Scutellaria barbata Inhibits Hepatocellular Carcinoma Tumorigenicity by Inducing Ferroptosis of Hepatocellular Carcinoma Cells

Ferroptosis is caused by accumulation of iron-dependent lipid peroxidation, which is characterized by reduction in cell volume and increase in mitochondrial membrane density. Studies have shown that ferroptosis contributes to the development and progression of numerous major diseases, including hepatocellular carcinoma (HCC). As a unique biomedical resource, Traditional Chinese Medicine (TCM) has been widely used in the treatment of HCC. In this present study, Scutellaria barbata was used to treat HCC cells in vitro, and the results revealed that S. barbata suppressed HCC cell growth through inducing ferroptosis. Next, the exploration of the molecular mechanism on how S. barbata induced ferroptosis in HCC cells suggested that S. barbata may induce ferroptosis by promoting iron perioxidation and lipid ROS metabolism. Finally, S. barbata also inhibited HCC tumorigenicity in vivo by inducing ferroptosis of HCC cells. These results provided theoretical basis for explaining the mechanism of TCM treatment for HCC and offered therapeutic opportunities for HCC patients.


INTRODUCTION
Despite the progress in diagnosis and treatment, hepatocellular carcinoma (HCC) is still the sixth most common malignancy and the third principal cause of cancer-related mortality worldwide (1). HCC is the result of chronic liver disease and associated with nonalcoholic steatohepatitis, alcoholic hepatitis, viral hepatitis, and cirrhosis (2). Due to the high incidence of hepatitis B virus (HBV) infection, HCC poses a serious threat to the health of the population in China and Africa, especially in the sub-Saharan region (3,4). Methods for the early diagnosis of HCC is limited, and hence, most people lose the opportunity of surgical therapy. Less than 18% of HCC patients remain to have a dismal 5-year overall survival (5). Due to the difficulties in the early diagnosis of HCC and poor prognosis for HCC patients, it is urgently needed to discover novel targets and develop novel therapies for HCC treatment.
Recently, increasing data shows Traditional Chinese Medicine (TCM) play an active role in the treatment HCC (6). TCMs have long been utilized in disease prevention, including HCC. Accumulated Chinese herbal compounds have been isolated and show preventive effects on the occurrence of HCC, such as Scutellaria barbata, ellagitannin, ardipusilloside-I, Annona squamosa seeds, Panax, and gypenoside (7)(8)(9)(10)(11)(12)(13). TCMs play an important role in ameliorating clinical symptoms, improving quality of life, preventing recurrence and metastasis, suppressing tumor progression, and prolonging survival period of HCC patients (14). As a critical medical resource for the development of novel treatments for HCC, TCM compound can cause HCC proliferation inhibition, cell apoptosis, cell cycle arrest, autophagy, cell aging (13,(15)(16)(17)(18).
As a new identified type of cell death, ferroptosis is probably induced by TCM. Ferroptosis is caused by accumulation of irondependent lipid peroxidation, and it is involved in many major diseases including neurodegenerative diseases, ischemiareperfusion injury, and a series of cancers (19)(20)(21)(22)(23)(24)(25)(26)(27). Ferroptosis is different from other major forms of regulated cell death (RCD) including apoptosis. It is an iron-dependent cell death with the accumulation of lipid peroxidation and regulated by a unique set of genes, such as iron-responsive element-binding protein 2 (IREB2), ATP synthase F0 complex subunit C3 (ATP5G3), citrate synthase (CS), acyl-CoA synthetase long chain family member 4 (ACSL4), solute carrier family 7 (SLC7A11), and glutathione peroxidase 4 (GPX4) (28). SLC7A11 is a member of the cystine-glutamate antiporter; the inhibition of SLC7A11 leads to the accumulation of reactive oxygen species (ROS) and subsequent ferroptosis (28). As a selenoprotein, the catalytic site of GPX4 protein contains a key selenocysteine residue, which can partially protect HCC cells from ferroptosis (29). However, the role of TCM in ferroptosis in HCC cells remains unclear. In this present study, we committed to screen the target TCM for inducing ferroptosis in HCC. In addition, we elucidated the molecular mechanism of S. barbata to induce ferroptosis in HCC, which may provide new treatments and targets for HCC.

Lactate Dehydrogenase and Cytotoxicity Test
Cells (adherent or suspended cells) were collected and washed with 1× medium (e.g., 1% serum or BSA) and subsequently seeded in 96-well plates, and blank control wells were added 200 µl medium to three repeating microporous plates. All other holes must be subtracted from the blank control value. A total of 2 × 10 4 cells/well were cultured in 200 µl of assay medium with three repeated micropores in low control wells, while 1-2 × 10 4 cells/ well were cultured in a 200-µl assay medium containing 1% Triton X-100 with three repeated micropores in high control wells. In addition, 1-2 × 10 4 cells/well were cultured in 200 µl of assay medium with three repeated micropores in sample wells. The cells were incubated for 20 min in an incubator of 5% CO 2 , 90% humidity, and 37°C. Then, the cells were centrifuged with a centrifugal force of 250g for 10 min. Suspended cells were added into the 96-hole plate. Next, 100 µl/well of reaction solution was added and incubated at room temperature (RT) in the dark for 30 min. The absorbance at wavelength of 490-500 nm of samples were measured, and the cell cytotoxicity was identified by the following formula: (OD of sample − OD of low control)/(OD of high control − OD of low control) × 100%.

Iron Ion Concentration Detection
For cells, drugs were dissolved in PBS and diluted with complete medium into their IC50 concentration. A total of 1 × 10 5 cells/ well were seeded in 24-well plates and treated with S. barbata for 48 h. Iron ion concentration was then determined using iron ion colorimetric detection kit following the manufacturer's protocol.
For tumor tissues, the tumor samples were washed with 2 ml of cold normal saline for two times, and then, the normal saline was suck up (if it is not tested immediately, it can be frozen at −20°C). Normal saline (200 ml/g) was added into tumor samples to prepare tissue homogenate, and tissue homogenate was centrifugated to the collected supernatant for iron ion colorimetric detection.

Mitochondrial Membrane Potential Detection
Scutellaria barbata was dissolved in PBS and diluted with complete medium into its IC50 concentration. A total of 1×10 6 cells/well were seeded in six-well plates and treated with S. barbata for 4 h. Mitochondrial membrane potential (MMP) was then determined using JC-1 Kit (Life Technologies Corporation, Jersey City, NJ, USA) following the manufacturer's protocol. Red fluorescence indicates high MMP, while green fluorescence represents the dissipation of MMP. The ratio of red to green fluorescence intensity demonstrates the total/monomer ratio of JC-1.

Reactive Oxygen Species Assay (C11-BODIPY Probe)
Scutellaria barbata was dissolved in PBS and diluted with complete medium into its IC50 concentration. A total of 1-2 × 10 6 cells/well were seeded in six-well plates and treated with S. barbata for 48 h. ROS was then determined using C11-BODIPY probe following the manufacturer's protocol. The ratio of emission fluorescence intensity at 590 to 510 nm gave a reading of ROS in the cell.

Realtime Polymerase Chain Reaction
After the treatment of S. barbata, real-time PCR was performed to verify the influence of S. barbata on GPX4, SLC7A11, IREB2, and ACSL4 in SMMC-7721 (or HepG2, Huh7) cells or in in vivo tumors. Total RNA was isolated from cells or tumor tissues using the Trizol reagent (Invitrogen) and then reversed transcribed by the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, Liaoning, China). Subsequently, real-Time PCR was performed by the StepOnePlus system (Applied Biosystem, Foster City, CA, USA) using TaKaRa SYBR Premix ExTaq II. Primer sequences are shown in Table 1.

EdU Assay
For EdU detection, first, each group of cells was pre-cultured with diluted EdU medium for 3 h according to the instructions. Then, cells were washed twice in PBS, then fixed in 4% paraformaldehyde for 30 min, infiltrated with 0.1% Triton X-100 for 10 min, washed with PBS, added with 100 ml Apollo staining solution to each well cells, incubated in the dark for 30 min, and then stained the nucleus. 4′,6-Diamidino-2phenylindole (DAPI) solution was applied, and then the results were visualized by a fluorescence microscope.

In Vivo Xenograft Model
Five-weeks-old male BALB/c nude mice used in this study were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China), and the housing conditions were as follows: a 12-h light/dark cycle, normal grade, and free to eat and drink. For the xenograft study, 1 × 10 7 cells in 100 µl PBS were inoculated subcutaneously into nude mice. The nude mice were randomly divided into two groups (intragastric administration of 140 g/10 g, S. barbata or PBS) with five mice in each group. The groups were labeled as administration group or unadministration group. Subcutaneous injection and administration were conducted according to the above groups. Cervical dislocation was performed in nude mice, and the tumor size and weight were measured. The tumor was collected for subsequent experimental detection. All animal experimental procedures were performed strictly in accordance with the Ethic Committee of The Third Affiliated Hospital of Sun Yat-Sen University.

Statistical Analysis
All experiments in this study were repeated at least two times, and average values of three experiments were presented as the mean standard deviation (SD) calculated by STDEV formula in Excel. The significance of all data was estimated by Tukey's multiplecomparison test in the ANOVA analysis by the Sigma Stat 3.5 software. Importantly, statistical significance was accepted when p < 0.05.

Scutellaria barbata Suppresses HCC Cell Growth
To evaluate the effect of S. barbata on HCC cell lines (including SMMC-7721, HepG2, and Huh7), CCK8 assay was performed to Results showed that S. barbata significantly inhibited the growth of HCC cells in a dose-dependent manner, and the inhibitory rate was enhanced by increasing S. barbata dosage ( Figure 1A). Moreover, the IC50 concentration of S. barbata was 44.26 mg/ml (SMMC-7721), 42.19 mg/ml (HepG2), and 52.01 mg/ml (Huh7), respectively, which was used for the subsequent experiments ( Figure 1B). Then, the marker of cell-death-released LDH was detected by ELISA. Results indicated that treatment of S. barbata dramatically increased the level of released LDH in HCC cells ( Figure 1C). The cytotoxicity was significantly increased in HCC cells treated with S. barbata ( Figure 1D). In addition, EdU staining assay was performed to detect the cell proliferation and revealed that the treatment of S. barbata significantly suppressed the growth of HCC cells ( Figure 1E). All these data suggested that S. barbata may suppress HCC cell growth through inducing HCC cell death.

Scutellaria barbata Induces Ferroptosis in HCC Cells
Ferroptosis is caused by the accumulation of iron-dependent lipid peroxidation, which is characterized by reduction in cell volume and increase in mitochondrial membrane density. Results showed that S. barbata significantly increased the iron concentration in HCC cell lines, including SMMC-7721, HepG2, and Huh7 cells (Figure 2A). Moreover, mitochondrial membrane potential identified by JC-1 staining was decreased in HCC cells after the S. barbata treatment (Figures 2B, C). Furthermore, the level of ROS detected by C11-BODIPY probe on flow cytometry was dramatically reduced in HCC cells treated with S. barbata (Figures 2D, E). Taken together, these results suggested that S. barbata induced ferroptosis in HCC cells.

Scutellaria barbata Induces Ferroptosis Through Regulating Genes Involved in Lipid ROS Metabolism and Iron Perioxidation in HCC Cells
Next, the molecular mechanism of S. barbata inducing ferroptosis in HCC cells was explored. Lipid ROS metabolismrelated genes GPX4 and SLC7A11 were ferroptosis inducer (30,31), whereas iron perioxidation-related gene IREB2 and CoA metabolism-related gene ACSL4 were ferroptosis inhibitor (32,33). The mRNA and protein levels of these four genes were detected in HCC cell lines (SMMC-7721, HepG2, and Huh7), respectively. Results revealed that both mRNA and protein levels of GPX4 and SLC7A11 were reduced after S. barbata treatment in HCC cells (Figures 3A, B). By contrast, both mRNA and protein levels of IREB2 and ACSL4 were elevated after S. barbata treatment ( Figures 3A, B). These data suggested that S. barbata may induce ferroptosis through regulating the expression of genes involved in lipid ROS metabolism and iron perioxidation in HCC cells.

Scutellaria barbata Inhibits HCC Tumorigenicity In Vivo
The above data suggested that S. barbata might be a valid choice to inhibit HCC tumorigenicity in vivo. Therefore, HCC cell lines (HepG2 and Huh7) were inoculated in nude mice separately, and subsequently, nude mice were treated by intragastric administration of S. barbata. The volume, size, and weight of the xenograft tumors in nude mice were measured. Results showed that most of the tumors without S. barbata treatment were significantly larger and heavier than those treated with S. barbata ( Figures 4A-C). Furthermore, the expression of Ki-67 protein in the xenograft tumors were measured by IHC. Results showed that Ki-67 protein in without S. barbata treatment was significantly higher than those treated with S. barbata ( Figure 4D). These data suggested that S. barbata suppressed HCC tumorigenicity in vivo.

Scutellaria barbata Inhibits HCC Tumorigenicity by Inducing Ferroptosis of HCC Cells In Vivo
To identify whether S. barbata inhibited HCC tumorigenicity by inducing ferroptosis in vivo, iron concentration in tumors was dramatically increased by S. barbata ( Figure 5A). Moreover, both mRNA and protein levels of SLC7A11 in tumors were reduced after S. barbata treatment (Figures 5B, C). These data together suggested that S. barbata inhibited HCC tumorigenicity in vivo by inducing ferroptosis of HCC cells.

DISCUSSION
Despite significant advances in clinical treatment of HCC in recent years, it remains one of the leading causes of cancer-related death worldwide. The difficulty in treatment and poor prognosis of patients with HCC lies in our limited choice of effective drugs for HCC, which suggests that we urgently need to develop new treatment methods for HCC. Ferroptosis is a new type of cell death characterized by accumulation of intracellular reactive oxygen species, which is iron dependent and non-apoptotic (34,35). It is related to the occurrence and development of a variety of diseases, including liver cancer (32). TCM is a valuable medical method and resource in China, which has a significant effect on the treatment of HCC (36). However, there are very few studies on the regulation of ferroptosis in TCM, and only one study shows that artemisinin can promote ferroptosis in cancer cells (37). Therefore, this present study focused on the effect of TCMinducing ferroptosis of HCC cells and provided theoretical basis for explaining the mechanism of TCM in treating HCC to find new drugs for treating HCC patients.
Scutellaria barbata has been wildly used in treatments for liver diseases. Several studies in human, cell, and animal have indicated the preventive effects of S. barbata on HCC. For example, a cohort study has revealed that S. barbata dramatically reduces HCC risk in patients with chronic hepatitis B, which is a major inducer of HCC. Moreover, total flavonoids of S. barbata prohibits HCC cell invasion through regulating TIMP expression (38). As the active component in S. barbata, pheophorbide a, C 35 H 36 N 4 O 5 (molecular weight, 593), attenuates multidrug resistance of HCC cell line (39). In animal experiments, S. barbata polysaccharides suppress HCC cell growth in the H22 hepatoma−bearing Kunming mice (40). Furthermore, S. barbata crude extract has a protective effect against liver tumorigenesis in rat. However, the role of S. barbata on ferroptosis is not clear. Results of our study indicated that S. barbata induced ferroptosis of HCC cells, suggesting that S. barbata may prevent HCC development through ferroptosis.
Recent studies have identified the genes and pathways related to ferroptosis in HCC. Therefore, we first screened the target genes and pathways of Scutellaria in HCC to clarify the related mechanism. We found out that S. barbata decreased significantly the mRNA and protein expression levels of antiferroptosis in lipid ROS metabolism-related genes, namely, GPX4 and SLC7A11; meanwhile, S. barbata increased significantly the mRNA expression levels of iron perioxidation-related gene IREB2 and CoA metabolism-related gene ACSL4, which both are ferroptosis-positive regulators. We came to the conclusion that S. barbata induced ferroptosis of HCC cells by promoting iron perioxidation and lipid ROS metabolism. In the end, we constructed an animal model of HCC through tumor formation in nude mice and found out that S. barbata significantly inhibited the growth of tumor cells in nude mice. Furthermore, S. barbata could significantly decrease the expression level of GPX4 and SLC7A11 and increase the expression level of IREB2 and ACSL4 in nude mice, which enabled the ability of S. barbata to increase iron concentration in nude mice. Specific inhibitors of ferroptosis have been shown to alleviate organ damage in several clinical models, including a model of heart disease. Fang et al. found that mice have altered cardiac iron homeostasis and develop mild cardiomyopathy with aging, which is rescued by treating the mice with the ferroptosis inhibitor ferrostatin-1 (41). Sorafenib, against advanced HCC, also exerts cytotoxic effects via the induction of ferroptosis (42). Next, we will explore the role of ferrostatin-1. Scutellaria barbata inhibits hepatocellular carcinoma tumorigenicity by inducing ferroptosis of hepatocellular carcinoma cells, which may be rescued by ferroptosis inhibitor. In summary, this present study demonstrated S. barbata induced HCC cells ferroptosis by promoting HCC cells' iron perioxidation and lipid ROS metabolism. In addition, we also found out that S. barbata inhibited HCC tumorigenesis in vivo by inducing ferroptosis of HCC cells. We provided theoretical basis for explaining the mechanism of a kind of TCM in treating HCC and offered therapeutic opportunities for a wide range of HCC patients.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by Ethic Committee of The Third Affiliated Hospital of Sun Yat-Sen University.

AUTHOR CONTRIBUTIONS
MD and Y-W L conceived and directed this research and contributed to the project design. YL and JZ conducted the experiments and analyzed the data. KZ, YC, WW, and HC helped provide data and comments on the manuscript. Reagents, materials, and contributions were contributed by ZZ. YL and JZ were responsible for drafting the manuscript. All authors contributed to the article and approved the submitted version.