Young Woo Kim,Seon Been Bak,Won-Yung Lee,Su Jin Bae,Eun Hye Lee,Ju-Hye Yang,Kwang Youn Kim,Chang Hyun Song,Sang Chan Kim,Un-Jung Yun,Kwang Il Park
Systemic and molecular analysis dissect the red ginseng induction of apoptosis and autophagy in HCC as mediated with AMPK
Red ginseng,Apoptosis,HCC,AMPK,Network pharmacology
Background Hepatocellular carcinoma (HCC) has a high incidence and is one of the highest mortality cancers when advanced stage is proceeded. However, Anti-cancer drugs available for treatment are limited and new anti-cancer drugs and new ways to treat them are minimal. We examined that the effects and possibility of Red Ginseng (RG, Panax ginseng Meyer) as new anti-cancer drug on HCC by combining network pharmacology and molecular biology. Materials and Methods Network pharmacological analysis was employed to investigate the systems-level mechanism of RG focusing on HCC. Cytotoxicity of RG was determined by MTT analysis, which were also stained by annexin V/PI staining for apoptosis and acridine orange for autophagy. For the analyze mechanism of RG, we extracted protein and subjected to immunoblotting for apoptosis or autophagy related proteins. Results We constructed compound-target network of RG and identified potential pathways related to HCC. RG inhibited growth of HCC through acceleration of cytotoxicity and reduction of wound healing ability of HCC. RG also increased apoptosis and autophagy through AMPK induction. In addition, its ingredients, 20S-PPD (protopanaxadiol) and 20S-PPT (protopanaxatriol), also induced AMPK mediated apoptosis and autophagy. Conclusion RG effectively inhibited growth of HCC cells inducing apoptosis and autophagy via ATG/AMPK in HCC cells. Overall, our study suggests possibility as new anti-cancer drug on HCC by proof for the mechanism of the anti-cancer action of RG.
Systemic and molecular analysis dissect the red ginseng induction of apoptosis and autophagy in HCC as mediated with AMPK

Hepatocellular carcinoma (HCC) has a high incidence and is one of the highest mortality cancers when advanced stage is proceeded. However, Anti-cancer drugs available for treatment are limited and new anti-cancer drugs and new ways to treat them are minimal. We examined that the effects and possibility of Red Ginseng (RG, Panax ginseng Meyer) as new anti-cancer drug on HCC by combining network pharmacology and molecular biology.

Network pharmacological analysis was employed to investigate the systems-level mechanism of RG focusing on HCC. Cytotoxicity of RG was determined by MTT analysis, which were also stained by annexin V/PI staining for apoptosis and acridine orange for autophagy. For the analyze mechanism of RG, we extracted protein and subjected to immunoblotting for apoptosis or autophagy related proteins.

We constructed compound-target network of RG and identified potential pathways related to HCC. RG inhibited growth of HCC through acceleration of cytotoxicity and reduction of wound healing ability of HCC. RG also increased apoptosis and autophagy through AMPK induction. In addition, its ingredients, 20S-PPD (protopanaxadiol) and 20S-PPT (protopanaxatriol), also induced AMPK mediated apoptosis and autophagy.

RG effectively inhibited growth of HCC cells inducing apoptosis and autophagy via ATG/AMPK in HCC cells. Overall, our study suggests possibility as new anti-cancer drug on HCC by proof for the mechanism of the anti-cancer action of RG.

The liver is known as a silent organ and thus most cases are diagnosed in advanced stages with few effective therapies. Hepatocellular carcinoma (HCC) is well documented as a main inducer for human death in the worldwide [1] and a result of hepatitis C infection and cirrhosis [2]. Despite efforts to understand mechanism in the development of progression of HCC for in the last few decades, the answer to that is still minimal [2]. In 2005, sorafenib was approved from Food and Drug Administration (FDA) and is only drug regarded as first-line chemotherapy for advanced hepatocellular carcinoma (HCC) [1,3]. Although sorafenib is beneficial in the survival of HCC patients, its efficacy is short and limited because of resistance development [4]. Therefore, studies for additional drug available after failure of sorafenib treatment are required to expand survival of patients. Red Ginseng (RG) is steamed and dried the root of Panax ginseng Meyer [5]. The steaming and drying procedure induces chemical change and its active ingredients have effective pharmacological effects [6]. RG and its components are traditionally used as an important herbal medicine in various diseases through vasorelaxation, anti-diabetic, antiaging and anti-cancer activities [[7], [8], [9]]. Especially, RG reduced growth of various cancer including breast, lung, colon, glioblastoma, lymphoma and lung [7,10,11] as well as the invasion and metastasis [12]. RG enhanced sensitivity to anti-cancer drug by P-glycoprotein inhibition [13]. In recent years, novel ginseng components including ginsenosides and polysaccharides continue to be characterized by virtue of technological advancement for purification and identification and have shown potential of anticancer drug [14]. Especially, ginsenosides can be classified into three groups based on structure of aglycones: (1) protopanaxadiol (PPD) group; (2) protopanaxatriol (PPT) group; (3) oleanane group [6]. Rg3 involved anti-tumor effect by inhibiting PI3K/Akt signaling pathways and inhibited invasion in the lung cancer [15,16]. In addition, Rg3 improved the cisplatin chemosensitivity [17]. Rb1 also inhibited hypoxia induced EMT in ovarian cancer cells and exerted anti-angiogenic activity via the up-regulation of PEDF [18,19]. Rh2 showed antitumor effect in the human HCT116 colorectal cancer cells by depression of PBK [20,21]. In cellular homeostasis, there are two major types, apoptosis and necrosis, although several other forms of cell death including autophagic cell death have highlighted discovering differing pathways in recent years [22]. Apoptosis, the most well-known form, is called as a programmed cell death, because it induces a number of characteristics such as morphological changes, compaction of chromatin and specific biochemical processes [22]. While the clearance of cells from the body by apoptosis leave minimal damage to surrounding tissues, Necrosis, uncontrolled death of the cell, results in detrimental damage by pervasion of the contents of the cell [22]. Failure of apoptosis accumulates damaged cells in the body and results in development of various forms of cancer. Therefore, an understanding of apoptotic pathways is important in developing efficient chemotherapeutics for cancer. Autophagy has a line of functions in the cell protection and evolutionally conserved cellular process [23]. Autophagosomal cargo is exposed to the lysosomal hydrolases and hence break-downed. The products of the autophagosomal cargo can be back to the cytoplasm and recycled cellular metabolism [24]. In various mouse models, defects in the autophagic machinery or knockout of autophagy genes promote oncogenesis [24]. However, several studies can explain the onco-suppressive roles of autophagy by prevention from genotoxic of reactive oxygen species (ROS), contribution to oncogene-induced cell death or senescence, degradation of oncogenic proteins, immune response to malignant cells, and anti-inflammatory effect [[24], [25], [26]]. Excessive and prolonged autophagy by anti-cancer drug can lead to tumor cell death, suggesting a novel therapeutic strategy [27]. Histone deacetylase inhibitors potentiate the anti-cancer effects via autophagy in hepatocellular carcinoma [28]. Fatty-acid derivative induced the cell death through autophagy and modulation of PKM in chronic myeloid leukemia [29]. AMP activated protein kinase (AMPK) promotes balance of ATP consumption and generation in the cells [30]. AMPK is a sensor of energy imbalance as mediated with regulation of AMP and ADP [31]. AMPK can induce autophagy through two different dual mechanism by inactivating mTOR and activating ULK1 phosphorylation [32]. In the last several decades, molecular machinery about autophagy has been characterized. The autophagy-related (ATG) genes highly controlled autophagy [33]. Although autophagy is considered as a survival mechanism in the stressed or nutrient deprived cells, it also functions an important pathway to induce cell death in tumor. ATG required for autophagy triggers apoptotic cell death in cancer cells [34]. Recently, it has been shown that apoptosis and autophagy have a common process in the regulation of cell maintenance [35]. In the present study, we constructed compound-target network of RG and identified potential pathways related to HCC using network pharmacological analysis. Here, we confirmed that RG and its active components successfully induces the autophagy as well as apoptosis in HCC through ATG/AMPK by network and molecular pharmacological approach. Overall, our results may supply a better understanding of an underlying mechanism of the effects of RG and its ingredients, 20S-PPD and 20S-PPT, on HCC.

Compound-target network was constructed by identifying RG ingredients and their protein targets. The ingredients of the RG were retrieved from Symmap [36], which provides integrated information including TCMID [37] and TCMSP [38]. For our analysis, we only considered ingredients that: 1) could be mapped in PubChem IDs, and 2) had protein target information with experimental evidence. The experimentally targets of RG ingredients were obtained from the data assembled by Huang et al [39]. They comprehensively collected three types of protein target information of natural products: 1) direct target database including ChEBMBL [40] and Binding DB [41], 2) indirect target databases including STITCH [42], herbal ingredient' target databases [43], or 3) manually curated information from literatures. HCC-related proteins and pathways were retrieved to understand the mechanisms of RG on the disease of interest. HCC-related proteins were obtained by selecting associations that were labeled with treatment or marker/mechanism in the comparative toxicogenetics database (CTD) [44]. HCC-related pathways were identified from KEGG and literature as follows [45,46]: Cell cycle and signaling pathways related with AMPK, Calcium, MAPK, p53, PI3K-Akt, TGF-beta and Wnt. The pathways associated with RG targets were identified using Gene set enrichment analysis (GSEA) provided by Enrichr [47]. The Bonferroni correction was applied to correct the family-wise error generated in multiple tests. A hypergeometric test was performed to evaluate whether the number of overlapped proteins between drug targets (i.e., ginseng target) and disease targets (i.e., HCC-related proteins) was significantly differ than chance level. P values are calculated by where refers the number of HCC-related proteins among drug targets, refers the total number of drug targets (the number of targets in the dataset collected by Huang et al,), refers the number of drug targets, refers the number of proteins related to the HCC, respectively.

Anti-PARP, caspase 3, Bcl-xL, LC3BI/II, Atg 5, Atg 7, Beclin-1, phospho-AMPKα, AMPKα, phospho-ACC, phospho-AKT and β-actin antibodies were purchased from purchased from Cell Signaling (Beverly, MA, USA). Horseradish peroxidase (HRP)-tagged goat anti-mouse and goat anti-rabbit IgGs were obtained from Novus Biologicals (Littleton, CO, USA). RG was from Korea Tobacco & Ginseng Corporation (Daejeon, Korea). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). RG was kindly provide as previously described [48].

HepG2, Hep3B and Huh7 hepatocyte-derived cells, and HeLa cells were purchased from the ATCC (Rockville, MD, USA). HepG2 cells and Hep3B (ATCC (Rockville, MD, USA) were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS and 50 units/ml penicillin and 50 μg/ml streptomycin, and Huh7 were grown in RPMI 1640 [48].

HepG2 and Hep3B cells were plated and cultured to confluency of 80-90 % and incubated in FBS free media for 12 h. Huh7 cells were plated in 5 % FBS added media and cultured to equal confluency [48]. Microplate reader (Tecan, Research Triangle Park, NC, USA) was used to assess. Absorbance was measured at 570 nm [48,49].

Huh7 cells were stained with annexin V / propidium iodine (PI) (BD Bioscience, San Jose, CA) and cell cycle detection (BD Bioscience, San Jose, CA) kits according to manufacturer's protocol, and analyzed by Accuri C6 flow cytometer (Accuri Cytometers Inc., Ann Arbor, MI, USA) [49].

Cell extracts from treated cells as described were prepared by incubating at 4 °C with Lysis Buffer (Thermo, Rockford, U.S.A.) as previously described [48].

Fixed cells by 4% paraformaldehyde for 10 min were washed with PBS for 3 times and stained with AO (10 mg/mL) solution for 15 min at room temperature. Followed by staining, culture slides were covered with mounting solution and observed under fluorescence microscope for detecting autophagy vacuole [49].

The experimental results were presented as mean ± standard deviation (S.D.) or S.E.M. For each significant treatment effect, the T-test or one-way ANOVA was utilized to compare multiple groups' means. The criterion for statistical significance was set at P < 0.05 or P < 0.01 [49].

We first identified RG ingredients and their protein targets from TCMSP and the dataset assembled by Huang et al, respectively. We initially found 1732 compound-protein interactions (CPIs) between 56 RG ingredients and 783 protein targets. Among protein targets of RG, CASP3, TNF, PTGS2, AKT1, and RELA showed the highest degree (18, 17, 16, 15, and 15, respectively). On the other hands, we found that 698 targets were interacting with only less than four of RG ingredients (4/153). To focus on protein targets associated with sufficient number of RG ingredients, we considered 85 targets interacting with five or more ingredients as key targets and conducted subsequent analysis focusing on them. To investigate the association between key targets and HCC, we compared overlapping numbers between key targets and HCC-related proteins, and found that approximately 40% of key targets (32/85) were overlapped with HCC. We conducted the hypergeometric test to check whether the observed number of overlapped targets was higher than random expectations. We found that the observed value was significantly higher (6.91 fold) than the random expectations (p-values < 10−15). This result indicates that the key targets of RG are closely associated with HCC-related proteins, highlighting the potential of RG as an anti-HCC agent. We then investigated which HCC-related signaling pathways are associated with RG targets. The results of enrichment analysis on the KEGG database revealed 5 pathways known to be associated with HCC were significantly associated with the key targets of RG (Fig. 1A). Specifically, we found that AMPK pathway, which can contribute to HCC treatment by inducing apoptosis, was also closely involved with key targets of RG [32]. By integrating information about the identified ingredients, protein targets, and the associated pathways of RG, we constructed and visualized the compound-target network of RG (Fig. 1B). The network consisted of 661 CPIs between 85 proteins and 49 RG ingredients. In the network, nodes represented RG ingredients and their targets, whereas edges represented the interactions between RG ingredients and protein targets. The number of related targets for the PI3K-Akt signaling pathway, MAPK signaling pathway, p53 signaling pathway, Cell cycle, and AMPK signaling pathway were 21, 15, 10, 8, and 7, respectively. Specifically, we found that CCND1, TP53, AKT1, CCNB1, CDK1, EGFR, and IGF1R were simultaneously involved in HCC and HCC-related pathways.

To investigate the cytotoxic effect of RG on human HCC cell lines, we evaluated effects of its on cell viability using the MTT assay. We treated different doses of RG with HepG2, Hep3B and Huh7 cell lines for 24h. As a result, 5 and 10 mg/ml of RG with three cell lines showed significant cytotoxicity by decreased cell viability compared to control (Fig. 2A–C). To confirm cell morphological changes by RG on human HCC cell lines, We used Huh7 cells, which decreased sensitively with increasing RG dose, in subsequent experiments. The results showed that treatment of the Huh7 with different doses of RG (1, 2 and 5 mg/ml) after 24h died the cell by changing cell morphology such as cell shrinkage and ballooning (Fig. 2D). RG inhibitions of viability was also confirmed by the fluorescent detection stained with Calcein AM and PI (Fig. 2D).

To determine whether RG inhibits cell survival by induction of apoptosis, we analyzed by flow cytometric analysis of annexin V and PI dual staining. Fluorochrome-labeled Annexin V can be used to specifically target and identify apoptotic cells. and PI is used to help differentiate between apoptotic and necrotic cells. Annexin + PI-stained cells percentage was increased in RG dose dependent manner. In 5 mg/ml RG treated cells, apoptotic cell percentage was significantly elevated (Fig. 3A and B). We further confirmed the RG-induced apoptosis by immunoblot to measure apoptosis related protein expression level, such as PARP, caspase3 and Bcl-xL in huh7 cells. RG dose- and time- dependently up-regulated the protein expression of cleaved PARP, which produced by cleavage of PARP when apoptosis. and, caspase3, which is activated after cleavage and then cleaves PARP, and Bcl-xL, which leads to the suppression of caspase, were dose- and time-dependent down-regulated (Fig. 3C and D). This suggests that RG induces cell apoptosis of Huh7 cells.

To confirm whether RG inhibit HCC cell migration, we assessed wound healing assay in RG-treated Huh7 cells. The motility of Huh7 cells was suppressed with treatment of 2 and 5 mg/ml RG for 24h (Fig. 3E). The wound width percentages of cells treated with 0, 1, 2 and 5 mg/ml RG were 1.66 ± 0.69, 43.05 ± 0.8.70, 54.37 ± 4.95 and 71.42 ± 2.64, respectively (Fig. 3F upper). The percentage of confluence of cells treated with 0, 1, 2 and 5 mg/ml RG were 98.34 ± 0.69, 56.95 ± 8.70, 45.63 ± 4.94 and 28.58 ± 2.26, respectively (Fig. 3F lower). In RG-treated huh7 cells, the wound width percentage increased in dose dependent manner, and the percentage of confluence decreased in dose dependent manner in dose dependent manner. This result suggests that RG inhibits cell growth via reducing the migration of HCC

As predicted in the network pharmacological analysis, RG-induced AMPKα activation was analyzed in Huh7 and HepG2 cells. Phosphorylation of AMPKα or ACC, a downstream molecule of AMPK, significantly increased with 2 mg/ml RG (Fig. 4A and C). The AMPKα was maximally activated at 4 h after treatment and maintained for 8 h (Fig. 4A). Upon treatment with RG, the expression of AMPKα activation also increased in a dose-dependent manner (Fig. 4B). Moreover, RG did not induce AMPK activation in the LKB1-deficient HeLa cells, which means the role of LKB1 for the RG induction of AMPK activation (Fig. 4D). These results showing here indicated that RG successfully increased the AMPKα activation. It has been shown that AMPKα induces autophagy, which inhibits cellular survival by inducing the apoptosis in cancer. To determine whether RG induced autophagy, we examined acridine orange (AO) staining to detect acidic autophagy vacuoles (AVOs) accumulated in Huh7 cells (Fig. 4E). An increase in AVOs is a phenomenon during cell autophagy. In 2 mg/ml RG treated cells, it was observed the increased AVOs formed under fluorescent microscope. Next, to further confirm the effect of RG-inducing autophagy in Huh7 cells, we analyzed expression of autophagy related proteins, such as LC3B I/II, Atg5, Atg7 and Beclin-1 in each dose and time dependent manner. LC3B is one of the factors used to confirm the increase in autophagy in cells, and autophagy can be confirmed by an increase in the ratio of LC3BII/LC3BI. We confirmed that LC3BI was converted to the form of LC3BII as the treated RG dose increased. Atg5, which induces autophagy, showed the highest expression level in 2 mg/ml RG treated cells for 24 hours. Moreover, the expression of Beclin-1, which is important in both autophagy and cell death, was elevated in 2 mg/ml RG treated cells compared non treated cells (Fig. 4F). Upon treatment with 2mg/ml RG, the expression of LC3BII, Atg7 and Beclin-1 was increased in a time-dependent manner. Atg5 increased time-dependently, but decreased at 48h (Fig. 4G).

We tried to identify the active compounds of RG focusing on ginsenosides, known as the major and unique phytochemical classes in the RG [50]. Therefore, we further investigated 11 compounds (Rb1, Rb2, Rg1, Rg3 20S, Rg3 20R, PPD 20R, PPD 20S, PPT 20S, compound K, Rc, Re) (Table 1). The active compounds of RG were determined through the cell viability of cells using MTT assay. MTT assay showed that 20S-PPD and 20S-PPT reduced cell viability compared to other compounds. In particular, 20S-PPT was most effective compound by reducing the cell viability to 60.2% compared with non-treatment (Fig. 5A).

We next examined the mechanisms of inhibition of cell viability of the active compounds, 20S-PPD and 20S-PPT. Treatment of 50μm of 20S-PPD and 20S-PPT each in huh7 cells showed increased expression of cleaved PARP related to apoptosis and increased expression of p-AMPKα and LC3B-II related to autophagy (Fig. 5D). Next, we performed Propidium iodide (PI) stain, which evaluates the cell viability by DNA staining of compromised cells, on huh7 cells each treated with 50μm of 20S-PPD and 20S-PPT, respectively. Both 20S-PPD and 20S-PPT treatments showed inhibition of cell viability through the increase of Sub-G1 in the cell cycle (Fig. 5C). In addition, Colony forming cell (CFC) assay was performed on huh7 cells treated with 50μm of 20S-PPD and 20S-PPT, respectively, to evaluate cell survival ability. CFC assay showed that 20S-PPD and 20S-PPT reduced cell survival compared to non-treat (Fig. 5B). Therefore, this result suggests that 20s-PPD and 20s-PPT of RG are effective compounds that inhibit cell growth.

Anti-cancer drugs and treatment strategies for the primary liver cancer, predominantly HCC, are very limited. It is urgent to develop new anti-cancer drugs and find new ways to treat them. In the present study, we suggested that RG can be a good candidate for new anti-cancer drug to inhibit growth of HCC through cell death by apoptosis based on both the network and molecular pharmacology. Here, we conducted a network pharmacological analysis and showed that RG has therapeutic potential against HCC by regulating various protein targets and signaling pathways. Then, we showed that RG inhibited growth of HepG2, Hep3B and Huh7 HCC cells. Cell death by RG was involved in apoptosis inducing cleavage of PARP and caspase 3 through activation of AMPKα and autophagy. Ginsenosides responsible for the anticancer effect may differ in pharmacology and mechanisms because of its chemical structure. Among the various ginsenoside of RG, PPD and PPT were effective in suppressing the growth of HCC in this study. PPD and PPT also induced apoptosis through activation of AMPKα and autophagy such as RG in HCC cells. Network pharmacology is one of the useful tools to predict the unknown efficacy of natural products or to explore the potential mechanisms of known efficacy [50]. Although its usefulness, the application could be limited in validating the molecular mechanisms between herbal components and target molecules as well as each herb's mode of action (MOA). Therefore, we thus leveraged network pharmacology with molecular experiment to systematically disect the efficacy and its mechanisms of RG on HCC. Our results proposed that the integrating these approaches could be an efficient way to identify system-level mechanisms of natural products. The various effects of natural products have been one of the important therapeutic means in combating human diseases. Among these natural products, red ginseng is one of the natural products recognized around the world for its particularly excellent efficacy. There are various effects of red ginseng, among which the anti-cancer effect is well known [51,52]. Previous finding showed that oral administration of RG extracts was suggested as cancer chemoprevention on spontaneous liver tumor formation [51]. In addition, RG efficaciously protects against the liver fibrosis [52]. However, studies of the effect of ginseng on human HCC were poorly carried out. This study describes potential effect as anti-cancer agent against HCC including some evidence of molecular mechanism. We first tested whether RG inhibits growth of HCC Cells by MTT analysis. As expected, RG inhibited growth in the HepG2, Hep3B, and Huh7 HCC cells. Especially, growth inhibition by RG treatment was shown the morphology of cell death. Consequently, we investigated mechanism of cell death by RG. RG significantly induced apoptosis judging from annexin V & PI staining, reduction of pro-caspase 3, and PARP cleavage. Apoptosis induced by RG might be related with downregulation of Bcl-xL. This is matched with that some anticancer drugs in the market are also used for therapeutic tools by inducing apoptosis [53]. Autophagy is a process which degrades and reuses the damaged organelles and waste protein. Autophagy is controversial that have been involved tumor suppression as well as tumor promoting function. Several cancer inhibitory agents have been reported to induce apoptosis and autophagy [23]. We showed that RG could induce autophagy by the formation of AVOs in the cell stained with acridine orage. In addition, RG significantly increased autophagy-related proteins Atg5, Beclin 1 and the ratio of LC3II/LC3I, autophagy marker, in the dose dependent manner. AMPK promotes autophagy, which is associated with many diseases, including cancer myophathy and neurogeneration [30]. Recent studies have been shown that AMPK is associated with autophagy by down regulation of mTOR, which mean that some factors in the autophagy could involve the AMPK/mTOR signaling [34]. In this study, RG increased the AMPK activation, which is in line with previous studies. The action mechanism of ginseng had known since ginsenosides were isolated. Various studies have reported that ginsenosides were responsible for the pharmacological and immunological activities for a long time. Especially, major ginsenosides, such as Rg1, Rg3 Rb1, Rb2, Re and Rc, exerted its anti-cancer effects on various cancer cells including breast, lung, ovarian, colon [54,55]. Especially, Rg1 showed effects as a potent candidate drug against nasopharyngeal cancer [54]. Clinical research demonstrated that ginsenosides have anti-cancer activities to a line of tumors [55]. PPD and PPT exerted its anti-cancer effects on various cancer cells. PPD acted as anti-cancer agent in MDA-MB-231 cells, and induced cell death in the colorectal cancer cells by induction of paraptosis through ROS production and NF-κB pathway [56]. PPT also inhibited the invasion in the lung cancer cells, and exhibited a synergistic effect on MDA-MB-231 cell proliferation through combination with Rh2 [57]. However, despite the excellent effects of PPD and PPT on cancer, research has been insignificant. Previous evidence suggested that Protopanaxadiol induces Sestrin2 expression and induces through AMPK activation [58]. 20S-PPD appears to induce apoptosis of hepatic stellate Cell (HSC) by activating LKB1–AMPK [59]. Consistent with this, we found that PPD and PPT induced apoptosis and autophagy through AMPK activation in Huh7 cells such as RG. Despite evidence of previous studies, the association between PPD/PPT and AMPK is not yet clear and poor. Especially, research on the effects of PPD/PPT on HCC cells is minimal and their activation of AMPK imply the possibility of the beneficial effects to prevent cancer. Besides, we showed that RG inhibited migration of HCC cells through wound closure analysis, indicating RG might prevent cancer metastasis. Unfortunately, we could not investigate crosstalk between apoptosis and autophagy by RG and PPD and/or PPT. The combination with autophagy inhibitor/activator and RG is expected to increase effectiveness of anti-cancer on HCC. Although the exact action of mechanisms by PPD and PPT, and effects of combination therapy remains to be investigated, we suggested that RG could reduce growth of HCC cells through AMPK activation by effect of PPD and/or PPT. Furthermore, combination therapy with RG and anti-cancer or autophagy inhibitor can accelerate effect on HCC cells. This finding provides new insights into chemotherapeutic effects of ginseng on cancer beyond the sorafenib [60].

Conceptualization, Park KI and Kim YW; Investigation, Lee WY, Yun UJ, Bak SB, Kim YW, Bae SJ, Song CH, Kim KY, Lee EH and Yang JH; writing—original draft preparation, Yun UJ, Kim YW, Bak SB and Lee WY; writing—review and editing, Kim YW, Bak SB, Lee WY and Park KI; Funding acquisition, Kim YW, Yang JH, Song CH, Kim SC and Park KI. All authors have read and agreed to the published version of the manuscript.

This research was also supported by a grant of the Korea Health Technology R&D Project through the 10.13039/501100003710Korea Health Industry Development Institute (10.13039/501100003710KHIDI), funded by the 10.13039/100009647Ministry of Health & Welfare, Republic of Korea (number: HF20C0212) and (number: HF21C0061). This work was also supported by 10.13039/501100001321National Research Foundation (NRF) grant funded by the Korean government (No. 2018R1A5A2025272) and (No. 2022R1I1A3053818). This research was supported by the 10.13039/501100008783National Research Council of Science & Technology (10.13039/501100008783NST) grant by the Korea government (10.13039/501100014188MSIT) (No. CPS22041-100).

The authors declare no conflict of interest.