Cistanche tubulosa phenylethanoid glycosides induce apoptosis in H22 hepatocellular carcinoma cells through both extrinsic and intrinsic signaling pathways
Cistanche tubulosa (Schenk) R. Wight is a traditional Chinese medicine that parasitizes the roots of the Tamarix plant and has been used to treat male impotence, sterility, body weakness, and as a tonic. However, its antitumor effect on hepatocellular carcinoma is still elusive. Here, we investigated the antitumor effect of C. tubulosa phenylethanoid glycosides (CTPG) on H22 hepatocellular carcinoma cells both in vitro and in vivo and its mechanisms.
The morphology, viability, apoptosis, cell cycle and mitochondrial membrane potential (Δψm) of H22 cells were analyzed by inverted microscopy, MTT assay and flow cytometry, respectively. The expression and activation of proteins in apoptosis pathway were detected by Western blot. The in vivo antitumor effect was evaluated in tumor mouse model established using male Kunming mice.
CTPG treatment significantly suppressed H22 cell growth in a dose- and time-dependent manner, which was correlated with the increased apoptosis and cell cycle arrest at G0/G1 and G2/M phases. Moreover, the chromosomal condensation was observed in CTPG-treated H22 cells. CTPG treatment significantly increased Bax/Bcl-2 ratio, reduced Δψm and enhanced the release of cytochrome c. The levels of cleaved caspase-8 and caspase-9 in both extrinsic and intrinsic signaling pathways were significantly increased that sequentially activated caspase-7 and -3 to cleave PARP. Finally, CTPG inhibited the growth of H22 cells in mice and improved the survival rate of tumor mice.
These results suggested that CTPG suppressed H22 cell growth through both extrinsic and intrinsic apoptosis pathways.
KeywordsCistanche tubulosa Phenylethanoid glycosides Apoptosis Signaling pathway Tumor mouse model
BCL-2-associated X protein
B cell lymphoma 2
Cistanche tubulosa phenylethanoid glycosides
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
DNA repair enzyme of poly (ADP-ribose) polymerase
traditional Chinese medicine
mitochondrial membrane potential
Liver cancer ranked sixth for cancer incidence and fourth for cancer deaths worldwide. Moreover, it ranked fourth for cancer incidence and first for cancer mortality in countries with low sociodemographic index . In China, liver cancer is the third leading cause of cancer-related death in 2015 . More than 90% of primary liver cancers are hepatocellular carcinoma (HCC) in the world . Currently, hepatic resection is the main option for the therapy of HCC. However, less than 30% of patients with HCC met the criteria of curative hepatic resection and the over-all 5-year survival rate is still as low as 35-50% due to the high recurrence rate [4, 5]. The availability of treatment options for the patients with intermediate to advanced HCC is very limited. Sorafenib, a molecular targeted drug, has been approved by FDA as the first-line treatment for advanced HCC. However, sorafenib only prolongs about 3 months of survival and the response rate is less than 4% [6, 7]. It is urgent to develop new drugs or strategies against HCC.
Traditional Chinese medicine (TCM) alone or combined with other strategies has been used to treat HCC and shown the clinical benefits including prolonged survival time, improved life quality, reduced adverse reactions, and so on [8, 9]. Cistanche, a kind of TCM, has various biological functions, such as anti-oxidation, anti-inflammation, anti-aging and neuroprotection [10, 11]. Phenylethanoid glycosides have been considered the major active components of Cistanche, which have diverse activities including anti-oxidation, anti-inflammation, hepatoprotection and neuroprotection [12, 13, 14, 15]. Our group has reported that Cistanche tubulosa phenylethanoid glycosides (CTPG) could induce apoptosis in melanoma B16-F10 cells and inhibited the growth of tumor in mice . In this study, we measured the antitumor effect of CTPG on HCC H22 cells both in vitro and in vivo and investigated its mechanisms. We found that CTPG induced apoptosis in H22 cells through both extrinsic and intrinsic signaling pathways and suppressed the growth of H22 tumor in mice.
The mouse H22 hepatocellular carcinoma 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) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Gibco) at 37 °C in a humidified atmosphere of 5% CO2.
CTPG was purchased from Hetian Dichen Biotech Co., Ltd. (Hetian, Xinjiang, China) and the major compounds of CTPG were qualified and quantified by high performance liquid chromatography . Cell viability was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA) assay. H22 cells were inoculated into 96-well plates at a density of 2 × 104 cells in 100 μl medium per well and cultured at 37 °C. After 24 h, cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) or 0.3% DMSO (equal to that in 400 μg/ml CTPG) for 24, 48 and 72 h, respectively. After centrifugation at 1000 rpm for 7 min, supernatant was discarded and 100 μl of MTT solution (5 mg/ml in PBS) was added to each well. The plates were incubated at 37 °C for 4 h and 100 μl DMSO was added to dissolve the formed formazan crystals. The OD490 values were detected by a 96-well microplate reader (Bio-Rad Laboratories, CA, USA). The cell viability was calculated according to the formula: Cell viability (%) = (ODtreated/ODuntreated) × 100%.
Detection of apoptosis
H22 cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) or 0.3% DMSO for 24 h, and then stained with Annexin V-FITC/Propidium iodide (PI) Apoptosis Detection Kit (YEASEN, China) according to the manufacturer’s instructions. Samples were analyzed by flow cytometry (BD FACSCalibur, USA).
Detection of mitochondrial membrane potential
H22 cells were treated with different concentrations of CTPG (0, 200 and 400 μg/ml) for 24 h, and then stained with the membrane-permeable JC-1 dye (Beyotime,China) for 20 min at 37 °C. After washing twice with JC-1 buffer, samples were resuspended with 300 μl of JC-1 buffer and analyzed by flow cytometry (BD FACSCalibur, USA).
Analysis of cell cycle
H22 cells were inoculated in 60 mm culture dishes and treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) or 0.3% DMSO for 24 h. All cells were collected and washed twice with PBS. Cells were fixed in 70% ice-cold ethanol at − 20 °C for 2 h and washed twice with PBS, then re-suspended in 300 μl Propidium iodide/RNase staining buffer (BD Biosciences). After 10 min at room temperature, samples were collected by flow cytometry (BD FACSCalibur, USA) and cell cycle distribution was analyzed with the ModFit LT 3.0 software.
Hoechst 33,258 staining
The morphological changes of H22 cell nuclei were analyzed by membrane-permeable DNA-binding dye Hoechst 33,258 staining. H22 cells were seeded in 6-well plate at the concentration of 1 × 105 cells/well in 2 ml medium. After 60%~ 70% confluence, the cells were treated with CTPG (0, 100, 200, 300 and 400 μg/ml) for 24 h. The cells were collected and fixed with 4% ice-cold Paraformaldehyde at 4 °C for 10 min. After washing with PBS, cells were stained with Hoechst 33,258 (Beyotime, China) at 4 °C for 10 min. Samples were observed by inverted fluorescence microscope (Nikon Eclipse Ti-E, Japan).
Anti-caspase-3, anti-cleaved caspase-3, Anti-Bcl-2 and anti-Bax were purchased from Beyotime Biotech Co., Ltd. (Shanghai, China). Anti-caspase-7, anti-cleaved-caspase-7, anti-caspase-8, anti-cleaved-caspase-8, anti-caspase-9, anti-cleaved-caspase-9, anti-PARP, anti-cleaved PARP, anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from Cell Signaling Technology. Anti-β-actin was purchased from Beijing ComWin Biotech Co., Ltd. (Beijing, China).
H22 cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) or 0.3% DMSO for 24 h. Cells were collected and lysed with the Cell Lysis Solution RIPA (Beijing ComWin Biotech Co., Ltd) for 30 min on ice. Samples were spun down (12,000 g for 15 min at 4 °C) to collect the supernatants and protein concentrations were measured by BCA Kit (Thermo Fisher Scientific, USA). Equal amount of protein in each sample was isolated by 12% SDS-PAGE and transferred to PVDF membranes (Biosharp, China). After blocking with TBST buffer contained 5% nonfat milk, membranes were incubated with corresponding primary antibodies and secondary antibodies conjugated to horseradish peroxidase (HRP), respectively. After washing with TBST, the target proteins were detected by ECL assay kit (Beyotime, China).
Animals and ethics statement
6-8 weeks old male Kunming mice were purchased from Animal Laboratory Center, Xinjiang Medical University (Urumqi, Xinjiang, China). Mice were kept in a standard temperature-controlled, light-cycled animal facility of Xinjiang University. All animal studies were carried out according to the guidelines of the Animal Care and Use Committee of Xinjiang University. The protocol was approved by the Committee on the Ethics of Animal Experiments of Xinjiang Key Laboratory of Biological Resources and Genetic Engineering (BRGE-AE001), Xinjiang University.
Tumor mouse study
For induction of tumor mouse model, male Kunming mice were subcutaneously injected with 1 × 106 H22 cells in 100 μl PBS into the right flank. After 3 days, mice were randomly divided into 3 groups (7 mice/group). Control group was injected with 0.1 ml DMSO subcutaneously around tumor. CTPG-200 and CTPG-400 groups were subcutaneously injected with 200 or 400 mg/kg CTPG in 0.1 ml DMSO around tumor. Mice were treated every 2 days for up to 21 days. Tumor sizes were measured using calipers up to 25 days and tumor volume was calculated according to the formula: tumor volume (mm3) = (length×width2)/2. After 25 days, survival of tumor mice was monitored every day until the end of this study.
Statistical significance was calculated by one-way analysis of variance among the treatment and control groups. All data were expressed as the mean ± standard deviation (S.D.). p < 0.05 was considered statistically significant.
CTPG reduced the viability of H22 cells in vitro
CTPG induced apoptosis in H22 cells
CTPG induces chromosomal condensation and cell cycle arrest in H22 cells
CTPG decreased mitochondrial membrane potential and increased the release of cytochrome c
CTPG activated caspase pathway and prevented DNA repair
CTPG suppresses the growth of H22 HCC in vivo and improves the survival rate of tumor mice
TCM has been used to treat various diseases including cancers for a long history. It has been reported that TCM can induce apoptosis in different types of tumor cells through both extrinsic (death receptor-mediated) and intrinsic (mitochondria-dependent) signaling pathways to exert antitumor effects [22, 23, 24, 25]. The two pathways can activate caspase-8 and -9, respectively [24, 26]. Here, we found that CTPG significantly suppressed H22 cell growth through induction of apoptosis and cell cycle arrest. The levels of cleaved caspase-8 and -9 were significantly up-regulated by CTPG treatment, suggesting that both extrinsic and intrinsic signaling pathways were involved in the induction of apoptosis. Our previous study showed that CTPG induced apoptosis in melanoma B16-F10 cells by mitochondria-dependent pathway that increased the level of cleaved caspase-9 but not caspase-8 . CTPG might activate distinct signaling pathways in different types of tumor cells.
Mitochondrial membrane integrity is tightly regulated by the members of the BCL-2 protein family including Bax and Bcl-2 [27, 28]. The ratio of Bax to Bcl-2 plays a critical role in mitochondria-dependent apoptosis pathway . In H22 cells treated by CTPG, Bax/Bcl-2 ratio was significantly up-regulated, which might cause the reduction of Δψm and the release of cytochrome c observed in this study. Consequently, pro-caspase-9 was cleaved and activated. Finally, the initiators of active caspase-8 and -9 activated the executioner of caspase-3 to cleave PARP to prevent DNA repair. Taken together, these results suggested that CTPG induced apoptosis in H22 cells through both extrinsic and intrinsic signaling pathways.
In tumor mouse model, CTPG significantly suppressed the growth of H22 HCC and greatly improved the survival of tumor mice. Interestingly, CTPG dose-dependently promoted the proliferation of splenocytes from Kunming mice, which is consistent with our previous study . These results suggested that CTPG might suppress the growth of H22 HCC in mice through both direct antitumor effect and indirect immune enhancement.
CTPG suppressed the growth of H22 cells both in vitro and in vivo and induced apoptosis in H22 cells through both extrinsic and intrinsic signaling pathways. These data indicated that CTPG might be a potential candidate for the treatment of HCC.
This work was supported by the High Level Talent Introduction Project of Xinjiang Uygur Autonomous Region to J.L., the Chinese National Natural Science Foundation Grant (31460241) to J.L. and the Doctoral Start-up Fund of Xinjiang University (BS160261 to X.W. and BS150236 to Y.L.).
Availability of data and materials
The raw data for this study are available upon reasonable request to the corresponding author.
JL and JL designed the experiments. PY, JL, AA and YY performed the experiments. LX, XW and YL analyzed the data. PY, JL and JL wrote the manuscript. All authors contributed and approved the final manuscript.
The animal study was approved by the Committee on the Ethics of Animal Experiments of Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Xinjiang University.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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