Part 2: Cistanche Tubulosa Phenylethanoid Glycosides Induce Apoptosis Of Hepatocellular Carcinoma Cells And Enhance Antitumor Effect

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

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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).

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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.

Figure 1. The effect of CTPG on the proliferation of HCC cells and splenocytes.

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 mitochondria-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.

We further evaluated the immunostimulatory effect of CTPG on mice. After i.p. injection, s.c. injection and i.g. administration of CTPG (200 and 400mg/kg), there was no significant change in the state of mice compared with the untreated group. The bodyweight of mice also had no significant difference among CTPG-treated groups and untreated groups (Figure 7A). The heart, liver, spleen, lung, kidney, and thymus indexes of mice were similar among all groups except the spleen indexes of mice in the 400mg/ kg CTPG i.p. injection group (Table 1). The results suggested that the selected doses of CTPG had no side effects in mice.

The spleen is an important immune defense organ in mammals, and its development directly affects the individual’s immune function and the ability to resist diseases. The immune organ index reflects the development and immune function of immune organs. The spleen contains a variety of immune cells, including T cells, B cells, natural killer cells (NK cells), macrophages, dendritic cells (DCs), and others, which play an important role in the immune system.30 It had been reported that polysaccharides can increase the number of immune cells to enhance the body’s immunity.31 As i.p. injection of CTPG significantly increased the spleen index (increased 0.55 times), we further analyzed the numbers of various immune cells in the spleen. The results showed that i.p. injection of CTPG significantly increased the numbers of T cells (CD3+CD19− cells) (increased 0.49 times), B cells (CD3−CD19+ cells) (increased 0.49 times), and NK cells (CD3−CD19−CD49b+ cells) (increased 1.63 times) compared with the untreated group. CD4+ and CD8+ T cells, as the main subtypes of T cells, play an important role in the antigen-specific cellular immune response.32 Therefore, we further analyzed the number and activation status of CD4+ and CD8+ T cells in the spleen. The results indicated that i.p. injection of CTPG not only significantly increased the numbers of CD4+ and CD8+ T cells (increased 0.55 and 0.34 times, respectively) but also enhanced the activation state of CD4+ T cells (CD4+CD44+ cells) (increased 0.84 times) (Figure 7B), these results suggesting that CTPG had immune enhancement function.

Figure 2. The effects of CTPG on cell cycle in HCC cells.

The Combination of CTPG and Cisplatin Exhibited Potent Antitumor Effect on H22 Tumor Mouse Model

It is generally accepted that chemotherapy or radiotherapy can lead to poor prognosis and induce numerous adverse events, which may be due to the suppression of the immune system and damage to normal tissues and organs. Polysaccharides from TCM have immune enhancement effects and alleviate the damage of immune organs and immune cells caused by chemotherapy, for example, chitosan33 and Lycium barbarum polysaccharides.34 The above result showed that CTPG could ameliorate the side effects of cisplatin on the immune system. Therefore, the antitumor effect of CTPG combined with cisplatin was evaluated in the H22 tumor mouse model. As shown in Figure 8A, CTPG alone, cisplatin alone, and the combination of CTPG and cisplatin (CTPG+cisplatin) significantly suppressed H22 tumor growth compared with the control group. CTPG+cisplatin showed a stronger inhibitory effect than that of CTPG and cisplatin alone. On the 20th day, the tumor volumes in the control, cisplatin alone, CTPG alone, and CTPG+cisplatin groups were about 1707.8, 722.5, 1033.8, and 367.5mm3, respectively. On the same day, the tumors were collected for visual observation (Figure 8B) and weight comparison (Figure 8C). CTPG+cisplatin showed better therapeutic effects on the H22 tumor mouse model than CTPG or cisplatin alone. The tumor weight in the CTPG+cisplatin group was lower than that in other groups. These results suggest that the anti-tumor activity of CTPG and cisplatin results in synergistic effects.

The body weights of mice were measured to reflect the health status of mice. The body weight remarkably decreased after cisplatin treatment, suggesting cisplatin-induced severe systemic toxicity.35 In comparison, CTPG combined with cisplatin significantly ameliorated body weight loss induced by cisplatin, while CTPG alone had no significant effect on mice body weight (Figure 8D). The organs including the spleen, heart, liver, lung, kidney, and thymus were also isolated to calculate organ indexes. Compared with the untreated group, spleen indexes were significantly decreased and increased by cisplatin and CTPG, respectively. CTPG + cisplatin recovered spleen indexes compared with cisplatin alone. Other organ indexes showed no significant difference among all groups (Table 2).

CTPG Combined with Cisplatin Enhanced the Immunity of Tumor Mice

The above results showed that CTPG enhanced the immunity of mice and inhibited the apoptosis of splenocytes induced by cisplatin. We further investigated whether the antitumor effect of CTPG+cisplatin was correlated with enhanced immunity. In the process of tumor occurrence and development, the body’s immune system plays an important immune surveillance function, and T cell-mediated cellular immunity plays a key role. CD4+ T cells have the function of assisting cellular immunity and humoral immune response, and CD8+ T cells are the main effector cells of the immune response, which can specifically kill target cells and play an important role in tumor immunity and antiviral immunity.32 The spleens of tumor-bearing mice were collected to detect the numbers of T cells and B cells by flow cytometry. The frequencies and numbers of T cells including CD4+ and CD8+ T cells in the CTPG+cisplatin group were significantly higher than those in the control (increased 0.55 and 0.7 times, respectively) and cisplatin groups (increased 0.92 and 1.16 times, respectively) (Figure 9A and B). Compared with the control group, CTPG alone also partly increased the numbers of CD4+ and CD8+ T cells although there was no significant difference. The frequencies and numbers of B cells had no significant difference among all groups.

Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) expand in tumors and inhibit antitumor immune responses; they suppress the activation and expansion of CD4+ T cells, the function of effector T cells, the activation and/or proliferation and cytokine formation of CD4+ and CD8+ T cells, the B-cell proliferation and immunoglobulin production and class switch, the cytotoxic functions of NK and natural killer T cells (NKT), and the function and maturation of DCs.36-38 The frequencies and numbers of MDSCs (CD11b+Gr-1+), macrophages (CD11b+Gr-1−), and Tregs (natural Tregs: CD4+CD25+Foxp3+; induced Tregs: CD4+CD25−Foxp3+) in the spleens of tumor mice were analyzed by flow cytometry. Compared with the control group, the frequencies and numbers of MDSCs decreased significantly after injection with cisplatin alone (decreased 1.56 times) or in combination with CTPG (decreased 0.62 times). The numbers of CD11b+Gr-1− macrophages also decreased significantly although there was no significant change in their frequencies (Figure 9C). In addition, CTPG alone and CTPG+cisplatin treatment significantly decreased the frequencies of induced Tregs (decreased 0.33 and 0.37 times, respectively) (Figure 9D). These results suggested that the antitumor effect of CTPG+cisplatin might be correlated with the enhanced immunity characterized by the increased numbers of T cells and the decreased numbers of MDSCs and Tregs.

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Discussion

It has been reported that TCM could induce apoptosis of various tumor cells through different pathways, including the extrinsic death receptor, intrinsic mitochondrial and endoplasmic reticulum (ER) stress pathways.39 Our previous study showed that N-butanol subfraction of Brassica rapa L. induced apoptosis of A549 lung adenocarcinoma cells16 and ethanol extracts of cultivated and wild Pleurotus ferulae induced apoptosis of H22 cells via ER stress- and mitochondria-dependent pathway.17 We also reported that CTPG inhibited the growth of melanoma B16-F10 cells and esophageal carcinoma Eca-109 cells through the induction of apoptosis via a mitochondrial-dependent pathway.13 Echinacoside is an important compound with many biological activities in PhGs. It had been reported that echinacoside exerted its antiproliferative and proapoptotic functions on the HepG2 HCC cell line via decreasing TREM2 expression and PI3K/AKT signaling.40 In the present study, we found that CTPG inhibited the growth of human HCC HepG2 and BEL-7404 cells through activation of the MAPK signaling pathway and induction of apoptosis via the mitochondria-dependent pathway. Bax and Bcl-2 play a critical role in the mitochondria-dependent apoptosis pathway.24 Here, we found that CTPG increased the expression of Bax and reduced the expression of Bcl-2 in HepG2 and BEL-7404 cells, which resulted in the reduction of Δψm and the release of cytochrome c to activate the caspase cascade. The up-regulated levels of cleaved caspase-9 and cleaved caspase-3 were observed after CTPG treatment, while the levels of cleaved caspase-8 were not changed by CTPG treatment, suggesting that the apoptosis of HepG2 and BEL-7404 cells induced by CTPG was not mediated by the extrinsic signaling pathway. However, our previous study showed that CTPG induced the apoptosis of H22 cells through both intrinsic and extrinsic pathways.15 These results suggested that CTPG might inhibit the growth of tumor cells through multiple targets and pathways.

TCM has been widely used for treating cancer through multiple signal pathways with or without minor side effects.41 The MAPK signaling pathway is closely related to the occurrence, invasion, and metastasis of tumor cells, which mainly involves the p38 pathway, JNK/SAPK pathway, and ERK1/2 pathway.20 The activation of p38 and JNK can induce apoptosis but the activation of ERK1/2 can be anti-apoptotic or pro-apoptotic depending on the cell properties.42 It has been reported that CHM induced the apoptosis of tumor cells by activating the p38, JNK, or ERK MAPK pathways.43,44 We found that CTPG up-regulated the phosphorylated levels of p38, JNK, and ERK, suggesting that the MAPK signaling pathway might be involved in the apoptosis of HCC cells induced by CTPG.

Chemotherapy and radiotherapy can cause various adverse events including the suppression of the immune system. In order to improve the clinical efficacy of chemotherapy, natural polysaccharides have been approved to be used as adjuvants for cancer treatment. It has been reported that the combination of lentinan and the chemotherapeutic agent tegafur improved the clinical efficacy of patients with esophageal carcinoma by enhancing the patient’s immune function.45 In our previous studies, the in vivo inhibitory effect of CTPG on tumor growth is related to not only its direct antitumor effect but also its immune enhancement function.13,15 The immune enhancement function of CTPG might be correlated with its polysaccharide content. Here, the in vivo antitumor effect of CTPG + cisplatin was investigated. In the H22 tumor mouse model, CTPG + cisplatin not only showed the best antitumor effect but also reduced the side effects of cisplatin. CTPG + cisplatin enhanced the immunity of tumor-bearing mice by increasing the numbers of CD4+ and CD8+ T cells and decreasing the numbers of MDSCs and Tregs, which participated in the tumor-induced inhibitory microenvironment, promoted tumor growth, and inhibited immune response.46 The increased numbers of T cells might be corrected with their proliferation promoted by CTPG and their decreased apoptosis protected by CTPG. However, the mechanisms of CTPG to reduce the numbers of MDSCs and Tregs need further investigation.

In conclusion, CTPG induced apoptosis in HCC cells through both the mitochondria-dependent and MAPK signaling pathways in vitro and inhibited the growth of HCC through direct antitumor effects and indirect immune enhancement in combination with cisplatin. CTPG might serve as a potential candidate for the treatment of HCC.

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