Alcohol extracts of Chinese bayberry branch induce S‐phase arrest and apoptosis in HepG2 cells

Abstract The alcohol extracts of Chinese bayberry (Myrica rubra) branches (MRBE) are rich in flavonoids which have a variety of medicinal benefits, but their effects on human HepG2 were unknown. In this study, the effects of MRBE on HepG2 cell growth and its potential for inhibiting cancer were explored. The results displayed that MRBE inhibited HepG2 proliferation both by arresting cells in S phase and promoting apoptosis. Quantitative reverse‐transcription PCR (qRT‐PCR), western blotting, and immunofluorescence showed that MRBE induced S‐phase arrest by upregulating p21, which in turn downregulated cyclin and cyclin‐dependent kinase messenger RNA (mRNA) and protein. Apoptosis was induced by blocking the expression of BCL‐2 and suppression of the Raf/ERK1 signaling pathways. These results indicated that MRBE may have the potential for treatment of human liver cancer, highlighting novel approaches for developing new pharmacological tools for the treatment of this deadly type cancer. Meanwhile, it provides a new direction for the medicinal added values of Chinese bayberry, which helped to broaden the diversified development of its industry chain.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is the most common variety of primary liver cancer (Kim et al., 2017). The 2021 cancer statistics showed that although liver cancer prevalence appears stable in males, it has risen in females, primarily due to obesity, hepatitis B and C viral infection, and other factors (Siegel et al., 2021).
Chemotherapy, surgical resection, and other treatment methods can improve patient survival, but up to 70% of these patients nonetheless experience recurrent disease within five years . Additionally, most patients are diagnosed when the cancer progressed and survived just 6-8 months on average (Gosalia et al., 2017). Owing to its initially indolent course, delayed symptom onset, and limited treatment options (Zhu et al., 2016), HCC remains a considerable threat, and its prevention and treatment are areas of active research interest.
Chinese bayberry (Myrica rubra Sieb. et Zucc.) is a famous subtropical fruit tree, which is mainly grown in Southern China, such as Zhejiang, Fujian, Yunnan provinces, etc., and it has important economic values. The cultivation of Myrica plants has been for more than 2000 years according to the historical records in China.
The bayberry fruit has a unique sweet/sour flavor and is beneficial to health, as it contains high levels of phenolic compounds, ascorbic acid, and anthocyanins (Sun et al., 2013). In addition, different organs of Chinese bayberry have high medicinal values. Previous studies have showed that active substance from bayberry fruit can protect against oxidative DNA damage (Chen, Liu, et al., 2015), exhibit robust antioxidant activity (Huang et al., 2014), and prevent hypoglycemia (Zhang et al., 2015). Bayberry leaf extracts offer protection against virus and bacterial infections as well as oxidative stress (Chen, Liu, et al., 2015;Zhang et al., 2015), and exhibit bacteriostatic (Zhang et al., 2017), antioxidant and antiproliferative (Zhang et al., 2016), neuroprotective (Li et al., 2018), and hypoglycemic activities (Wang, Jiang, et al., 2019;Zheng et al., 2021). In the cultivation of Chinese bayberry, the tree needs to be trimmed 2-3 times/year, and the trimmed branches become waste. Old trees need to be cut down, and are also discarded or burned, resulting in both waste and pollution. With the continuous improvement of cultivation efficiency and the continuous expansion of the planting area, the potential resources of branches are also accumulating and getting wasted. The functions of ethanol extracts prepared using bayberry branches, however, have not been reported to date.
Most anticancer drugs were originally from natural plants (Li & Martin, 2011;Rawat et al., 2018). Traditional Chinese medicine has utilized natural products to treat diseases for thousands of years, with most plant parts being used as sources of these natural compounds, which largely derive their pharmacological activity from plant secondary metabolites (Beyoğlu & Idle, 2020;Yu et al., 2021). Many drugs and natural products used in the treatment of liver cancer modulate enzyme levels or cell signaling pathways. For example, Tatariside F (TF), which is extracted from the roots of Fagopyrum tataricum (L.)Gaertn, exhibits significant antitumor activity against HCC through a mechanism that may be related to increased expression of p53 and BAX and reductions in Bcl-2 (B-cell lymphoma 2) protein levels (Peng et al., 2015). Piperlongumine (PL) is a natural extract of piperlongumine that selectively kills HCC cells and preferentially inhibits both invasion and migration of HCC cells via the ROS-ER-MAPK-CHOP (reactive oxygen species-endoplasmic reticulum-mitogen-activated protein kinase-c/EBP homology protein) signaling pathway (Chen, Zhou, & Zheng, 2015). The ethanol extract of Artemisia capillaris leaves has been shown to not only effectively promote apoptosis, but to also reduce human HCC cell growth by suppressing PI3K/ AKT (phosphoinositide 3-kinase/protein kinase B) signaling activity (Kim et al., 2018). Other studies have explored the effects of proanthocyanidins on proteins associated with apoptosis, the cell cycle, and MAPK signaling and on the expression of NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1) in HepG2 cells (Wang et al., 2020). In addition, natural extracts from plants, such as Beilschmiedia tsangii root , Kigelia africana (Lam.) Benth. (Wambua Mukavi et al., 2020), Diospyros kaki leaves (Ko et al., 2020), Licorice (Wang, Luo, et al., 2019), Phoenix dactylifera L. (Khan et al., 2017), germ and bran of red rice (Upanan et al., 2019), Coptis chinensis (Kim et al., 2021), and Juniperus communis Linn. (Huang et al., 2021), have been found to induce the apoptotic death of HepG2 cells via the exogenous, mitochondriamediated endogenous pathway, MAPK signaling pathway, and ER signaling pathway. Thus, these extracts have the potential for being developed into drugs that specifically target HCC.
In this study, MRBE was extracted from Myrica rubra branches via an ultrasonic ethanol extraction method. To explore the molecular mechanisms whereby it induces apoptosis, the application of different concentrations of the extracts to HepG2 cells and related experiments were carried out.

| MRBE preparation and targeted metabolite analysis
MRBE was extracted from Chinese bayberry waste branches collected after pruning. The trees were planted in the International Research Center of Chinese bayberry (latitude 29.30°N, longitude 119.59°E), Jinhua, Zhejiang Province, China. This center was cofounded by our institute and local government, and the age of most trees used in this study was over 15 years.
The fresh branches of "Dongkui" bayberry were selected and removed from leaves, and dried to a constant weight at 60°C.
Samples were crushed and ground, and then filtered using 70% ethanol for ultrasonic extraction at room temperature 4 times (filter pore size 80-120 μm). After repeated evaporation and freezedrying, MRBE was obtained from these extracts for subsequent study. All experiments were performed using three biological duplicate samples.
Next, 50 mg of the MRBE sample was added to 600 μl of water:methanol (v:v = 1:2) containing succinic acid-2,2,3,3-d4 (50 ng/ml), after which 400 μl of chloroform was added. Two steel balls were then added, and the samples were pulverized in a grinder at 60 Hz for 2 min (60 Hz). The material was then sonicated on ice for 20 min.
The peak area of each chromatographic peak corresponded to the relative content of the corresponding metabolite, with the integral peak area for each metabolite being used to calculate the concentration thereof based on a standard curve. The absolute content of each metabolite in the actual sample was then determined as follows: Metabolite content (ng/g) = C × V/M × N, where C = the metabolite concentration as calculated with a standard curve based on the peak area value (ng/ml), V: constant volume (0.2 ml), M: sample mass (g), and N: dilution multiplier (5 times).

| Cell proliferation assay
Logarithmic-phase cells were cultured in 96-well plates for 24 h, after which 100 μl of MRBE stock solution was added to obtain final concentrations of 100, 200, 300, and 400 μg/ml. The control was 0.1% dimethyl sulfoxide (DMSO) in water. The cells were grown for 24, 36, and 48 h, the medium was removed, and the cells were trypsinized. Approximately 20 μl of cell suspensions was mixed with an equal volume of trypan blue solution (0.4%) and counted (IC1000; Countstar; ALIT Life Science Co., Ltd.). Proliferation was measured in an MTT assay and calculated according to the formula "Inhibitory ratio (%) = [1 − absorbance (test)/absorbance (control)] × 100%" (Zhong et al., 2020).

| qRT-PCR
Total RNA was isolated from cells using the TaKaRa MiniBEST Universal RNA Extraction Kit (Takara). A PrimeScript RT reagent kit with gDNA Eraser (Takara) and SYBR® Fast qPCR Mix (Takara) were used for PCR amplification using a CFX96 real-time PCR instrument (Bio-Rad) with the following thermocycler settings: 95°C for 30 s; 40 cycles of 95°C for 5 s; and 60°C for 30 s. β-Actin served as a normalization control, and the comparative 2 −∆∆Cq method was used to compare gene expression using primers detailed in a prior study (Yu et al., 2021). All primers used were presented in Table S1.

| Western blotting
Total protein was extracted from cells using SD-001 buffer (Invent

| Immunofluorescence
Cells were grown on slides, fixed for 30 min with 4% paraformaldehyde in PBS at room temperature, and washed in PBS. After blocking with 3% BSA for 50 min, the slides were probed overnight with pri-

| Statistical analysis
Data were expressed as means ± SD and analyzed with SPSS 16.0.
One-way analyses of variance (ANOVAs) and Tukey's post hoc test were used to compare groups. A value of p < .05 was the significance threshold.
These components may thus be bioactive compounds that shaped the activity of MRBE. showed that the MRBE application caused S-phase arrest, seen in increased numbers of cells in S and fewer in G0/G1 (Figure 2). This suggests that MRBE is effective in reducing proliferation in HepG2 cells.

| MRBE induces cell S-phase cycle arrest and apoptosis
Cell cycle is the central process underlying cellular replication, and various stages of this cycle were regulated by different genes. The mammalian cell cycle is divided into five sequential stages (G0, G1, S, G2, and M), and it normally proceeds through a series of strict regulatory checkpoints including the G1/S, S, G2/M, and mid-and spindleassembly checkpoints (Harashima et al., 2013). Only when the preceding phase is complete can the next cell cycle stage proceed.
In this study, to further study the inhibitory mechanisms induced The effects of MRBE on apoptosis were also investigated with flow cytometry (Figure 3). MRBE treatment augmented the frequencies of both early and late apoptotic cells, as well as necrotic cells relative to controls. These findings indicate that S-phase arrest and apoptosis may be the primary mechanism whereby MRBE suppresses the proliferation of HepG2 cells.

| MRBE induces S-phase arrest via p21-cyclin-CDK complex signaling pathway
To further elucidate the antiproliferative actions of MRBE, quantitative polymerase chain reaction (qPCR) was used to investigate changes in the expression of several key cell cycle related genes. To confirm the correctness of gene expression by qPCR, we performed western blotting and immunofluorescence analysis. As seen in Figure 4b   TGFβ has been linked to HCC development after liver injury (Dituri et al., 2019;Fabregat & Caballero-Díaz, 2018). The activation of TGFβ signaling regulates gene encoding proteins involved in the cell cycle through Smad-dependent transcriptional mechanisms, including the retinoblastoma (RB) gene and CDK inhibitors (Laiho et al., 1990;Polyak et al., 1994). CDK inhibitors include p21, p27, and p53, which belong to the CIP/Kip family and play a negative regulatory role in the cell cycle alone or in combination with one another (Orlando et al., 2015). CDK4 and CDK6 form kinase complexes with Cyclin D to phosphorylate retinoblastoma (Rb) family proteins, promoting cellular progression from G0 to G1, driving the release of E2F, Cyclin A, and Cyclin E from Rb protein inhibition, and thereby promoting transcription. CDK2 facilitates cell progression from G1 to S by complexing with cyclins A and E, while CDK1 forms kinase complexes with cyclins A and B, resulting in progression from S to M phase (Sherr & Roberts, 1999).

| MRBE induces apoptosis by downregulating Bcl-2 and ERK1 expression
Next, qPCR was used to examine the levels of several apoptotic Levels of the anti-apoptotic genes BCL-2 and ERK1 were markedly reduced after MRBE treatment for 48 h (Figure 5a). Western blotting confirmed that these alterations were in accordance with the protein levels ( Figure 5b). Moreover, immunofluorescent staining for Bcl-2 showed a marked reduction in cell density after application of 400 μg/ml MRBE for 48 h, with a similar decline in the green-fluorescent Bcl-2 signal (Figure 5c). Together, these findings indicated that MRBE treatment may lead to apoptosis by downregulating the expression of BCL-2 and ERK1 in HepG2 cells.
Apoptosis refers to the programmed death of cells via a regulated pathway that maintains the stability of the internal environment, and many cancer cells fail to normally undergo apoptotic death, leading to uncontrolled proliferation (Wu et al., 2008).
The process is largely controlled by Bcl-2 proteins, which are separated into three primary groups. Group 1 includes the antiapoptotic Bcl-w, Bcl-xl, and Bcl-2 proteins, while Group 2 includes the pro-apoptotic Bax and Bak proteins, and Group 3 includes the

| CON CLUS IONS
This investigation demonstrated that alcohol extracts of Chinese bayberry branch were able to effectively inhibit HepG2 cell proliferation through S-phase arrest. This action appears to be accomplished by the activation of p21--Cyclin-CDK complex signaling pathway. MRBE also promoted apoptosis by attenuating expression of Bcl-2 and the proteins of the Raf/ERK1 (extracellular signal-regulated kinase 1) signaling pathway. This suggests that MRBE may be a useful candidate for drug development efforts aimed at preventing and treating HCC. This study provided the potential roles of waste branches of Chinese bayberry, and it could not only realize the rational utilization of resources, turn waste into treasure, and reduce pollution, but also expand the bayberry industry chain.

This work was supported by the Key Research and Development
Project of Zhejiang Province (No. 2021C02066-2). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data will be available on request.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.