Hedyotis diffusa Willd. Suppresses Hepatocellular Carcinoma via Downregulating AKT/mTOR Pathways

Objective Hedyotis diffusa Willd. (HDW) is a famous Chinese herbal medicine, traditionally used to treat cancer in China. Currently, the clinically used drugs for the treatment of hepatocellular carcinoma (HCC) still have poor efficacy and have many side effects. HDW has fewer side effects after taking it, so this study explores the inhibitory effect of HDW on HCC, which may become a promising drug for the treatment of HCC. Methods HCC cell lines such as SMMC-7721, SK-hep1, and Hep-G2 were treated with Hedyotis diffusa Willd. (HDW), after which migration was detected via transwell, while the proliferation of these cells was detected via MTT, CCK-8, and colony formation assays. Furthermore, protein levels were evaluated by western blotting, and Hep-G2 cells were implanted in nude mice to establish a xenograft model to evaluate the antitumor effect of the drug. Results HDW exhibited the ability to inhibit the proliferation and migration of HCC cells. And its anticancer mechanism in hepatocellular carcinoma may be via AKT/mTOR pathway. Moreover, the drug use of HDW in the mouse model system has achieved a good effect. Importantly, it did not cause significant weight loss or hepatorenal toxicity. Conclusion HDW can suppress the activation of the AKT/mTOR pathway in HCC cells, which may bring new light for the treatment of this kind of malignant tumor, but its exact mechanism still needs to be further explored.


Introduction
Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world and the third most common cause of cancer death [1]. e current conventional treatment method is still surgical resection; the drug treatment method is still unsatisfactory; and the side effects of the currently used drugs are relatively large [2]. With the development of research, natural products and their structural analogs have made significant contributions to the drug treatment of cancer and are expected to provide a new path for drug treatment of cancer [3]. In a retrospective analysis of natural products used as the source of new drugs in the past four decades, it is shown that natural products are still the best choice for finding new drugs and can be effective drugs for the treatment of many human diseases [4]. Usually, the patients diagnosed with liver cancer miss the opportunity of operation, so it is urgent to seek alternative therapy for HCC.

Hedyotis diffusa Willd. (HDW) is a famous traditional
Chinese herbal medicine with thousands of years of history. It is usually used to clear away heat, detoxify, and remove blood stasis, especially in the treatment of various types of inflammation [5]. Traditional Chinese medicine (TCM) has fewer side effects and has been clinically used to treat various diseases including cancer. Among them, HDW has long been used as an important ingredient in a variety of traditional Chinese medicine formulations to treat various types of cancer [6]. As an antitumor Chinese herbal medicine, it has been studied in the treatment of colorectal cancer [7,8], breast cancer [9], and prostate cancer [10]. However, the effect of HDW on HCC and the potential mechanism of its antitumor effect have not been fully explored. A study based on network pharmacology found that the potential mechanism of HDW's anticancer may be due to its coordinated regulation of multiple cancer-related pathways, such as angiogenesis, cell differentiation, migration, apoptosis, invasion, and proliferation [10].
AKT/mTOR signaling pathway plays an important role in tumorigenesis, and abnormal AKT/mTOR pathways can be found in almost all cancers [11]. Overactivation of AKT/ mTOR is associated with carcinogenesis and has become a potential molecular therapeutic target in cancer therapy. For example, trisubstituted imidazoles exert their anticancer activity through the PI3K/AKT/mTOR signaling pathway in breast cancer cells [12]. Moreover, mTOR is a drug target related to the development of cancer, and its inhibitors are usually used for the eradication of cancer and tumor stem cells [11]. e purpose of this study is to explore the antitumor effect of HDW on HCC, which may provide an effective alternative therapy for liver cancer.

Preparation of Hedyotis diffusa Willd. (HDW).
A certain weight of HDW was weighed, after which PBS was added to form 20 mg/ml mother liquor, which was divided into multiple tubes and stored at −20°C. We took out one tube of mother liquor each time and diluted to the corresponding target concentration for the experiment.

Cell Culture.
Human Hep-G2 HCC cells and SK-hep1 cells were provided by the Basic Medical College of Xiangya Medical College (Changsha, Hunan, China), while SMMC-7721 cells were provided by the Medical College of Hunan Normal University (Changsha, Hunan, China). Hep-G2 HCC cells and SMMC-7721 cells were grown in Dulbecco's modified Eagle's medium (HyClone, Logan, UT, USA), while SK-hep1 cells were grown in RPMI medium modified (HyClone, Logan, UT, USA). All these mediums contained 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (both from HyClone). And the culture temperature was 37°C, and the humidity was 5% CO 2 .

MTT Assay.
MTT assay was used to evaluate cell viability. In short, cells were added to a 96-well plate (8 × 10 3 cells/well) for 24 h, followed by a series of HDW concentrations for 72 h. en MTT tetrazolium salt was added to each well (50 μL; Sigma) for 5 h, after which 150 μL of DMSO (Sigma) was added per well, and the absorbance was at 490 nm was evaluated via microplate reader (BioTek, Synergy HTX, VT, USA). SPSS 16.0 (IBM, IL, USA) was used to calculate the half-maximal inhibitory concentration (IC50) values from dose-response curves.  Assay. CCK-8 assay was also used to evaluate cell viability. According to the manufacturer's instructions, the CCK-8 Kit (AccuRef Scientific) was used to measure the survival rate of cells under the specified conditions. In short, cells were added to a 96-well plate (8 × 10 3 cells/well) for 24 h, followed by a series of HDW concentrations for 72 h. en, CCK-8 reagent was added to the wells, and the absorbance of each well at 450 nm was quantified using an automatic microplate reader (Bio-Rad, Hercules, CA, USA).

Colony Formation Assay.
e ability of HDW to inhibit HCC cell proliferation was evaluated by colony formation assay. In short, cells were added to 24-well plates (8 × 10 3 cells/well) for 24 h; then they were incubated with a range of HDW concentrations for another 5-7 days. Finally, cells were fixed with 10% formaldehyde, which were then stained with 0.1% crystal violet for 1 h at room temperature. en absorbance at 550 nm was evaluated via a microplate reader.

Transwell Assay.
Polycarbonate transwell filters were used to assess cellular migration. In short, 4 × 10 4 cells in 200 μL of serum-free medium were added to the upper chamber, while medium containing 10% FBS was placed in the lower chamber. After adding an appropriate amount of HDW solution for 24 hours, the upper chamber cells were removed, and the remaining cells were fixed with 10% formaldehyde for 30 minutes, stained with 0.1% crystallizer for 2 hours, and observed under a microscope.
2.8. siRNA Transfection. HCC cells were transfected with commercially AKT siRNA (Sense strand: 5ʹ-CGCCAUG-GAUUACAAGUGUTT-3ʹ; Ribobio, China) or negative control (NC) siRNA (Sense strand: 5ʹ-UUCUCCGAAC-GUGUCACGUTT-3ʹ; Ribobio, China) with the transfection reagent Lipofectamine 2000 (Invitrogen); they were divided into siAKT group (AKT siRNA transfection) and siCtrl group (NC siRNA transfection) according to the different transfection siRNA. In short, cells were seeded in a six-well plate at a density of 3 × 10 5 cells per culture dish, and cells at 30%-50% conflueny were first transfected with AKT siRNA or NC siRNA using Lipofectamine 2000 for 6 hours. After washing in PBS, the medium was replaced with a medium containing 10% serum for 36 hours. Finally, cell proteins are collected and confirmed by Western blot analysis to confirm specific silencing.

Western Blotting.
e protein samples were separated by SDS-PAGE, transferred to membranes, and incubated with appropriate primary antibodies in a buffer containing bovine serum albumin (BSA) overnight at 4°C. e blots were then washed three times with PBS containing 0.1% Tween-20 (PBST) for 15 minutes each time, stained with a secondary antibody for 1 h at room temperature, and then washed three times in PBST for 15 minutes each time. e protein bands were then detected with Pierce Super Signal chemiluminescent substrate (Rockford, IL) and imaged with a ChemiDoc system (Bio-Rad). ImageJ (NIH, Bethesda, MD) was used for densitometric analyses; β-actin was used for normalization; and protein expression was evaluated relative to the untreated control group.

Murine Xenograft Models.
To evaluate HDW's antiproliferative effects on HCC tumors, female BALB/c-nu mice (4-6 weeks old) from Hunan SJA Laboratory Animal Co. Ltd. (Changsha, Hunan, China) were used to establish a xenograft nude mouse model. ese experiments were consistent with the guidelines of the Institutional Animal Care and Use Committee at Hunan Normal University. Mice were housed under sterile conditions with access to food and water. A total of 12 mice were randomly selected, and 5 × 10 6 Hep-G2 cells were subcutaneously implanted in their right flank. When the tumors grew to 50-80 mm 3 in size, the mice were randomized into control group (100 µl of 1% dimethyl sulfoxide (DMSO) plus 5% Tween and 5% neutral resin) and HDW (100 mg/kg/day) group (n � 6 animals/group). All treatments were performed in the stomach, and the administration was continued for 2 weeks. Tumor volumes and body weight were measured every two days, and tumor volume was calculated as follows: volume � 1/2 (length × width2). After two weeks, mice were euthanized with sodium pentobarbital, and tumor tissues were collected. e study was approved by the Institutional Research Ethics Committee of Xiangya Medical College (Hunan, China).

Histologic and Immunohistochemistry Analysis.
After the animals were euthanized, the organisms including the liver and kidney were collected and fixed with 4% neutralbuffered formalin to prepare histologic slides. en the samples were stained with hematoxylin and eosin (H&E), and 7-μm tissue sections were analyzed via standard light microscopy (DFC450 C; Leica, Wetzlar, Germany). For immunohistochemical staining (IHC), antigen retrieval was performed by boiling in citrate antigen recovery solution (p0081; Beyotime) for 15 min. en endogenous peroxidase activity was blocked by 3% H2O2. Ki67 (Abcam, ab15580, 1:200) antibody and a VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA) were used for staining, followed by the DAB substrate kit (Vector Laboratories) according to the manufacturer's instructions.

Statistical Analyses.
Data were mean ± SD, and data were given with 95% confidence intervals and reported with corresponding P-values ( * P < 0.05, * * P < 0.01, and * * * P < 0.001). e independent Student's t-test was used to analyze the significant differences between the two groups. For comparisons between multiple groups of samples, oneway analysis of variance (ANOVA) was used together with Tukey's multiple comparisons post hoc test unless otherwise specified. GraphPad Prism 6 and SPSS 13.0 were used for all statistical analyses.

HDW Inhibits HCC Proliferation and Colony Formation
Activity. To evaluate the effect of HDW on the proliferation of HCC cells, SMMC-7721, SK-hep1, and Hep-G2 cells were treated with a series of HDW doses (Figure 1(a)), revealing a dose-dependent inhibition of the proliferation of the three cell types after 72 hours via MTT assay. e IC50 value is 5.045 mg/ml (SMMC-7721), 1.345 mg/ml (SK-hepG1), and 2.961 mg/ml (Hep-G2). Similarly, the CCK-8 experiment was used to detect the effects of different concentrations of HDW on the proliferative activity of three different liver cancer cell lines (Figure 1(b)). HDW also inhibited the colony formation of HCC cells in a dosedependent manner, especially in Hep-G2 and SK-hepG1 cells (Figure 1(c)).

HDW Suppresses HCC Cell Migration.
A transwell assay system was used to evaluate the ability of HDW to inhibit the migration of SMMC-7721, SK-hep1, and Hep-G2 cancer cells, which showed that the drug could inhibit the migration activity of HCC (Figures 2(a)-2(b)).

HDW Induces HCC Cells Apoptosis and Knockout of AKT Affects Cell Clone Formation.
ree different HCC lines were treated with different concentrations of HDW (0, 0.5, 1, and 2 ug/ml) for 24 hours to detect the expression of apoptotic proteins Bcl-2 and Bax by western blotting (Figures 4(a) and 4(b)). ese results strongly proved that HDW induced cell apoptosis. Subsequently, AKT was knocked out in the cells, and the transfection efficiency was verified by western blotting (Figures 4(c) and 4(d)). Furthermore, knockdown of AKT expression reduced colony formation of HCC cells (Figure 4(e)). ese results found that knockdown of AKT expression significantly reduced the cell growth rate compared with the siCtrl group by the colony formation assay.

HDW Inhibits Xenograft HCC Tumor Growth in Mice.
To explore the efficacy of HDW treatment of HCC in vivo, nude mice were implanted subcutaneously with Hep-G2 cells, and then the mice were treated with the drug. Experiments showed that HDW treatment showed a good effect and significantly inhibited tumor growth (Figures 5(a)  and 5(b)). Importantly, we observed a significant difference in tumor weight between the control group and the HDW treatment group (783 mg and 283 mg, respectively; P < 0.01; Figure 5(c)). And no significant weight loss was observed in Evidence-Based Complementary and Alternative Medicine any treatment group, which is in line with the safety of drug treatment ( Figure 5(d)). en the tumor suppressor effect of HDW was studied. Immunohistochemistry was performed on the xenograft. Compared with the control group, the cells of HDW-injected xenograft showed significantly lower Ki67 expression (Figures 5(e) and 5(f )). HDW treatment also did not cause any kidney or liver damage to treated mice ( Figure 5(g)). All in all, these data indicate that HDW treatment in vivo can effectively inhibit the growth of HCC tumors.

Discussion
HCC is the fourth most deadly cancer and the fifth most common cancer in the world [13]. Although great efforts have been made to improve HCC treatment measures, the mortality rate of liver cancer patients is still very high, which is a serious threat to human life and health [14,15]. As one of the common diseases that plague the whole world, HCC still has poor treatment effects [16]. At present, the commonly used therapeutic drugs have poor effects and large side effects, so it is urgent to find an alternative drug. e development of cancer is strictly regulated by a variety of intracellular signaling pathways, such as AKT [17], mitogen-activated protein kinase (MAPK) [18], and STAT3 [19] pathways. e AKT signaling pathway is related to various types of cancer and is often related to anticancer therapy. When the AKT signal transduction pathway is activated, the mTOR signal transduction pathway is then activated, leading to abnormal cell proliferation, apoptosis, and differentiation [20]. ese molecules play an important role in the induction, progression, and metastasis of HCC [21]. TCM has long been used to treat a variety of diseases, including cancer [22]. HDW is a natural plant extract, which has been used as a common ingredient in TCM for antibacterial and anti-inflammatory, enhancing immunity, antitumor, and other effects [23,24]. With the progress of research, the anticancer effect of HDW has been paid more and more attention [25]. erefore, this article studies the effect of HDW on HCC. In this study, it was demonstrated that HDW reduced cell viability in different HCC cell lines in a dose-dependent manner. It is confirmed that HDW can inhibit cell proliferation and colony formation. It is worth noting that the downregulation of P-ERK, P-AKT, P-mTOR, and P-4EBP1 also provides further support, indicating that HDW can have an impact on HCC. HDW can exert anticancer activity through the AKT/mTOR pathway. It has also been observed that in vivo experiments HDW can significantly reduce the weight and volume of tumors and has low liver and kidney toxicity and side effects.
In short, HDW has good anticancer activity and few side effects. erefore, HDW may be developed as a promising and potential anticancer drug for the clinical treatment of HCC.  Data Availability e data used to support the findings of this study are included within the article.

Disclosure
Lingli Huang and Hui Xu are co-first authors.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
LH was involved in the writing of the original draft and running part of the experiments. HX was involved in the conceptualization of the study, as well as in the preparation of the figures, and the completion of the experiments. TW took part in revising the manuscript. GL was involved in the conceptualization of the study, project administration, study supervision, and writing, reviewing, and editing of the manuscript. .