Part 1: Cistanche Tubulosa Phenylethanoid Glycosides Induce Apoptosis Of Hepatocellular Carcinoma Cells And Enhance Antitumor Effect
Mar 25, 2022
Contact: ali.ma@wecistanche.com
Pengfei Yuan, BSc1, Changshuang Fu, MSc1, Yi Yang, PhD1, Aipire Adila, PhD1, Fangfang Zhou, MSc1, Xianxian Wei, MSc1, Weilan Wang, PhD1, Jie Lv, PhD1, Yijie Li, PhD1, Lijie Xia, PhD1, and Jinyao Li, PhD1
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 direct antitumor effect and indirect immunoenhancement effect, and improved the antitumor efficacy of cisplatin.
Keywords Cistanche tubulosa phenylethanoid glycosides, apoptosis, MAPK pathway, mitochondria-dependent pathway, cisplatin, immunoenhancement
Cistanche has the anti-tumor effect.
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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.
Cistanche enhances immunity.
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.
echinacoside
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 12000rpm 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 1hour 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 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).
cistanche extract
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) were 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 was 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).
Cistanche enhances the anti-tumor effect and improves immunity.
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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.
