-PE, anti-CD49bFITC or anti-CD4-APC, anti-CD44-PE, anti-CD8-FITC (BD Biosciences). Samples were detected by flow cytometry (BD FACSCalibur).

Antitumor Efficacy of CTPG Combined with Cisplatin in H22 Tumor-Bearing Mice

About 6 to 8 week-old male BALB/c mice were subcutaneously injected with H22 cells (1.0 × 106 per mouse in 100 μL PBS). When the tumor volumes reached approximately 60mm3, tumor-bearing mice were randomly divided into 4 groups (6 mice/group) and treated with cisplatin (4mg/kg), CTPG (400mg/kg), cisplatin (4mg/kg) plus CTPG (400mg/kg), or without treatment (control group), respectively. Tumor mice were intraperitoneally injected with CTPG on the 5th, 7th, 9th, 11th, and 13th days, respectively. Cisplatin was intravenously injected on the 7th and 11th days. The tumor volume and body weight were measured every other day. Tumor volume was calculated as the following: Tumor volume (V) = a × b2 /2, in which a and b represent the longest and shortest diameter of a tumor measured by vernier caliper, respectively. On the 20th day, organs and tumors were isolated and weighted. The splenocytes were collected, counted, and stained with anti-CD3-APC and anti-CD19-PE or anti-CD4-FITC and anti-CD8-APC or anti-CD11b-PE and anti-Gr-1-APC or anti-CD4-FITC, anti-CD25-APC, and anti-Foxp3-PE (BD Biosciences). Subsequently, the frequencies and the numbers of the cells were analyzed by flow cytometry (BD FACSCalibur).

Statistical Analysis

All data were expressed as mean±standard error of the mean (SEM). Statistical analysis was conducted using one/ two-way analysis of variance (ANOVA) using Prism5.0 software. P<.05 was considered statistically significant.

Results

CTPG Inhibited the Proliferation of HCC Cells In Vitro

The components of CTPG were qualified and quantified by HPLC using echinacoside and acteoside standards (Supplemental Figure 1A), which were the main components of phenylethanoid glycosides from Cistanche. 18 According to the peak retention times and the peak areas, CTPG contained 28% echinacoside and 9.9% acteoside. Besides, the content of polysaccharides in CTPG was 34.8% by the phenol-sulfuric acid method.19 The MTT assay showed that CTPG significantly reduced the viability of BEL-7404 and HepG2 cells in a dose- and time-dependent manner (Figure 1A). Consistently, the proliferation of BEL-7404 cells was significantly inhibited by CTPG treatment, which was analyzed by Ki-67 staining (Figure 1B). The effect of CTPG on the proliferation of splenocytes in vitro was also analyzed by MTT assay. We observed that CTPG enhanced the proliferation of splenocytes in a dose-dependent manner (Figure 1C).

The mitogen-activated protein kinase (MAPK) signaling pathway plays a pivotal role in the survival, differentiation, and drug resistance of human cancer cells.20 In order to investigate whether the MAPK signaling pathway was involved in the inhibitory effect of CTPG on the proliferation of HCC cells, phosphorylation levels of various proteins from the MAPK signal pathway were analyzed in HepG2 cells after treatment with CTPG at different concentrations and different time points. The phosphorylation of JNK (P-JNK) and p38 (P-p38) was dose-dependently enhanced by CTPG treatment. The phosphorylation of ERK (P-ERK) was down-regulated by 200 and 400μg/mL CTPG treatment, while P-ERK was up-regulated under 600μg/mL CTPG treatment (Figure 1D). Moreover, the levels of P-JNK, P-p38, and P-ERK were up-regulated in a time-dependent way (Figure 1D). These results suggested that CTPG might inhibit the proliferation of HCC cells through the MAPK signaling pathway

CTPG Induced HCC Cell Cycle Arrest at S Phase

We further analyzed whether CTPG inhibited HCC cell proliferation through induction of cell cycle arrest. After treatment with CTPG, an accumulation of BEL-7404 cells at the S-phase was observed in a dose-dependent manner. Similarly, CTPG also induced HepG2 cell cycle arrest at S-phase and the frequencies increased from 40.66% in the control group to 61.90% in the 600μg/mL CTPG treated group (Figure 2A). Cyclins and cyclin-dependent kinases (CDKs) play important roles in cell division control and development,21 of which were associated with the G2/M phase.22 The expression level of Cyclin D1 was dose-dependently decreased by CTPG treatment but promoted G1 to S phase progression. The expression levels of Cyclin B1, CDK1, and CDK2 were also reduced; these proteins were associated with the G2/M phase (Figure 2B). These results suggested that CTPG suppressed HCC cell proliferation by inducing cell cycle arrest.

CTPG Activated Mitochondria-Dependent Apoptosis Pathway in HCC Cells

We also detected whether CTPG triggered apoptosis in HCC cells and found that CTPG induced apoptosis in HepG2 and BEL-7404 cells in a dose-dependent manner (Figure 3A). In addition, the expression levels of Bax and Bcl-2 were increased and decreased by CTPG treatment in a dose-dependent manner, respectively (Figure 3B). The apoptosis of HepG2 cells was further determined by Hoechst 33342 staining after treatment with CTPG for 24 hours. The nuclear morphology was observed by an inverted fluorescence microscope. As shown in Figure 3C, the control HepG2 cells were homogeneously stained while HepG2 cells treated with CTPG showed chromatin condensation and fragmentation in a dose-dependent manner, which was similar to HepG2 cells treated with cisplatin. These results indicated that CTPG induced apoptosis of HCC cells.

The integrity of the outer mitochondrial membrane is strictly regulated by the Bcl-2 family and the reduction of Δψm promotes the release of cytochrome c that activates the caspase cascade to induce apoptosis.23,24 When Δψm decreases, the JC-1 polymer (red fluorescence) decomposes into monomers (green fluorescence).25 Therefore, the Δψm of HCC cells was detected by JC-1 staining after CTPG treatment for 24hours. As shown in Figure 4A, the green fluorescence intensity of the FL-1 channel was dose-dependently increased by CTPG treatment while the red fluorescence intensity of the FL-2 channel was dose-dependently decreased in both BEL-7404 and HepG2 cells. Inverted fluorescence microscope observation exhibited a similar result (Figure 4B), indicating that CTPG reduces the Δψm of HCC cells. Subsequently, we observed that levels of cytochrome c were increased in both BEL-7404 and HepG2 cells after CTPG treatment (Figure 4C), which further confirmed the reduction of Δψm in CTPG treated HCC cells.

The release of cytochrome c can activate the initiator caspase-9 to induce apoptosis.26 The results of the Western blot assay suggested that levels of cleaved caspase-3, -7, and-9 were increased while the level of cleaved caspase-8 was not changed (Figure 5). The activated caspase-3 can cleave the DNA repair enzyme of poly (ADP-ribose) polymerase (PARP) to prevent DNA repair and accumulate DNA damage.27 We also observed up-regulated levels of cleaved PARP. The role of the caspase cascade in HCC cell apoptosis induced by CTPG was further determined by using caspase inhibitor (Z-VAD-FMK) and caspase-3 inhibitor (Ac-DEVD-CHO) respectively. Z-VAD-FMK significantly reversed the apoptosis of BEL-7404 and HepG2 cells induced by CTPG (Supplemental Figure 1B). Similarly, Ac-DEVD-CHO also significantly reversed the apoptosis of HepG2 cells induced by CTPG (Supplemental Figure 1C). The results demonstrated that CTPG induced apoptosis of HCC cells by a mitochondrial-dependent pathway.

CTPG Suppressed HCC Cell Migration In Vitro

Cancer cell migration is considered one of the critical processes in tumor metastasis. To determine whether CTPG affects HCC cell migration, HepG2 cell motility was evaluated by the wound-healing assay. As shown in Figure 6A and B, HepG2 cell migration was significantly inhibited by CTPG treatment in a dose-dependent manner. MMP family and VEGF play critical roles in the migration of tumor cells.28 After CTPG treatment for 24 hours, the levels of MMP-2 and VEGF were significantly decreased (Figure 6C), suggesting that CTPG might suppress the invasion and metastasis of HCC.

CTPG Enhanced the Immunity of Mice

It has been reported that Cistanche deserticola polysaccharides have immunomodulatory functions including promoting lymphocyte proliferation and activating macrophages.29 T cells and B cells are the main lymphocytes, which mediate cellular and humoral immune responses, respectively. CTPG contained 34.8% polysaccharide content. Therefore, we examined the effects of CTPG on the proliferation of T cells and B cells. As shown in Supplemental Figure 2A, CTPG significantly increased the proliferation of T cells and B cells in a dose-dependent manner, even in the presence of cisplatin. Interestingly, CTPG significantly inhibited the apoptosis of splenocytes induced by cisplatin (Supplemental Figure 2B), indicating that CTPG could ameliorate the side effects of cisplatin on the immune system of mice.

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