Inhibitory Effect of Hernandezine on the Proliferation of Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) causes 830000 deaths every year and is becoming the third malignant tumor worldwide. One of the primary reasons is the lack of effective drugs. Hernandezine (HER), a bisbenzylisoquinoline alkaloid of Thalictrum simplex, has been confirmed to have antitumor activity. But there are few reports about its effect on HCC and the underlying mechanisms still remain unclear. Therefore, the antitumor effects and mechanisms of HER on HCC were evaluated in HepG2 and Hep3B cells. The in vitro experiments demonstrated that HER significantly induced G0/G1 phase arrest, inhibited the proliferation and promoted cell apoptosis in liver cancer cell lines. In the mechanisms, the antitumor effects of HER on liver cancer cells were mediated by phosphatidylinositol 3-kinase (PI3K)–protein kinase B (AKT) pathway and reactive oxygen species (ROS), simultaneously. In one way, HER inhibited the activities of PI3K–AKT pathway, which interrupt the dimer formation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1) and result to G0/G1 phase arrest. In another way, after HER treatment, ROS accumulated in liver cancer cells and caused mitochondria injury which further influenced the expression of apoptosis-related proteins and eventually resulted to HepG2 and Hep3B cell apoptosis. In addition, HER showed a tumor restrain function in HepG2 and Hep3B bearing nude mice. Overall, these findings indicated that HER is a promising antitumor drug, which may provide a new direction for clinical HCC treatment.

INTRODUCTION

As a major primary hepatic carcinoma, hepatocellular carcinoma (HCC) is the third cancer to cause mortality in the world.^1,2) HCC incidence displays an upward trend every year due to the undruggable nature of HCC mutations and the unresponsiveness of this tumor to existing antitumor therapies.³⁾ On one side, surgical resection is the current standard curative avenue for HCC patients in early stage. However, high frequency of postoperative recurrence after surgery seriously weakens the therapeutic efficacy on patients.⁴⁾ On the other side, although sorafenib, a broad spectrum kinase inhibitor, has been approved for treating advanced HCC by U.S. Food and Drug Administration (FDA), but it provides only modest benefit to HCC patients.^2,3) Therefore, the inefficiency of surgery and the low drug response rate highlight the urgency to develop novel treatment methods for HCC.

Tumorigenesis is a complex process that is closely related to the balance between the proliferation and apoptosis of tumor cells.⁵⁾ And multiple signal pathways and endogenous active compounds are involved in tumorigenesis, even in HCC.⁶⁾ For example, phosphatidylinositol 3-kinase (PI3K)–protein kinase B (AKT) pathway was verified participant in drug resistance,⁷⁾ tumor growth,⁸⁾ and metastases⁹⁾ in HCC via modulating phosphofructokinase (PFK), forkhead box O6 (FoxO6), and mothers against decapentaplegic homolog 7 (SMAD7), respectively. In addition, the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway can also induce tumor cell G0/G1 phase arrest in breast cancer,¹⁰⁾ colorectal cancer,¹¹⁾ and endometrial carcinoma,¹²⁾ but still unknown in HCC.

Traditional Chinese medicine (TCM) has been identified to possess effective roles in restraining tumor development and preventing the metastasis and recurrence of liver cancer.^13,14) It has been proven that the functions of TCM on preventing HCC recurrence and liver fibrosis after surgical operation in clinical trials. Huaier granule and Jiedu granule have been proven to efficiently prolong recurrence-free survival and prevent HCC recurrence in patients after surgical operation.^15–17) Some natural compounds isolated from TCM also have been used for treating HCC patients with significant therapeutic effects and fewer side effects.^18,19) Hernandezine (HER) is a bisbenzylisoquinoline alkaloid with multiple biological functions, which could be mainly extracted from Thalictrum species including Thalictrum simplex, Thalictrum podocarpum, Thalictrum fendleri, Thalictrum hernandezii, Thalictrum rochebrunianum.^20,21) It is reported that HER has a number of biological functions e.g., cancer resistance and anti-inflammation.^22,23) HER has been exhibited powerful protection hair cells against aminoglycoside-induced damage,²⁴⁾ inhibition of protein kinase C signaling in human peripheral blood T cells²⁴⁾ and repression of lipopolysaccharide (LPS) induced tumor necrosis factor-α (TNF-α) generation in human macrophage cells by activating AMP-activated protein kinase (AMPK).²²⁾ HER could inhibit neuronal nicotinic acetylcholine receptors (nAChRs).²⁵⁾ HER also could block non-voltage manipulated Ca²⁺ entry in human leukemic HL-60 cells and rat glioma C6 cells activated by depletion of intracellular Ca²⁺ store evoked by bombesin and thapsigargin.^26,27) Moreover, HER was confirmed recently to be the most effective isoquinoline alkaloid to cause autophagy and autophagic cell death in apoptosis resistant cells via activating AMPK pathway directly.²³⁾ Bisbenzylisoquinoline alkaloid such as tetrandrine has potent anticancer function. Several studies showed that tetrandrine inhibited HCC proliferation by suppressing of cell cycle progression at the G2/M phase, or increasing sorafenib sensitivity for HCC treatment.^28,29) However, as another important representative of bisbenzylisoquinoline alkaloid, the function of HER on HCC are still unclear.

In this study, the function of HER against HCC was evaluated in vivo and in vitro. It was identified that HER extremely inhibited the proliferation of HCC cell lines HepG2 and Hep3B in vitro. In addition, HER exhibited a HCC resistance function in HepG2 and Hep3B bearing nude mice models. The specific functional mechanism of HER on HCC was by inhibiting the PI3K/AKT signal pathway which disturbed the interaction between cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1) as well as induced Bcl2-associated X protein (Bax) expression and activities of caspase-3 and caspase-9. The data has provided a new direction for the clinical HCC therapy.

MATERIALS AND METHODS

Cell Line and Cell Culture

HepG2 (Cat. No. CL-0103) and Hep3B (Cat. No. CL-0102) cell lines were purchased from Procell Life Science & Technology, and cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium (Gibco, Invitrogen, CA, U.S.A.) containing with 10% fetal bovine serum (FBS) (AusGeneX, Brisbane, Australia) and 1% penicillin–streptomycin (P/S) (Solarbio, Beijing, China) in a CO₂ incubator at 37 °C. These two cell lines were separately treated with different concentrations of HER (hernandezine, CAS No. 6681-13-6) (ChemFaces, Chengdu, China; purity ≥98.0%).

Cell Viability Assay

HepG2 and Hep3B were plated at a density of 4 × 10³ cells per well of 96 well plates and attached overnight. Then the cell cultural medium was replaced with fresh DMEM medium containing indicated concentrations of HER (10 to 100 µM) dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich Co., St. Louis, MO, U.S.A.). After 24 and 48 h treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich Co.) was added into the cells and further cultured for another 4 h. Then supernatants were discarded and replaced with DMSO. The absorbance at 570 nm was read using a microplate reader (Infinite M200 Pro, TECAN, Switzerland). The IC₅₀ values of HER against HepG2 and Hep3B were recorded and calculated by GraphPad Prism 5.0.

Colony Formation Assay

HepG2 and Hep3B cells were respectively seeded in six-well plates at a density of 200 cells per well. After 24 h of HER (30, 40, 50 µM) intervention, the cells were further cultured for 7 d in fresh medium. The medium was renewed every three days. Then, after washing twice with phosphate buffered saline (PBS), cells were fixed with 4% formaldehyde solution for 15 min, and stained using crystal violet staining solution for 10 min. After PBS washing, colonies number was counted under an inverted microscope.

Cell Cycle Detection

Cell cycle detection was carried out using propidium iodide (PI) staining. Briefly, HepG2 and Hep3B cells were cultured in six well plates at a density of 1.0 × 10⁵ cells per well and treated with HER. After incubation with 0, 30, 40, or 50 µM HER for 24 h, cells were collected by 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) digestion and centrifugated at 1200 × g for 3 min, then subsequently fixed using 70% precooled ethanol at 4 °C for 3 h. After incubation with ribonuclease (RNase) A at 37 °C for 30 min, PI (25 µg/mL) was added to the cell suspension and incubated with cells for 30 min at 4 °C. The cell cycle of HepG2 and Hep3B was subsequently detected by flow cytometry using FACSCanto II Flow cytometer (Becton Dickinson, Franklin Lakes, NJ, U.S.A.).

Cell Apoptosis Analysis

Apoptotic cells were analyzed using an AnnexinV Alexa Fluor488/PI kit (Solarbio) according to the manufacturer’s instructions. HER treated HepG2 and Hep3B cells were harvested and adjusted the concentration of cells to 3 × 10⁶ cells/mL. Five microliters Annexin V-Alexa Fluor488 was incubated with 100 µL of cell suspension for 5 min in dark. 10 µL PI (20 µg/mL) was then added into cells, followed by 400 µL PBS was added. Finally, cells were detected immediately by FACSCanto II Flow cytometer.

Western-Blot Analysis

HepG2 and Hep3B cells were cultured in six well plates, and treated with different concentrations of HER for 24 h. Total protein was prepared using radio immunoprecipitation assay (RIPA) lysis buffer. Mitochondrial and cytoplasmic proteins were separated with a Cell Mitochondria Isolation Kit (Beyotime, Shanghai, China). Protein concentration was determined using a bicinchoninic acid (BCA) protein concentration determination kit (Solarbio). Western blots were performed as previously described using primary antibodies against β-actin (1 : 5000, TransGen Biotech, Beijing, China), CDK4 (1 : 2000, Abcam, Cambridge, U.K.), CCND1 (1 : 2000, Abcam), Bcl-2 (1 : 1000, Abcam), Bax (1 : 1000, Abcam), Caspase-3 (1 : 500, Abcam), Cleaved Caspase-3 (1 : 3000, CST, Boston, MA, U.S.A.), Caspase-9 (1 : 1000, Abcam), Cleaved Caspase-9 (1 : 3000, CST), Cytochrome C (1 : 1000, Abcam), AKT (1 : 1000, CST), p-AKT (1 : 1000, Abcam), PI3 Kinase p85 (1 : 5000, Proteintech, Wuhan, China), and PI3 Kinase p85 α (1 : 1000, Abcam).

Hoechst 33258 Staining

HepG2 and Hep3B cells were cultured and incubated with HER in six well plates for 24 h. After fixation with 4% formaldehyde for 30 min and PBS washing, cells were then stained with 100 µL Hoechst 33258 staining solution (Solarbio) for 10 min at room temperature. PBS washed three times, the cells were photographed under a fluorescence inverted microscope (Leica, Germany).

Mitochondrial Membrane Potential (MMP, ∆ᴪm) Measurement

The MMP (∆ᴪm) was measured using JC-1 mitochondrial membrane potential assay kit (Beyotime) according to the manufacturer’s protocol. HepG2 and Hep3B cells were separately incubated with HER for 24 h, and then stained with JC-1 for 30 min at 37 °C. After JC-1 staining buffer washing, the cells were resuspended in 300 µL JC-1 staining buffer. And MMP was detected using flow cytometry. Cellular fluorescent photographs were obtained with microscope.

Immunofluorescence

Briefly, HepG2 and Hep3B cells were fixed in 4% paraformaldehyde for 20 min at room temperature after PBS washing. Then cells were subjected to 0.1% Triton-X-100 for 5 min at room temperature, and blocked with 5% goat serum. Primary antibodies including anti-p-PI3K, p-AKT, Ki67 antibodies diluted at 1 : 100, 1 : 100 and 1 : 200 was combined with adequate Alexa488 labelled secondary immunoglobulin G (IgG) (Molecular Probes) in PBS. Nuclei was stained with Hoechst.

Reactive Oxygen Species (ROS) Detection

The intracellular ROS was detected using 10 µM of 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA), a cell-permeable probe. Following HepG2 and Hep3B cells treated with HER, they were then incubated with the fluorescent probe at 37 °C for 30 min. Then the cells were analyzed using flow cytometry.

Antioxidant N-Acetyl-L-cysteine (NAC) Pretreatment

The antitumor effect of HER in HCC cells was ascertained by NAC, a widely-used antioxidant. In briefly, both HepG2 and Hep3B cells were cultured in medium with or without NAC (5 mM) and HER (40 µM) for 24 h. Then, the changes of ROS level, MMP, cell cycles distribution and apoptotic rate were examined by respective methods.

Animal Experiment

All the animal experiments complied with the National Institutes of Health guide for the care and use of Laboratory animals (http://oacu.od.nih.gov/regs/index.htm). The experiment approach was approved by the Research Committee of Macao Polytechnic University (11-2019). Given the in vitro anti-liver tumor cell activity of HER, tumor xenograft models were established to evaluate its in vivo effect. Briefly, forty nude mice were randomly divided into five groups, including control group, HepG2 model group, Hep3B model group, HepG2 treated by HER group and Hep3B treated by HER group. Mice were subcutaneously injected in the right fossa axillaries of nude mice with 1 × 10⁷ HepG2 or Hep3B cells. In both drug treatment groups, the mice were administrated with 50 mg/kg of HER by intragastric administration. Then tumor volume and mice body weight were recorded. Finally, the mice were euthanized and tumor tissue was collected for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and Ki67 detection by immunohistochemical method.

Statistical Analyses

The results were expressed as the mean ± standard deviation (S.D.). Student’s t-test or one-way ANOVA was employed for statistical analysis with SPSS 21.0 software. p < 0.05 was considered to be statistically significant.

RESULTS

The Anti-proliferative Effects of HER on HCC Cell Lines Were Manipulated by PI3K–AKT Pathway

To screen appropriate anti-proliferative concentrations of HER to HCC cells, HepG2 and Hep3B cells were incubated with various concentrations of HER (from 10 to 100 µM), and the MTT assay was performed after 24 h. The results showed that HER significantly inhibited proliferation of both HepG2 and Hep3B cells, the 24 h-IC₅₀ were 44.17 and 41.26 µM, respectively (Fig. 1A). Three concentrations, 30, 40, and 50 µM were used for further experiments to investigate the dose-dependent antitumor effects of HER on HCC cells. Light microscopy showed that the cell numbers in HER-treated groups were significantly decreased compared with the DMSO control group (Fig. 1B). In addition, effects of HER on cell colony formation were also investigated. Compared with the DMSO control group, numbers of cell colonies in the HER treatment group were declined in both HepG2 and Hep3B cells (Figs. 1C, D). And the expression of Ki67 was also decreased in both two cell lines treated with HER (Figs. 1E, F). Furthermore, considering that PI3K/AKT signaling pathway plays an essential role in cancer cell progression, expressions of PI3K and AKT were determined by western blots. The data showed that after being treated by different concentrations of HER, the phosphorylated PI3K and AKT in both HepG2 and Hep3B were much lower than the control group (Figs. 1G, H). And these phenomena were verified by immunocyte fluorescent staining which p-PI3K (Fig. 1I) and p-AKT (Fig. 1J) protein levels were inhibited after HER treatment in both HCC cells.

Fig. 1. HER Inhibits the Proliferation and Colony Formation of HCC Cells

(A) The cell viability of HepG2 and Hep3B cells treated with HER for 24 h was measured by MTT. (B) Morphological changes of HepG2 and Hep3B cells under microscope. Scale, 100 µm. (C, D) A representative cell colony formation image of HepG2 or Hep3B cells treated with HER for 24 h. (E, F) Ki67 staining and quantitative analysis were used to detect the morphological changes of apoptosis in HCC cells treated with HER. (G, H) Expression of p-PI3K, PI3K, p-AKT, and AKT in HER-treated HepG2 and Hep3B cells. (I, J) Cellular immunofluorescence assay of p-PI3K and p-AKT in HCC cells treated with HER. All data were expressed as the mean ± S.D. of three independent experiments. Compared with the control group, * p < 0.05.

HER Induces G0/G1 Phase Arrest in HCC Cells

Subsequently, the roles of HER in HCC cell cycle were detected by flow cytometry assay. As suspected, HER induced G0/G1 phase arrest in HepG2 and Hep3B cells (Figs. 2A, B). The proportion of HepG2 (and Hep3B) cells in the G0/G1 phase increased from 55.2% (43.5% of Hep3B) to 63.4% (53.5% of Hep3B), 69.2% (60.2% of Hep3B), and 73.4% (64.1% of Hep3B) after treated by 30, 40, and 50 µM HER respectively. In contrast, G2/M phase was decreased in dose-dependent manners (Figs. 2C, D). However, the next results demonstrated that the expression of G0/G1 phase related proteins including CDK4, CDK6 and CCND1 in neither HepG2 nor Hep3B were influenced by HER (Figs. 2E, F). Next, the Co-IP assay was performed to detect the binding status between CDK4 and CCND1. After CDK4 antibody was used to precipitate CDK4 alone or CDK4-binding proteins in HepG2 and Hep3B cells, the CDK4 and CCND1 levels in precipitates were detected by immunoblot assay. The results demonstrated that the CDK4 and CCND1 levels were uninfluenced with or without HER treatment in ‘input’ group, which consist with the above Western blot assays (Fig. 2G). In addition, although the CDK4 protein levels precipitated by CDK4 antibodies showed no differences among all groups, the amounts of CCND1 that coprecipitated with CDK4 were significantly decreased compared control group to HER-treated groups (Figs. 2G–I). The above results demonstrated that HER induced G0/G1 phase arrest in HCC cell lines resulted from HER inhibited the heterodimer formation of CDK4 and CCND1 proteins.

Fig. 2. HER Induces Cycle Arrest of HCC Cell in G0/G1 Phase

The cell cycle distribution of HepG2 (A) and Hep3B (B) cells treated with HER was measured by flow cytometry. (C, D) Statistical analysis of cell cycle distribution in HCC cells treated with HER. (E, F) The protein expressions of CDK4/6 and cyclin D1 in HCC cells treated with HER were measured by Western blot. (G–I) The protein binding between CDK4 and CCND1 by Co-IP assay. All data were expressed as the mean ± S.D. of three independent experiments. Compared with the control group, * p < 0.05.

HER Induces Apoptosis in HCC Cells via Modulating Bax/Bcl-2 and Caspase-3/9

Furthermore, Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis kit was used to further determine the pro-apoptotic effects of HER on HCC cells by flow cytometry assay. After 24 h stimulation, the apoptotic rates were dramatically increased in HCC cells (Figs. 3A, B). For HepG2 cells, the apoptotic rates were 21.4 ± 3.7, 31.7 ± 4.6, and 43.9 ± 5.3% after stimulation with 30, 40, and 50 µM HER, respectively (Fig. 3C). For Hep3B cells, the apoptotic rates were 22.7 ± 4.8, 35.9 ± 4.1, and 46.6 ± 5.8% after stimulation with 30, 40, and 50 µM HER, respectively (Fig. 3D). Meanwhile, the Hoechst 33258 staining demonstrated that the nuclei in the HER-treated groups exhibited chromatin condensation, increased fluorescence intensity, and formation of apoptotic bodies (Figs. 3E, F). Western blot detection showed that the expression of Bcl-2, an intracellular apoptosis-related signaling molecule, was significantly down-regulated, while Bax expression was significantly up-regulated in HER-treated groups. The expressions of caspase-3 and caspase-9 were both significantly reduced in the cells of the treated group, furthermore the expressions of cleaved caspase-3 and cleaved caspase-9 were markedly elevated in both HCC cell lines treated with HER (Figs. 3G–I).

Fig. 3. HER Induces Apoptosis in HCC Cells

The apoptosis of HepG2 (A) and Hep3B (B) cells treated with HER was detected by Annexin V-Alexa Fluor488/PI staining. (C, D) Statistical analysis of apoptosis rate of HCC cells treated with HER. (E, F) Hoechst staining was used to detect the morphological changes of apoptosis in HCC cells treated with HER. (G) The protein expressions of Bcl-2, Bax, caspase-3, cleaved caspase-3, caspase-9, and cleaved caspase-9 in HepG2 and Hep3B cells treated with HER were measured by Western blot. (H, I) Quantitative analysis of relative protein levels in HCC cells treated with HER. All data were expressed as the mean ± S.D. of three independent experiments. Compared with the control group, * p < 0.05.

Mitochondrial Injury and ROS Accumulation May Be Involved in Apoptosis in HCC Cells Induced by HER

The staining with DCFH-DA and JC-1 was conducted to further determine whether HER-induced apoptosis of HCC cells was related to the intracellular ROS level and the mitochondrial injury. Compared with the DMSO control group, ROS levels and fluorescence intensity in HCC cells were gradually increased following 30, 40, and 50 µM HER treatment (Figs. 4A–D). In contrast, the MMP in both HepG2 and Hep3B were decreased, which implied that HER led to mitochondrial injury of HCC cells (Figs. 4E, F). Cell staining with JC-1 kits confirmed HER-induced mitochondrial injury due to the fluorescence ratios of red to green were significantly decreased by HER (Figs. 4G, H).

Fig. 4. HER Affects ROS and Mitochondrial Membrane Potential (MMP, ∆ᴪm) Levels in HCC Cells

(A–D) Intracellular reactive oxygen levels in HCC cells treated with HER. (E, F) Changes in mitochondrial membrane potential (MMP, ∆ᴪm) in HepG2 and Hep3B cells treated with HER. (G, H) Representative images of changes in mitochondrial membrane potential (MMP, ∆ᴪm) of HCC cells treated with HER. Scale, 50 µm. All data were expressed as the mean ± S.D. of three independent experiments.

NAC Attenuated HER-Induced Cell Cycle Arrest and Cell Apoptosis in HCC Cells

To further verify whether ROS involved in HER-induced cell cycle arrest and cell apoptosis in HCC cells, HepG2 and Hep3B cells were stimulated by HER (40 µM) with or without NAC (5 mM). Indeed, compared with the HER-treated group, NAC significantly attenuated HER-induced the increased ROS level in HER + NAC-treated HepG2 (Figs. 5A, C) and Hep3B cells (Figs. 5B, D). Moreover, NAC obviously reversed the reduced MMP in HER + NAC-treated HepG2 (Figs. 5E, G) and Hep3B cells (Figs. 5F, H) when compared to the corresponding HER-treated group. Consistently, the cell cycle arrest at G0/G1 phase and the increased cell apoptotic rate induced by HER were significantly attenuated in HER + NAC-treated HCC cells (Figs. 5I–L). All these results implied that ROS at least partially participated in the HER-induced cell cycle arrest and cell apoptosis in HCC cells.

Fig. 5. NAC Mediated HER-Induced Pro-apoptotic Effect on HCC Cells

(A–D) Intracellular reactive oxygen levels in HCC cells treated with HER (40 µM) with or without NAC (5 mM). (E–H) Changes in mitochondrial membrane potential (MMP, ∆ᴪm) in HepG2 and Hep3B cells treated with HER (40 µM) with or without NAC (5 mM). (I–L) The distribution of cell cycle (I, J) and apoptosis (K, L) in HepG2 and Hep3B cells treated with HER (40 µM) with or without NAC (5 mM). All data were expressed as the mean ± S.D. of three independent experiments. Compared with the HER-treated group, * p < 0.05.

HER Inhibited HCC Cells Proliferation in Vivo

To further verify the antitumor effects of HER on HCC cells, HepG2 or Hep3B cells (1 × 10⁷ cells per mouse) were injected into fossa axillaries of nude mice treated with or without HER (50 mg/kg, intragastric administration). The tumor volume and weight of each group were calculated and recorded after 28 d. The results showed that, after HER treatment, the body weights were slightly higher in both HepG2 and Hep3B-bearing mice compared with respective control group (the body weights in HepG2-bearing mice were 21.5 ± 4.6 g in HER-treated group vs. 20.3 ± 3.2 g in control group; the body weights in Hep3B-bearing mice were 22.6 ± 3.5 g in HER-treated group vs. 21.1 ± 4.5 g in control group) (Fig. 6A). Compared with the control group, the tumor volume (Figs. 6B, C) and weight (Fig. 6D) in both HepG2 and Hep3B cell bearing mice were notably suppressed after HER administration. Meanwhile, Ki67 and TUNEL staining in both HCC cell bearing mice confirmed that, compared with the control group, HER suppressed the numbers of Ki67- (Figs. 6E, F), and promoted TUNEL-positive cells (Figs. 6G, H). The in vivo investigations demonstrated that HER indeed had anti-proliferative activity in HCC cells.

Fig. 6. HER Inhibited HCC Cells Proliferation in Vivo

The body weight (A), image (B), tumor volume (C), and weight (D) in HepG2 and Hep3B bearing mice with or without HER administration. (E, F) Ki67 staining and quantitative in HepG2 and Hep3B bearing mice with or without HER administration. (G, H) TUNEL staining and quantitative in HepG2 and Hep3B bearing mice with or without HER administration. (n = 8). All data were expressed as the mean ± S.D. of three independent experiments. Compared with the control group, * p < 0.05.

DISCUSSION

HCC is characterized by high malignant degree and drug resistance. Although there exist diverse therapeutical strategies treating HCC including radiotherapy/chemotherapy intervention, early surgical treatment, liver transplantation, the 5-year survival rate is still lower than 5%.³⁰⁾ Therefore, searching for new effective antitumor drugs is necessary. TCM has been widely acceptable in the world mainly due to its efficacy and safety in clinical treatment and prevention of cancer.³¹⁾ Natural small molecule products isolated from Chinese herbs exhibit low cytotoxicity and antitumor activity.

The earliest description of HER can be traced back to 1990 which found the potential antitumor effect of HER in various cancer cell lines.³²⁾ After that, multiple biological activities of HER were verified including anticancer property, antioxidant and anti-inflammatory activity, antiviral activity, antibacterial activity and so on.^33–35) In terms of mechanism, HER acts as an inhibitor of ATP-binding cassette (ABC) drug transporter ABCB1, and involves in multidrug resistance in epidermal carcinoma and ovarian carcinoma cell lines.³⁶⁾ Additionally, previous studies demonstrated that HER was a novel AMPK activator, which inhibits lipopolysaccharide-induced TNF-α production in macrophage cells²²⁾ and efficiently induced cytotoxicity against HCC cell lines,²³⁾ respectively. Unfortunately, these researches did not clarify the specific mechanism of HER. Present study showed that HER extremely inhibited HCC cell growth, significantly caused cell cycle arrest at G1 phase and eventually resulted to cell apoptosis through promoted ROS levels and inhibited PI3K/AKT pathway, simultaneously.

PI3K/AKT signaling pathway has been identified to affect tumor progression and invasion.³⁷⁾ AKT is a key downstream effector of PI3K, and once inactivated it can inhibit cell proliferation and mediate apoptosis.³⁸⁾ Previous studies demonstrated that inactivated PI3K/AKT is associated with G0/G1 phase arrest of tumor cells, and CDK4/CCND1 may involve in this process. CCND1 is a regulator of the cyclin-dependent kinases CDK4/6 which modulates cell proliferation and growth via its transcriptional regulator role.³⁹⁾ It has been identified that CCND1 is related to tumor invasion and metastasis in clinical studies and in in vivo experiments.⁴⁰⁾ A dual PI3K/mTOR pathway inhibitor BEZ235 was reported to reduce the expression of cyclin D1 and caused G1 phase arrest in lung cancer cells.^41,42) P27, a downstream factor of PI3K/AKT, have been shown to inhibit CDK4/6 activity and stabilize cyclin D-CDK4 dimer in breast cancer.⁴²⁾ And this study showed that HER reduced the phosphorylation of PI3K and AKT in HepG2 and Hep3B cells, but did not affect CDK4 and CCND1 levels, which suggested that there was another pathway regulating CDK4 and CCND1 expression due to HER indeed induced the cells to undergo cycle arrest in G0/G1 phase. The Co-IP assay was performed subsequently with CDK4 antibody to detect whether HER affected the dimer formation of CDK4-CCND1. We found that compared with the control group, HER gradually inhibited the dimer formation of CDK4-CCND1 in dose-dependent manner, which explained HER induced G0/G1 phase arrest in HCC cells without influencing the expression of cell-cycle related proteins.

Apoptosis is an important pathway of cell death in the organism, and the whole process is regulated by a variety of factors. Numerous studies have confirmed that the Bcl-2 family is closely related to cell apoptosis, and the imbalance between anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax can damage the mitochondrial membrane and induce cell apoptosis.⁴³⁾ Apoptosis is usually characterized by Bcl-2 expression decline, Bax expression promotion, Bcl-2/Bax heterodimers decrement, and Bax/Bax homodimers increase.¹²⁾ The mitochondrial membrane permeability increases and transmembrane potential decreases; thus, mitochondrial membrane ion channels that mediate cytochrome c release are formed, which in turn triggers the caspase cascade to promote apoptosis. Consistent with the above findings, HER decreased the mitochondrial membrane potential by upregulating the Bax level, downregulating Bcl-2 expression, and promoting cytochrome c to enter the cytoplasm to activate caspase-9 and caspase-3, thereby initiating the HepG2 and Hep3B cells apoptotic program.

An increasing number of studies have demonstrated that ROS have an important role in cell cycle regulation and the occurrence of apoptosis.⁴⁴⁾ Studies have confirmed that elevated ROS reduce the expression of Bcl-2, promote the expression of proapoptotic proteins (e.g., Bax),⁴⁵⁾ and reduce mitochondrial membrane potential, thereby regulating apoptosis. Upon detection, HER increased intracellular ROS level, decreased JC-1 level, and induced cell apoptosis in the two cell lines. In addition, HER exhibited antitumor function in mice model by a mechanism inducing HepG2 and Hep3B apoptosis. In short, HER promoted ROS production and the latter influenced the levels of BAX/Bcl2, cleaved caspase-3 and -9, resulting in HCC cell apoptosis. In additional, HER inhibited phosphorylation of PI3K which disturbed the interaction between CDK4 and CCND1 and caused G0/G1 phase arrest (Fig. 7). All the results hint that HER is a potential drug candidate for HCC treatment.

Fig. 7. Schematic Diagram of the Proposed Mechanism of Antiproliferative and Cell Apoptosis Activity of HER

HER simultaneously inhibited PI3K/AKT activities and promote ROS production in HCC cells. In one-way, accumulated ROS in HCC cells caused mitochondria injury and further influenced the expression of Bax/Bcl2, caspase-3 and caspase-9. In another way, HER and ROS coordinated inactivate PI3K/AKT and interrupt CDK4-CCND1 dimer formation, which result to G0/G1 phase arrest. which eventually resulted to HCC cell apoptosis.

In summary, HER regulates cell cycle arrest to inhibit HepG2 and Hep3B cell proliferation and activates mitochondrial apoptosis pathway to induce cell apoptosis by promoting ROS-mediated PI3K/AKT signaling pathway. These findings offer new ideas and directions for the research of HER as a clinical antitumor drug.

Acknowledgments

This study was funded by the Foundation of Macao Polytechnic University (RP/ESS-01/2016, RP/ESCSD-02/2019).

Author Contributions

C.K. and L.M. conceived and designed this study. Q.W. and Y.Q. conducted the experiments. C. K., L. M. and P.F. interpreted and analyzed the data. Q.W. and C. K. prepared the draft manuscript. L. M. and P.F. reviewed the manuscript. All authors have read and agreed with the published version of the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Liu MX, Jin L, Sun SJ, Liu P, Feng X, Cheng ZL, Liu WR, Guan KL, Shi YH, Yuan HX, Xiong Y. Metabolic reprogramming by PCK1 promotes TCA cataplerosis, oxidative stress and apoptosis in liver cancer cells and suppresses hepatocellular carcinoma. Oncogene, 37, 1637–1653 (2018).
• 2) Qiu Z, Li H, Zhang Z, et al. A Pharmacogenomic landscape in human liver cancers. Cancer Cell, 36, 179–193.e11 (2019).
• 3) Zucman-Rossi J, Villanueva A, Nault JC, Llovet JM. Genetic landscape and biomarkers of hepatocellular carcinoma. Gastroenterology, 149, 1226–1239.e4 (2015).
• 4) Reddy SK, Steel JL, Chen HW, DeMateo DJ, Cardinal J, Behari J, Humar A, Marsh JW, Geller DA, Tsung A. Outcomes of curative treatment for hepatocellular cancer in nonalcoholic steatohepatitis versus hepatitis C and alcoholic liver disease. Hepatology, 55, 1809–1819 (2012).
• 5) Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell, 144, 646–674 (2011).
• 6) Luo Y, Liu L, Zhao J, Jiao Y, Zhang M, Xu G, Jiang Y. PI3K/AKT1 signaling pathway mediates sinomenine-induced hepatocellular carcinoma cells apoptosis: an in vitro and in vivo study. Biol. Pharm. Bull., 45, 614–624 (2022).
• 7) Icard P, Simula L, Wu Z, Berzan D, Sogni P, Dohan A, Dautry R, Coquerel A, Lincet H, Loi M, Fuks D. Why may citrate sodium significantly increase the effectiveness of transarterial chemoembolization in hepatocellular carcinoma? Drug Resist. Updat., 59, 100790 (2021).
• 8) Zuo X, Chen Z, Cai J, Gao W, Zhang Y, Han G, Pu L, Wu Z, You W, Qin J, Dai X, Shen H, Wu J, Wang X. 5-Hydroxytryptamine receptor 1D aggravates hepatocellular carcinoma progression through FoxO6 in AKT-dependent and independent manners. Hepatology, 69, 2031–2047 (2019).
• 9) Xia H, Ooi LL, Hui KM. MicroRNA-216a/217-induced epithelial-mesenchymal transition targets PTEN and SMAD7 to promote drug resistance and recurrence of liver cancer. Hepatology, 58, 629–641 (2013).
• 10) Miller TW, Rexer BN, Garrett JT, Arteaga CL. Mutations in the phosphatidylinositol 3-kinase pathway: role in tumor progression and therapeutic implications in breast cancer. Breast Cancer Res., 13, 224 (2011).
• 11) Zhang Y, Chen G, Zhuang X, Guo M. Inhibition of growth of colon tumors and proliferation of HT-29 cells by warburgia ugandensis extract through mediating G0/G1 cell cycle arrest, cell apoptosis, and intracellular ROS generation. Oxid. Med. Cell. Longev., 2021, 8807676 (2021).
• 12) Yang X, Zhang M, Wei M, Wang A, Deng Y, Cao H. MicroRNA-216a inhibits neuronal apoptosis in a cellular Parkinson’s disease model by targeting Bax. Metab. Brain Dis., 35, 627–635 (2020).
• 13) Tang KY, Du SL, Wang QL, Zhang YF, Song HY. Traditional Chinese medicine targeting cancer stem cells as an alternative treatment for hepatocellular carcinoma. J. Integr. Med., 18, 196–202 (2020).
• 14) Li XJ, Jia MM, Li YS, Yang YL, Mao XQ, Tang HB. Involvement of substance p/neurokinin-1 receptor in the analgesic and anticancer activities of minimally toxic fraction from the traditional Chinese medicine Liu-Shen-Wan in vitro. Biol. Pharm. Bull., 37, 431–438 (2014).
• 15) Wang Z, Yu XL, Zhang J, Cheng ZG, Han ZY, Liu FY, Dou JP, Kong Y, Dong XJ, Zhao QX, Yu J, Liang P, Tang WZ. Huaier granule prevents the recurrence of early-stage hepatocellular carcinoma after thermal ablation: a cohort study. J. Ethnopharmacol., 281, 114539 (2021).
• 16) Chen Q, Shu C, Laurence AD, et al. Effect of Huaier granule on recurrence after curative resection of HCC: a multicentre, randomised clinical trial. Gut, 67, 2006–2016 (2018).
• 17) Zhao HT, Meng YB, Zhai XF, Cheng BB, Yu SS, Yao M, Yin HX, Wan XY, Yang YK, Liu H, Shen F, Ling CQ. Comparable effects of Jiedu Granule, a compound Chinese herbal medicine, and sorafenib for advanced hepatocellular carcinoma: a prospective multicenter cohort study. J. Integr. Med., 18, 319–325 (2020).
• 18) Hu F, Han J, Zhai B, Ming X, Zhuang L, Liu Y, Pan S, Liu T. Blocking autophagy enhances the apoptosis effect of bufalin on human hepatocellular carcinoma cells through endoplasmic reticulum stress and JNK activation. Apoptosis, 19, 210–223 (2014).
• 19) Yang F, Li J, Zhu J, Wang D, Chen S, Bai X. Hydroxysafflor yellow A inhibits angiogenesis of hepatocellular carcinoma via blocking ERK/MAPK and NF-kappaB signaling pathway in H22 tumor-bearing mice. Eur. J. Pharmacol., 754, 105–114 (2015).
• 20) Song Y, Wang Z, Zhang B, Zhang Y, Zhang W, Yang C, Meng F, Feng X. Determination of a novel anticancer AMPK activator hernandezine in rat plasma and tissues with a validated UHPLC-MS/MS method: Application to pharmacokinetics and tissue distribution study. J. Pharm. Biomed. Anal., 141, 132–139 (2017).
• 21) Umarov KS, Telezhenetskaya MV, Ismailov ZF, Yunusov SY. Alkaloids of Thalictrum simplex. Chem. Nat. Compd., 6, 219–220 (1970).
• 22) Li P, Li X, Wu Y, Li M, Wang X. A novel AMPK activator hernandezine inhibits LPS-induced TNFalpha production. Oncotarget, 8, 67218–67226 (2017).
• 23) Law BY, Mok SW, Chan WK, Xu SW, Wu AG, Yao XJ, Wang JR, Liu L, Wong VK. Hernandezine, a novel AMPK activator induces autophagic cell death in drug-resistant cancers. Oncotarget, 7, 8090–8104 (2016).
• 24) Kruger M, Boney R, Ordoobadi AJ, Sommers TF, Trapani JG, Coffin AB. Natural bizbenzoquinoline derivatives protect zebrafish lateral line sensory hair cells from aminoglycoside toxicity. Front. Cell. Neurosci., 10, 83 (2016).
• 25) Virginio C, Graziani F, Terstappen GC. Differential inhibition of rat alpha3* and alpha7 nicotinic acetylcholine receptors by tetrandrine and closely related bis-benzylisoquinoline derivatives. Neurosci. Lett., 381, 299–304 (2005).
• 26) Leung YM, Berdik M, Kwan CY, Loh TT. Effects of tetrandrine and closely related bis-benzylisoquinoline derivatives on cytosolic Ca²⁺ in human leukaemic HL-60 cells: a structure–activity relationship study. Clin. Exp. Pharmacol. Physiol., 23, 653–659 (1996).
• 27) Imoto K, Takemura H, Kwan CY, Sakano S, Kaneko M, Ohshika H. Inhibitory effects of tetrandrine and hernandezine on Ca²⁺ mobilization in rat glioma C6 cells. Res. Commun. Mol. Pathol. Pharmacol., 95, 129–146 (1997).
• 28) Yu VW, Ho WS. Tetrandrine inhibits hepatocellular carcinoma cell growth through the caspase pathway and G2/M phase. Oncol. Rep., 29, 2205–2210 (2013).
• 29) Niu B, Wei S, Sun J, Zhao H, Wang B, Chen G. Deciphering the molecular mechanism of tetrandrine in inhibiting hepatocellular carcinoma and increasing sorafenib sensitivity by combining network pharmacology and experimental evaluation. Pharm. Biol., 60, 75–86 (2022).
• 30) Huang D, Yang B, Yao Y, Liao M, Zhang Y, Zeng Y, Zhang F, Wang N, Tong G. Autophagic inhibition of caveolin-1 by compound phyllanthus urinaria L. Activates ubiquitination and proteasome degradation of beta-catenin to suppress metastasis of hepatitis B-associated hepatocellular carcinoma. Front. Pharmacol., 12, 659325 (2021).
• 31) Tao L, Zhu F, Xu F, Chen Z, Jiang YY, Chen YZ. Co-targeting cancer drug escape pathways confers clinical advantage for multi-target anticancer drugs. Pharmacol. Res., 102, 123–131 (2015).
• 32) Xu CX, Lin L, Sun RH, Liu X, Han R. Anti-tumor effect of hernandezine and other components extracted from Thalictrum glandulosissimum. Yao xue xue bao, 25, 330–335 (1990).
• 33) Peng L, Kang S, Yin Z, Jia R, Song X, Li L, Li Z, Zou Y, Liang X, Li L, He C, Ye G, Yin L, Shi F, Lv C, Jing B. Antibacterial activity and mechanism of berberine against Streptococcus agalactiae. Int. J. Clin. Exp. Pathol., 8, 5217–5223 (2015).
• 34) Law BY, Chan WK, Xu SW, Wang JR, Bai LP, Liu L, Wong VK. Natural small-molecule enhancers of autophagy induce autophagic cell death in apoptosis-defective cells. Scientific Reports, 4, 5510 (2014).
• 35) Li Z, Geng YN, Jiang JD, Kong WJ. Antioxidant and anti-inflammatory activities of berberine in the treatment of diabetes mellitus. Evid. Based Complement. Alternat. Med., 2014, 289264 (2014).
• 36) Hsiao SH, Lu YJ, Yang CC, Tuo WC, Li YQ, Huang YH, Hsieh CH, Hung TH, Wu CP. Hernandezine, a bisbenzylisoquinoline alkaloid with selective inhibitory activity against multidrug-resistance-linked ATP-binding cassette drug transporter ABCB1. J. Nat. Prod., 79, 2135–2142 (2016).
• 37) Zheng W, Li S, Huang J, Dong Y, Zhang H, Zheng J. Down-regulation of ubiquitin-specific peptidase 9X inhibited proliferation, migration and invasion of osteosarcoma via ERK1/2 and PI3K/Akt signaling pathways. Biol. Pharm. Bull., 45, 1283–1290 (2022).
• 38) Chen H, Zhou L, Wu X, Li R, Wen J, Sha J, Wen X. The PI3K/AKT pathway in the pathogenesis of prostate cancer. Front. Biosci. (Landmark Ed.), 21, 1084–1091 (2016).
• 39) Shan K, Wang Y, Hua H, Qin S, Yang A, Shao J. Ginsenoside Rg3 combined with oxaliplatin inhibits the proliferation and promotes apoptosis of hepatocellular carcinoma cells via downregulating PCNA and cyclin D1. Biol. Pharm. Bull., 42, 900–905 (2019).
• 40) Fusté NP, Fernández-Hernández R, Cemeli T, Mirantes C, Pedraza N, Rafel M, Torres-Rosell J, Colomina N, Ferrezuelo F, Dolcet X, Gari E. Cytoplasmic cyclin D1 regulates cell invasion and metastasis through the phosphorylation of paxillin. Nat. Commun., 7, 11581 (2016).
• 41) Wu YY, Wu HC, Wu JE, Huang KY, Yang SC, Chen SX, Tsao CJ, Hsu KF, Chen YL, Hong TM. The dual PI3K/mTOR inhibitor BEZ235 restricts the growth of lung cancer tumors regardless of EGFR status, as a potent accompanist in combined therapeutic regimens. J. Exp. Clin. Cancer Res., 38, 282 (2019).
• 42) Goel S, Bergholz JS, Zhao JJ. Targeting CDK4 and CDK6 in cancer. Nat. Rev. Cancer, 22, 356–372 (2022).
• 43) Pan Y, Ye C, Tian Q, Yan S, Zeng X, Xiao C, Wang L, Wang H. miR-145 suppresses the proliferation, invasion and migration of NSCLC cells by regulating the BAX/BCL-2 ratio and the caspase-3 cascade. Oncol. Lett., 15, 4337–4343 (2018).
• 44) Zhu X, Chen X, Wang G, Lei D, Chen X, Lin K, Li M, Lin H, Li D, Zheng Q. Picropodophyllin inhibits the proliferation of human prostate cancer DU145 and LNCaP cells via ROS production and PI3K/AKT pathway inhibition. Biol. Pharm. Bull., 45, 1027–1035 (2022).
• 45) Mehmeti I, Lenzen S, Lortz S. Modulation of Bcl-2-related protein expression in pancreatic beta cells by pro-inflammatory cytokines and its dependence on the antioxidative defense status. Mol. Cell. Endocrinol., 332, 88–96 (2011).