Cistanche tubulosa phenylethanoid glycosides induce apoptosis in H22 hepatocellular carcinoma cells through both extrinsic and intrinsic signaling pathways

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 [16]. 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 (%) = (OD_treated/OD_untreated) × 100%.

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).

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).

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.

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).

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.

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.

In order to explore antitumor effect of CTPG on HCC, H22 cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) in vitro. After 24 h, the morphology of H22 cells was observed using inverted microscope. We found that the morphology of H22 cells was dramatically changed by CTPG treatment. With increasing CTPG concentration, cells became small and round and cell number was also greatly reduced (Fig. 1a). MTT assay was used to analyze the viability of H22 cells after CTPG treatment for 24, 48 and 72 h, respectively. CTPG significantly reduced H22 cell viability in a dose-dependent and time-dependent manner (Fig. 1b). CTPG at 300 μg/ml arrived at the best inhibitory rate (Fig. 1c). The values of IC₅₀ of CTPG for H22 cells are 236 μg/ml at 24 h and 169.8 μg/ml at 48 h.

To investigate whether the decreased viability of H22 cells is mediated by the induction of apoptosis, H22 cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) for 24 h and stained with PI and Annexin V. The flow cytometry results showed that CTPG significantly induced apoptosis of H22 cells (including early and late apoptosis) in a dose-dependent manner (Fig. 2a). Although the high dose of CTPG also significantly increased necrosis of H22 cells, necrosis plays a minor role in the inhibition of H22 cell growth due to its lower proportion (8.3%) compared to that of apoptosis (52.6%). Further, total proteins of H22 cells were isolated after CTPG treatment and the expressions of anti-apoptotic B cell lymphoma 2 (Bcl-2) and pro-apoptotic BCL-2-associated X protein (Bax) were detected by Western blot. Grayscale scanning data showed that the expression levels of Bax and Bcl-2 were increased and decreased, respectively. The Bax/Bcl-2 ratio was significantly increased (Fig. 2b). These results suggest that CTPG induces apoptosis in H22 cells.

It has reported that DNA damage and cell cycle arrest induced by drugs can inhibit tumor cell growth and cause apoptosis in tumor cells [17, 18]. To detect the morphology of nuclei in H22 cells after CTPG treatment for 24 h, H22 cells were stained by Hoechst 33,342 and observed using inverted fluorescent microscopy. CTPG-treated cells showed a dose-dependent increase of brightly condensed chromatin of nuclei, while the untreated cells showed the homogeneously stained nuclei (Fig. 3a). Cell cycle distribution in H22 cells was further analyzed by PI staining after CTPG treatment for 24 h. As shown in Fig. 3b, CTPG treatment significantly increased the proportion of G0/G1- and G2/M-phase cells and significantly decreased the proportion of S-phase cells, suggesting that CTPG induced G0/G1 and G2/M-phase arrest in H22 cells. The high dose of CTPG also significantly increased the proportion of sub G1 cells.

Mitochondrial-dependent pathway plays an important role in the induction of apoptosis [19, 20]. The changes of mitochondrial membrane potential (Δψm) can be monitored by JC-1 staining due to JC-1 aggregate (red fluorescence) can disintegrate into monomer (green fluorescence) with the reduction of Δψm [21]. After CTPG treatment for 24 h, H22 cells were stained by JC-1 dye. The flow cytometry data showed that the red fluorescence in FL-2 channel and green fluorescence in FL-1 channel were significantly decreased and increased upon CTPG treatment. The proportion of PE⁻FITC⁺ cells were significantly increased (Fig. 4a), suggesting that CTPG reduced the Δψm in H22 cells. This is consistent with the increased Bax/Bcl-2 ratio. Consequently, we observed the release of cytochrome c was significantly increased upon CTPG treatment (Fig. 4b). These results indicated that CTPG might partially induce apoptosis in H22 cells via mitochondrial-dependent (intrinsic) pathway.

Next, the activation of caspase induced by CTPG via both extrinsic and intrinsic signaling pathways was analyzed. After CTPG treatment for 24 h, total proteins were isolated from H22 cells and the levels of pro- and cleaved-caspases were detected by Western blot. Compared with the untreated or the DMSO control, CTPG treatment significantly up-regulated not only the level of cleaved caspase-8 (extrinsic pathway) but also the level of cleaved caspase-9 (intrinsic pathway) (Fig. 5). Sequentially, activated caspase-8 and -9 cleaved the downstream pro-caspase-3 and -7 that was observed in Fig. 5. Activated caspase-3 cleaved the DNA repair enzyme of poly (ADP-ribose) polymerase (PARP) to prevent DNA repair and accumulate DNA damage as observed in Fig. 3a. These results indicated that CTPG induced apoptosis in H22 cells through both extrinsic and intrinsic signaling pathways.

Finally, the antitumor effect of CTPG on HCC was evaluated in tumor mouse model, which was established by subcutaneous injection of H22 cells. After 3 days of H22 cell injection, tumor mice were treated with CTPG for 8 times. The body weight of mice and tumor sizes were monitored at indicated time points. As shown in Fig. 6a, the body weight of mice in each group has no significant difference, suggesting that the selected doses of CTPG have no obvious side effect. Interestingly, the tumor growth in mice treated with both 200 mg/kg and 400 mg/kg of CTPG was significantly inhibited (Fig. 6b). Moreover, the two doses of CTPG treatment greatly improved the survival of tumor mice (3/7, 3/7) compared with control group (0/7) at the end of the experiment (Fig. 6b). We also found that CTPG significantly enhanced the proliferation of splenocytes isolated from male Kunming mice in a dose-dependent manner (Fig. 6c), suggesting that CTPG has immunostimulatory effect.