Anthocyanins from Lycium ruthenicum Murray Inhibit HepG2 Cells Growth, Metastasis and Promote Apoptosis and G2/M Phase Cycle Arrest by Activating the AMPK/mTOR Autophagy Pathway

Among the most common malignancies in humans, liver cancer ranks third in terms of mortality in the world. Seeking new anticancer drugs or adjuvant chemotherapy drugs from natural products has attracted the attention of many researchers. Lycium ruthenicum Murray (LR), a health food and traditional Chinese medicine, exerts extensive pharmacological properties, of which anthocyanins are one of the key active components. In this research, we explored the antitumor activity and autophagy regulation mechanism of anthocyanins from Lycium ruthenicum Murray (ALR) in HepG2 cells. Our results found that ALR profoundly reduced the cell viability, clone formation, migration, and invasion and promoted apoptosis and G2/M phase arrest of HepG2 cells in a dose-dependent pattern. Further studies confirmed that ALR treatment significantly increased the number of autophagic vacuoles and autophagosomes, upregulated the expression of Beclin-1, p62, LC3-II/LC3-I, and p-AMPK, and concomitantly downregulated the expression of p-mTOR. When autophagy was inhibited by 3-methyladenine (3-MA), ALR-induced proliferation inhibition, invasion, and migration capabilities, as well as apoptosis rate and G2/M phase arrest, were all reversed, and the activities of key proteins in the AMPK/mTOR pathway were all constrained. In summary, the results presented here indicate that ALR may be effective as a natural antitumor agent by activating AMPK and inhibiting the mTOR autophagy pathway in HepG2 cells.


Introduction
Te majority of liver cancers in the digestive system are hepatocellular carcinoma (HCC), which accounts for 75-85% of cases [1]. By 2030, liver cancer will cause approximately 1,000,000 deaths, according to the World Health Organization [2]. In clinical practice, surgery, chemotherapy, and radiotherapy are the conventional treatment approaches for HCC [3]. However, these treatments have limited efcacy for advanced HCC and have serious side efects due to their weak selectivity for cancer cells [4]. Hence, it is urgently necessary to develop efcient and safe new antitumor drugs.
Natural products are an important source for discovering anticancer or adjuvant chemotherapy agents. At present, many natural or mimicked natural products have been approved for clinical use as anticancer drugs, such as irinotecan, vincristine, etoposide, and paclitaxel [5]. LR is vividly known as "the king of anthocyanins" because of its high content of anthocyanins [6]. Its fruit has become an indispensable ingredient in people's daily diets [7]. Numerous researchers have noted that anthocyanins play a signifcant role in cancer prevention and management [8]. For example, anthocyanins derived from black chokeberries exhibited anticancer activity by preventing the growth, adhesion, and metastasis of SK-Hep1 cells [9]. In addition, anthocyanin supplements as a dietary strategy may possess potential anticancer activity by suppressing the viability of cancer cells, but epidemiological studies in humans still need further confrmation [10]. Mechanistically, the anticancer efects of anthocyanins are mainly involved in apoptosis and autophagy [11,12]. Unlike apoptosis, which ultimately results in cell death, autophagy determines cell fate in a bidirectional manner, relying on the strength and duration of irritants [13]. Moderate autophagy can maintain cell homeostasis to promote cell survival, while excessive autophagy impairs essential cellular components and processes to activate apoptosis or necrosis, ultimately leading to cell death [14]. A study found that mulberry anthocyanins may be used as adjuvant therapy in patients with thyroid cancer by activating autophagic cell death [12], while another study confrmed that anthocyanins in black soybean prevented apoptosis by inducing autophagy in U2OS cells [15]. Tese studies reveal that anthocyanin-induced autophagy plays a completely diferent role in distinct tumors. A growing body of literature has demonstrated that the AMPactivated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway plays an important role in the regulation of autophagy [16]. AMPK, an energy sensor, is a positive regulator of autophagy, and its activation can lead to decreased phosphorylation of mTOR, thereby promoting cellular autophagy [17]. Nevertheless, the exact role of ALR in HCC and whether it has a selective toxic or protective efect on HepG2 cells in terms of autophagy remain unclear. Terefore, it is crucial to clarify the role and regulatory mechanism of autophagy in ALR anti-HCC.
In this research, we frst evaluated the antitumor properties of ALR in HepG2 cells and then further explored its possible antitumor mechanism. Our study is the frst to demonstrate that ALR plays an anti-HCC role by inhibiting HepG2 cell proliferation and metastasis and promoting apoptosis and G2/M phase cycle arrest by activating the AMPK/mTOR autophagy pathway. According to our fndings, ALR can be used as a potential candidate or adjuvant agent for treating HCC.

Cell Culture and Sample Treatment.
Human hepatocellular carcinoma cell lines (HepG2) and human normal hepatocyte cell lines (LO2) were bought from the Shanghai Institute of Cell Biology, China. Cells were grown in DMEM containing 10% FBS and 1% penicillin/streptomycin (P/S, Solarbio, Beijing, China) at 37°C and 5% CO 2 humidifed atmosphere (Termo, Waltham, USA). Te 3-MA was dissolved in serum-free DMEM medium to prepare 20 mM mother liquor, fltered, aliquoted, and stored at −20°C for subsequent assays. Te ALR was refrigerated and packed in tin foil, dissolved in serum-free DMEM medium, and used immediately after aseptic fltration with a 0.22 μm syringe flter.

Cell Viability Assay.
HepG2 and LO2 cells were planted on a 96-well plate overnight and then stimulated with varying doses of ALR with or without 3-MA (2.5 mM) for another 24 or 48 h. After that, the CCK-8 reagent (Beyotime, Shanghai, China) was appended to each well (10 μL/well), and then incubated for additional 30 min at 37°C, followed by measuring the optical density (OD) value at 450 nm using a microplate reader (Multiskan GO, Termo, USA), with 5 duplicates in each group. Cell viability (%) � (OD treatment − OD blank )/(OD control − OD blank ) × 100%.

Colony Formation Assay.
HepG2 cells were planted on a 6-well plate overnight and then stimulated with varying doses of ALR for another 24 h. After that, the cells were grown in DMEM medium containing 0.5% FBS and 1% P/S for approximately 16 days until colonies were evident. Ten, the colonies were fxed and colored using 4% paraformaldehyde and 0.1% crystal violet, respectively, and fnally, pictures were captured under natural light.

Cell Scratch Assay.
HepG2 cells were planted on a 6-well plate until the density reached 95%. Ten, the cells were scratched using 200 μL sterile tips and cultured for 24 h with diferent doses of ALR with or without 3-MA (2.5 mM). Te wound width of each group was observed and captured under a microscope. By comparing the average area of migration to the starting scratch area, the migration ratio was calculated.

Cell Migration and Invasion Assays.
After treatment with varying doses of ALR with or without 3-MA (2.5 mM) for 24 h, HepG2 cells were resuspended in DMEM medium without FBS and planted into a 24-well transwell (upper chamber, pore size 8 μm, Corning) at a density of 2 × 10 4 cells/ well (precoated with 0.5 mg/mL Matrigel (Corning, NY, USA) for invasion assay, not for migration assay). Te lower chamber was flled with the DMEM complete medium. After 24 or 48 h, the cells in the upper chamber were scrubbed with cotton swabs, while the cells that migrated or invaded the lower chamber were fxed and colored using 4% paraformaldehyde and 0.1% crystal violet, respectively, and fnally, pictures were captured using an inverted microscope.

AO/EB, AO, MDC Staining.
HepG2 cells were planted on a 12-well plate overnight and stimulated with varying doses of ALR with or without 3-MA (2.5 mM) for another 24 h. After that, the cells were incubated separately with 5 μL acridine orange (AO) solution (Sangon Biotech, Shanghai, China) or 5 μL AO/EB (Sangon Biotech, Shanghai, China) at room temperature, protected from light for 5 min, or with 25 μL monodansylcadaverine (MDC, Solarbio, Beijing, China) at room temperature, protected from light for 15 min. Finally, the images were photographed under an inverted fuorescence microscope.

mGFP-LC3-Adenovirus
Staining. HepG2 cells were planted on a 12-well plate overnight and transduced with 10 MOI mGFP-LC3-adenovirus (HanBio, Shanghai, China) for 48 h. After that, the cells were stimulated with varying doses of ALR with or without 3-MA (2.5 mM) for another 24 h. Finally, the images were captured under an inverted fuorescence microscope.
2.9. Flow Cytometry Assay. HepG2 cells were planted on a 6-well plate overnight and then stimulated with varying doses of ALR with or without 3-MA (2.5 mM) for another 24 h. After that, the cells were treated with trypsin without EDTA and divided into two parts. One part was stained using 5 µL annexin V-FITC and 5 µL propidium iodide (KeyGen, Nanjing, China) for analysis of apoptosis. Te other part was fxed in chilled 70% ethanol overnight, followed by incubation with 100 μL RNase A solution and 400 μL propidium iodide (Solarbio, Beijing, China) for analysis of the cell cycle by fow cytometry (Beckman, SA, USA).

Western Blot Assay.
After treatment with varying doses of ALR with or without 3-MA (2.5 mM) for 24 h, HepG2 cells were collected and lysed, and the total protein concentration was detected by the BCA protein detection kit (KeyGen, Nanjing, China). Equal concentrations of protein were subjected to 12% SDS-PAGE gels and transferred to a PVDF membrane (Millipore, MA, USA). Ten, the membrane was blocked with skim milk for 60 min and incubated with primary antibodies overnight at 4°C, followed by rinsing the membrane and incubating with secondary antibodies for 60 min. Finally, the protein bands were immersed in the enhanced chemiluminescence (ECL) substrate solution (Termo, Carlsbad, USA) in the dark for 1 min and then analyzed using a protein chemiluminescence detector (Bio-Rad, USA). As a loading control, β-actin was detected simultaneously.
2.11. Statistical Analysis. All data in this study were analyzed with one-way analysis of variance (ANOVA) by using SPSS 26.0 (IBM, NY, USA), and were expressed as mean ± SD of three independent experiments. Diferences were considered signifcant at P < 0.05, P < 0.01, and P < 0.001.

ALR Suppresses the Viability and Proliferation of HepG2
Cells. To investigate whether ALR afects the viability and proliferation of HepG2 cells, the CCK-8 and colony formation experiments were carried out. After treatment with ALR (0, 250, 500, 750, 1000 and 1500 μg/mL) for 24 h and 48 h, CCK-8 results showed that the viability of HepG2 cells was suppressed in a time-and dose-dependent pattern (Figure 1(a)). Following 24 h of treatment, ALR at 500 μg/ mL or above had a signifcant inhibitory efect on HepG2 cell activity, while ALR at 1000 μg/mL or below had no significant inhibitory efect on LO2 cells (Figures 1(a) and 1(b)). Terefore, the low concentration (500 μg/mL) and high concentration (1000 μg/mL) of ALR for 24 h were selected for follow-up experiments. Te cell morphological changes were also observed by inverted microscopy, as shown in Figure 1(c). ALR treatment signifcantly decreased the cell numbers and adsorption capacity of HepG2 cells after 24 h, and the changes became more apparent with the increase in ALR concentration. In addition, the proliferation capacity of HepG2 cells was signifcantly inhibited in a dose-dependent pattern, as confrmed by the clone formation assay (Figure 1(d)). Te above results suggest that ALR can suppress the viability and proliferation of HepG2 cells.

ALR Suppresses the Migration and Invasion of HepG2
Cells. To further evaluate the impact of ALR on the metastatic potency of HepG2 cells, the scratch assay, longitudinal migration, and invasion assays were performed. Te scratch assay result (Figure 2(a)) showed that after 24 h of ALR treatment, the migration area on HepG2 cells was signifcantly decreased, and the migration ability was suppressed in a dose-dependent pattern. Te transwell assays further indicated that the numbers of migratory and invasive cells were signifcantly reduced after ALR treatment for 24 h (Figures 2(b) and 2(c)). Tese fndings reveal that ALR can inhibit the migration and invasion abilities of HepG2 cells.

ALR Promotes the Apoptosis and Cycle Arrest of HepG2
Cells. To illustrate whether ALR inhibition of HepG2 cell proliferation is related to apoptosis and the cell cycle, we frst detected the apoptosis rate by AO/EB and V-FITC/PI staining. Te AO/EB result showed that abundant normal cells stained green in the control group, with intact nuclei and clear boundaries. However, with the increase of ALR concentration, some cells showed apoptosis characteristics such as chromosome shrinkage and fragmentation, and the numbers gradually increased, presenting bright orange (Figure 3(a)). Te fow cytometry result further showed that the apoptotic rate of HepG2 cells increased signifcantly with the increase of ALR concentration, as shown in Figure 3(b). Next, the cell cycle was further measured, and in comparison with the control group, ALR at 500 and 1000 μg/mL blocked the G2/M phase of HepG2 cells in a dose-dependent pattern (Figure 3(c)). Te above results suggest that ALR can promote HepG2 cell apoptosis and G2/M phase cycle arrest in a dose-dependent pattern.

ALR Induces the Autophagy Morphological Changes of
HepG2 Cells. During tumorigenesis and development, autophagy plays a crucial role. To afrm whether ALR induces autophagy in HepG2 cells, the morphological changes of autophagy were detected by the following experiments: AO and MDC are two kinds of autophagic fuorescent dyes used to label autophagic vacuoles. When autophagy occurs, AO staining cells release orange-red fuorescence, while MDC staining cells show blue fuorescence under a fuorescence microscope. GFP-LC3-adenovirus is used to label and track LC3, which is an autophagosome indicator, and the number of green spots represents the strength of autophagic fow. Figure 4(a) suggests that increasing the concentration of ALR signifcantly increased the number of fuorescent cells, indicating that ALR can induce the occurrence of autophagic fux.
To further confrm the autophagy of HepG2 cells induced by ALR, 3-methyladenosine (3-MA), which inhibits autophagy by blocking the formation of autophagosomes, was applied to observe the changes in autophagy's morphology. As shown in Figure 4 detection. Te above results suggest that ALR can induce autophagy morphological changes in HepG2 cells.

ALR Afects Autophagy-Related Proteins of HepG2 Cells.
Te occurrence of autophagy depends on the expression of related proteins; therefore, the protein levels of Beclin-1, LC3-I, LC3-II, and p62 were further examined by western blot before and after 3-MA intervention. Figure 5(a) indicates that the Beclin-1, p62, and LC3-II/LC3-I were signifcantly upregulated with increasing ALR concentration. After intervention with 3-MA in HepG2 cells, compared with the ALR alone, ALR combined with 3-MA signifcantly downregulated Beclin-1 and LC3-II/LC3-I levels, whereas p62 was still upregulated (Figure 5(b)). Te increase of p62 predicts that the cells may develop defective autophagy for the blockage, which will be further verifed in later experiments. Taken together, the above results once again prove that ALR does cause autophagy in HepG2 cells.

Blocking Autophagy Impairs the Proliferative Activity and
Metastasis Potential of ALR-Treated HepG2 Cells. To reveal whether the inhibition of HepG2 cells activity and function by ALR is related to the occurrence of autophagy, the CCK-8, scratch assay, migration, and invasion assays were again

Blocking Autophagy Impairs the Apoptosis and Cycle of ALR-Treated HepG2 Cells.
We have previously confrmed that the ALR can induce apoptosis, autophagy, and cycle arrest in HepG2 cells, but whether the apoptosis and cycle are related to autophagy is unknown. To this end, we explored the efects of inhibiting autophagy on apoptosis and the cell cycle. As shown in Figures 7(a) and 7(b), the apoptotic cells in the ALR combined with 3-MA treatment  group were less signifcant than those in the ALR alone group, as confrmed by the assays of AO/EB staining and V-FITC/PI staining. A similar trend was observed that the percentage of G2/M phase in the combined group was reversed compared with the ALR group (Figure 7(c)). Te above results show that the ALR regulates apoptosis and cycle of HepG2 cells through autophagy.

ALR Regulates Autophagy of HepG2 Cells by AMPK/ mTOR Pathway.
Considering the important role of the AMPK/mTOR signaling pathway in autophagy, we examined by western blot whether this cascade is involved in ALR-induced autophagy. As illustrated in Figure 8(a), in comparison with the control group, with the increase in ALR concentration, the p-AMPK/AMPK protein level was markedly upregulated, while the p-mTOR/mTOR level was signifcantly downregulated. However, the p-AMPK/AMPK and p-mTOR/mTOR protein levels were all reversed in the ALR combined with 3-MA group compared with the ALR alone group (Figure 8(b)). Taken together, these results reveal that ALR can induce autophagy in HepG2 cells by activating the AMPK/mTOR signaling pathway.

Discussion
In recent years, with the enhancement of health consciousness, people have paid more and more attention to dietary intervention for cancer prevention and management, and some natural products with wide sources and low or no toxicity have shown promising efects in this regard [18,19]. LR (also called black goji berry) is a wild prickly shrub, which is mainly planted in northwestern China such as Ningxia, Qinghai, and Xinjiang due to its unique drought-resistant Evidence-Based Complementary and Alternative Medicine 7 and salt-resistant characteristics [20]. Te fruit of LR, with a violet-black appearance, has been broadly utilized in China as nutraceutical food and folk medicine for at least 2000 years [21]. LR contains a variety of bioactive ingredients, e.g., favonoids, anthocyanins, polysaccharides, alkaloids, phenolic acids, etc., which make it exhibit antiinfammatory, antitumor, antiaging, anti-infertility, and other biological functions [7]. LR contains a large amount of anthocyanins, a class of water-soluble favonoid compound widely existing in plants, and is an ideal plant source for anthocyanin extraction [6]. Te basic structural unit of anthocyanins is favylium, and the main components include pelargonidin, cyanidin, delphinidin, petunidin, malvidin, etc. [22]. Although multiple studies have demonstrated the anticancer efects of anthocyanins extracted from strawberries, grapes, corn, etc. [8], there is still a lack of investigation on the anti-HCC activity and exact molecular mechanism of ALR. Terefore, in this research, we frst investigated the efects of ALR on the proliferation, migration, and invasion of HepG2 cells. Our results (Figures 1  and 2) verifed that ALR signifcantly suppressed the viability, clone formation, migration, and invasion of HepG2 cells in a dose-dependent pattern, which showed consistent biological functions with other sources of anthocyanins [11]. A study reported that apoptosis induction and cycle arrest were closely related to tumor proliferation inhibition [23]. Terefore, we further analyzed the apoptosis and cycle of ALR-treated HepG2 cells. Te result suggested that the apoptotic cells signifcantly increased with increasing ALR concentration, and the cells were arrested in the G2/M phase, as indicated by AO/EB staining, V-FITC/PI staining, and cycle analysis (Figure 3). Li et al. also demonstrated that anthocyanins derived from Lycium ruthenicum Murray induced apoptosis in DU-145 prostate cancer cells [24]. However, studies found that anthocyanins have diferent targets on the cell cycle for diferent cancer cells. For example, anthocyanins from blue corn and tortillas could arrest the cycle of breast and prostate cancer cells at the G1 phase [11].
Research has demonstrated that autophagy plays a pivotal role in regulating cell survival [25]. Autophagy is a selfprotective mechanism for cells to adapt to various environmental changes and maintain homeostasis [26]. Te occurrence of autophagy is regulated by a series of  Figure 5: ALR afects the changes of autophagy-related proteins of HepG2 cells. Western blot assay to examine the protein changes of Beclin-1, p62, LC3-I, and LC3-II without 3-MA intervention (a) and with 3-MA intervention (b) * P < 0.05, * * P < 0.01, and * * * P < 0.001, vs. the control group. # P < 0.05, ALR vs. the ALR+3-MA group. 8 Evidence-Based Complementary and Alternative Medicine autophagy-related proteins [27]. For example, Beclin-1 participates in the beginning of autophagy and the maturation of autophagosome [28]. During the autophagosome formation, LC3-I is transformed to LC3-II, and the LC3-II/ LC3-I ratio is usually used to assess autophagy levels [29]. Te autophagy substrate p62 is fused into the autophagosome by interacting with LC3 and then gradually degraded by the autophagolysosome [30]. A variety of natural compounds, such as silibinin and resveratrol, have been proven to promote autophagy in various cancer cells [31,32]. We also discovered a signifcant aggregation of autophagic vacuoles and autophagosomes with the increase in ALR concentration, as confrmed in Figure 4. Furthermore, the Beclin-1, p62, and LC3-II/LC3-I protein levels were signifcantly increased in ALR-treated HepG2 cells ( Figure 5). Notably, p62, an autophagy negative regulator, was not reduced; instead, p62 expression was signifcantly upregulated in ALR-treated HepG2 cells, especially when ALR was combined with 3-MA ( Figure 5(b)). Tis phenomenon indicated that ALR blocked autophagy fux, which meant autophagosomes were formed but had not been degraded.
Clinical studies have confrmed that aggregation of p62 could lead to poor prognosis for HCC patients [33]. Studies suggested that the aggregation of p62 could activate the Nrf2 antioxidant system and resist ferroptosis in liver cancer cells, thereby promoting the growth of HCC [34]. Whether ALRinduced p62 aggregation is related to ferroptosis remains to be further explored.
Given that the role of autophagy in tumors sometimes involves paradoxical functions, we further analyzed the relationship between autophagy and antitumor activity. We observed that combined treatment with 3-MA attenuated the proliferation inhibition, invasion, migration, apoptosis, and G2/M phase cycle block of ALR-induced HepG2 cells, as shown in Figures 6 and 7, suggesting that autophagy plays a cytotoxic role. However, Chen et al. found that autophagy inhibition promoted the apoptosis and antiproliferative activity of delphinidin-induced breast cancer HER-2 cells, suggesting that delphinidin induces protective autophagy [17]. Tese studies indicate that anthocyanin-induced autophagy plays diferent roles in diferent tumors. To further illuminate the mechanism of autophagy regulation, in view of the crucial role of mTOR in regulating autophagy, we examined the expression levels of mTOR and its upstream molecule AMPK. Our results confrmed that ALR induces autophagy in HepG2 cells by activating AMPK and inhibiting the mTOR pathway ( Figure 8). A similar fnding was reported that meoru anthocyanins, activators of AMPK α1, could prevent colon cancer cell growth by inhibiting mTOR phosphorylation [35]. To summarize, this work sheds light on the anticancer efects of ALR and suggests that it may have therapeutic potential as an autophagy inducer in HepG2 cells. However, the susceptibility to oxidation and low bioavailability of anthocyanins restrict their anticancer potential and clinical application. Nonetheless, ALR is still highly recommended as a nutritional supplement for cancer prevention and management without side efects.

Conclusions
Taken together, our study, summarised in Figure 9, clearly confrms that ALR induces autophagy via the AMPK/mTOR pathway and that autophagy inhibition impairs the proliferation inhibition, invasion, and migration capabilities as  Figure 8: ALR regulates autophagy of HepG2 cells by the AMPK/mTOR pathway. Western blot assay to analyze the protein changes in p-AMPK, AMPK, p-mTOR, and mTOR without 3-MA intervention (a) and with 3-MA intervention (b) * P < 0.05, * * P < 0.01, and * * * P < 0.001, vs. the control group. # P < 0.05 and ### P < 0.001, ALR vs. the ALR+3-MA group. well as apoptosis and G2/M phase arrest of ALR-treated HepG2 cells. Te in vivo anti-HCC efect of ALR will require further evaluation and exploration. In a word, ALR may be considered a promising natural product for the design of novel anticancer agents.

Data Availability
Te data used to support the fndings of this study are available from the corresponding authors upon request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.