α‐Pinene inhibits the growth of cervical cancer cells through its proapoptotic activity by regulating the miR‐34a‐5p/Bcl‐2 signaling axis

Among gynecological tumors, cervical cancer (CC) has the second‐highest prevalence and mortality rate. α‐Pinene is a bicyclic monoterpenoid compound extracted from pine needles that carried promising anticancer properties. Nevertheless, its effect on CC and the underlying mechanism has not yet been elucidated. Therefore, we investigated the effect of α‐Pinene on apoptosis in CC via in vitro assays of flow cytometry (FCW), terminal deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL) assay, quantitative real‐time polymerase chain reaction (qRT‐PCR), and Western blot. Following that, we detected the proapoptotic function of α‐Pinene on HeLa cells in vivo by TUNEL assay and immunofluorescence staining. Our results displayed that the α‐Pinene inhibited the growth of HeLa cells and stalled the cells in the G0/G1 phase. Interestingly, we also detected that α‐Pinene induced HeLa cells to apoptosis. The results investigated that α‐Pinene induced HeLa cells apoptosis along with up‐regulating the expression of Bax, Bid, caspase‐9, caspase‐3, miR‐34a‐5p, and down‐regulating the expression of Bcl‐2 in vitro. At the same time, the expression levels of target genes in vivo were consistent with those in vitro. Our experiment proved that α‐Pinene promoted apoptosis, which will be used to hopefully maximize the therapeutic strategies in clinical studies in CC.


| INTRODUCTION
Cervical cancer (CC) is the second most common cancer among females worldwide, with a disproportionately high burden in low/middle-income countries of the world. Although with the rapid development of the human papillomavirus (HPV) vaccine, it was seen that the incidence and mortality of CC did not decrease and it was still regarded as a severe public health problem. Moreover, rates of HPV vaccination have not been popularized in less developed countries, resulting in missed or delayed vaccinations of most females- Xia et al. (2019). It was estimated that the mortality of CC in less-developed regions was still 13.2 per 100,000 women and resulted in nearly 604,127 new cases and 341,831 deaths globally recorded annually (Hoque et al., 2021). Unfortunately, the incidence and mortality rates are increasing year by year and the population is getting younger (Pistritto et al., 2016). Notwithstanding these therapeutic and preventive measures of the HPV vaccine, surgery, radiotherapy, and chemotherapy, women's health continues to be jeopardized by the ongoing burden of the disease. Chemotherapy is used primarily to prolong the life expectancy of patients, including recurrence and metastatic disease. However, its resistance severely compromises treatment causing relapse and metastases for CC patients.
Meanwhile, the drugs for CC including 5-FU, cisplatin, paclitaxel, and platinum are expensive and have several negative effects and high recurrence rates. For the reasons stated above, there is consequently an urgent need for seeking new therapeutic drugs with effective, cheap, and fewer side effects for CC.
Nowadays, a substantial body of evidence suggests that natural materials may be a good source for the development of a new generation of anticancer drugs. Natural compounds have the ability to include anticancer capacity, the potential to overcome drug resistance, and safety. α-Pinene, extracted from pine needles, possessed powerful anticancer and anti-inflammation activities (Chen, Liu, et al., 2015;X. J. Li et al., 2016). We and some previous mechanistic investigations demonstrated that α-Pinene was able to block the cell cycle in cancers (Chen, Liu, et al., 2015;Chen, Solomides, et al., 2015;Zhao et al., 2018). Recently, interest in exploring the anticancer mechanisms of α-Pinene appears to be rising. Nevertheless, to date, little is known about the effect and mechanism by which α-Pinene regulates CC.
To the best of our knowledge, miRNAs can accelerate the degradation and/or block the translation of their target genes, induce posttranscriptional gene expression, and thus engage in the regulation of various biological processes such as cell proliferation, metabolism, and apoptosis (J. Xu et al., 2012), as well as in tumor cell development. miR-34a-5 is one of the important miRNAs. Several studies have shown that upregulation of miR-34a-5p inhibits cell proliferation and promotes apoptosis (Hu et al., 2021;E. Ma et al., 2020;Xiao et al., 2020). There was however, it remains unclear whether α-Pinene has a regulatory effect on miR-34a-5p in HeLa cells.
Apoptosis is generally regarded as one of the prominent signal pathways that triggered cancer cell death, and it played a pivotal part in both drug sensitization and drug resistance in cancer Zong et al. (2019).
Apoptosis also could be triggered in cancer cells through death receptors and mitochondrial pathways, as well as caspase-mediated extrinsic or intrinsic pathways, and so on. The Bcl-2 family proteins, pivotal regulators of mitochondria-dependent apoptosis, have been sorted as pro-and antiapoptotic factors based on their different features (Z. W. Ma & Liu, 2018). Bcl-system, which includes oncoproteins affecting proapoptotic (Bax, Bid, Bim, Bak) and antiapoptotic (Bcl-2, Bcl-xL, RaJ, Mcl-1) is a key factor in the regulation of the apoptotic processes Pal'tsev et al. (2000). A mounting number of investigations demonstrated that the Bcl-2 signaling pathway exerts a considerable contribution to cell proliferation and apoptosis (Ashkenazi et al., 2017;Berrak et al., 2016). Moreover, Bcl-2 is an antiapoptotic gene and its downregulation favors the reduction of tumor cell resistance to radiotherapy and the induction of apoptosis. The Bax gene belongs to the same family as the Bcl-2 gene and shares 21% of the amino acid sequence with Bcl-2. The Bax gene not only accelerates apoptosis directly but also antagonizes to some extent the function of the Bcl-2 gene in inhibiting apoptosis Gahl et al. (2016). Bid proteins are relevant in checking mitosis and maintaining genomic stability, among other things, and are characterized by enhanced apoptosis Yin (2000).
Thus, our study explored how the expression of proapoptotic proteins (e.g., Bax and Bid), and antiapoptotic proteins (e.g., Bcl-2) in HeLa cells after α-Pinene treatment. Caspases 3,6,8,9,10, and so on are among the 14 caspases involved in cell apoptosis (Pistritto et al., 2016). Caspase-3 is a downstream apoptosis execution factor in the caspase protein family, and its elevated activity induces irreversible apoptosis (Huang et al., 2011). Caspase-9 is an important indicator of the mitochondrial pathway that induces apoptosis. When activated, caspase-9 induces apoptosis by initiating the downstream caspase-3 pathway (Wei et al., 2020). Consequently, we also examined whether the caspase family (caspase 9, 3) was affected in HeLa cells after treatment with α-

Pinene.
Our discoveries exhibited that α-Pinene induced apoptosis in CC by activating death signals including miR-34a-5p, Bcl-2 family members, and caspases in vitro and in vivo, which could provide theoretical support for the fight against CC with α-Pinene and thus enhance patient survival. This study will provide greater insight into the causes of α-Pinene-induced HeLa cell apoptosis.

| Cell culture and cell treatment
Human cervical cancer HeLa cells were acquired from Suyan Biotechnology and cultured in RPMI-1640 medium (Gibco-BRL) mixed with 10% fetal bovine serum, and 1% streptomycin/penicillin (Solarbio Corp.). The cells were cultured in a constant-temperature incubator with 5% CO 2 at 37°C.

| Cell viability
The cell viability of the HeLa cells was assessed using the methyl thiazolyl tetrazolium (MTT) assay. 5-FU is widely used in the treatment of cervical cancer, and was known to downregulate the Bcl-2 family and induce caspases (Yim et al., 2004), so we used it as a positive control and set the concentration at 50 μmol/L according to Yi et al. (2020). For drug treatment experiments, the HeLa cells (4 × 10 3 cells/well) were firstly seeded into 96-well plates according to the report by P. Zhang et al. (2019), then treated with α-Pinene (10,20,40,80,160,320, 640 μmol/L) according to our previous studies of Q. Xu et al. (2018) and Ye et al. (2020) for 24 h. After that, an MTT assay was conducted. Finally, the automated microplate reader (Sunrise) was utilized to detect the absorbance at 570 nm.

| Cell cycle analysis
For the cell cycle, HeLa cells were harvested by P. Zhang et al. (2019) and fixed in 75% ethanol overnight at 4°C, then resuspended in precooling phosphate-buffered saline (PBS) containing 10 μl RNase and 0.02 mg/ml propidium iodide (PI) for 30 min at 37°C. At last, stained cells were detected by flow cytometer (Beckman), and data analysis was performed with ModFit LTTM software (Beckman).

| Cell apoptosis assay
Cell apoptosis was detected via Annexin V-FITC/PI Apoptosis Detection kit (BD Biosciences) according to the manufacturer's instructions. Briefly, the HeLa cells treated with α-Pinene were harvested and incubated in a solution mixed with 5 µl/ml Annexin V-FITC dye solution and 5 µl/ml PI dye solution at 4°C in the dark for 10 min. Immediately cell apoptosis was detected via flow cytometer (Beckman). Flowjo software (Beckman) was applied to analyze apoptotic cells.

| Terminal deoxynucleotidyl transferasemediated nick end labeling (TUNEL) assay
TUNEL staining kit (Beyotime) was used to verify the α-Pinene induced apoptosis in HeLa cells (1 × 10 5 cells/well) following the manufacturer's protocol and previously (Ye et al., 2020). The main steps are as follows, after 24 h incubation, the attached cells were fixed with 4% paraformaldehyde for 30 min. Next, cells were stained with 50 μl of TUNEL detection solution and incubated for 1 h at 37°C. Finally, the labeled cells were observed with a fluorescence microscope (Olympus; IX53), and the fluorescence density value was processed with Image J software.
2.6 | Quantitative real-time polymerase chain reaction (qRT-PCR) qRT-PCR was used to detect the messenger RNA expression of apoptosis-related genes. Total RNA was extracted from cells with Trizol reagent (Invitrogen). Then, qRT-PCR was performed using a sequence detection system with site-specific primers. The primer sequences were presented in (Table 1). qRT-PCR amplification was performed on a CFX96 real-time PCR detection system (Bio-Rad).
Samples were normalized to the control and ploidy changes were calculated according to equation 2 C -ΔΔ t .

| Western blot analysis assay
Furthermore, the protein expression of the apoptosis-related molecules was detected by Western blot. HeLa cells (1 × 10 6 cells/well) were treated with 25, 50, and 100 μmol/L of α-Pinene for 24 h. The total cellular proteins were isolated using radio-immunoprecipitation assay buffer (Sigma-Aldrich) containing protease inhibitors (Roche). The protein concentration was detected using the bicinchoninic acid assay protein assay kit (Beyotime Biotechnology). Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The proteins were blotted onto a polyvinylidene fluoride membrane (Millipore), the membrane was washed with tris buffered saline with tween (TBST, Millipore), then incubated with primary antibodies at 4°C overnight, washed three times withTBST for 15 min, and finally incubated with anti-rabbit/mouse immunoglobulin G antibodies (110,000 dilution) in 5% BSA buffer for 1 h at room temperature. The band intensity was visualized via ECL light-emitting liquid (Meilun Biotechnology Co.) and Image Lab™ Software (Bio-Rad).
2.8 | miR-34a-5p mimics and inhibitor transfection miR-34a-5p mimics, inhibitors, and control were synthesized by Ruibo Biotechnology. HeLa cells (1 × 10 6 cells/well) in the logarithmic growth phase were cultured in six-well plates and cell transfection was performed when the confluent of cells reached 80%. Subsequently, miR-34a-5p mimics and inhibitors were transfected into HeLa cells by lipofectamine 3000™ (Invitrogen) following manufacturer protocol. After transfection of 24 h, cells were treated with α-Pinene, and qRT-PCR and Western blot assay were employed to detect the related molecular expression.

| Animal experiments
To previously (Ye et al., 2020). Correspondingly, the proteins associated with cell apoptosis were detected through the immunofluorescence staining in tumor tissues.

| Statistical analysis
All the experiments were performed in triplicate. The data were presented as the mean ± standard deviation (SD). Differences between groups were evaluated by one-way analysis of variance.
Statistical significance was defined as p < .05. The analyses were performed using GraphPad Prism 8.0 statistical analyses.

| α-Pinene markedly induced apoptosis in HeLa cells
The TUNEL assay was conducted to further confirm the effects of 24 h treatment with α-Pinene (25, 50, and 100 μmol/L) on apoptosis in HeLa F I G U R E 1 Survival rate of HeLa cells was detected from 24 h, after treatment with α-Pinene using the methyl thiazolyl tetrazolium array. IC 50 , concentration that inhibits 50% of cell growth. Figure 4a, TUNEL assay results illustrated that α-Pinene treatment increased the proportion of apoptotic cells. Interestingly, as illustrated in Figure 4b, the TUNEL assay results also demonstrated that increased the proportion of apoptotic cells increased in a concentrationdependent manner under α-Pinene administration. Hence, consistent with Annexin V-FITC/PI Apoptosis assay results, these findings demonstrated that α-Pinene promoted apoptosis in HeLa cells.

| α-Pinene influenced the expression of apoptosis-related genes
The expression levels of five genes (Bcl-2, Bax, Bid, Caspase-9, and Caspase-3) were examined by quantitative polymerase chain reaction (qPCR) and Western blot to confirm whether α-Pinene could affect the expression changes of apoptosis-related genes. As shown in Figure 5a,

| α-Pinene induced HeLa cells apoptosis in vivo
The growth of xenogeneic tumors was inhibited after treatment ( Table 2). The difference in tumor weight between groups was not statistically significant before treatment, whereas the tumor weight of α-Pinene and 5-FU groups was significantly smaller than that of the PBS group after treatment (Table 2) Notably, the fluorescence intensity of the 5-FU group and the α-Pinene group was stronger than that of the control group (Figure 7a).
It indicated that α-Pinene led to apoptosis of HeLa cells in vivo. This was consistent with the in vitro result, including Annexin V-FITC/PI Apoptosis and TUNEL assay. It was noteworthy that the expression levels of Bcl-2 in both the 5-FU group and α-Pinene group were significantly lower than those in the control group ( Figure 7b).
Conversely, the expression level of Bax, Bid, caspase-9, and caspase-3 in both the 5-FU group and α-Pinene group were significantly higher than those in the control group, which revealed their roles in α-Pinene indued HeLa cells apoptosis (Figure 7c-f).

| DISCUSSION
CC is a great threat to women's health worldwide and more than 30% of patients develop recurrence or metastasis after treatment worldwide. In China, the 5-year survival rate is a mere 36.9% (Saleh et al., 2020). Chemotherapy resistance is the most serious obstacle to cancer treatment which is also the most frequent factor leading to clinical failure in cancer therapy Zheng (2017). Therefore, it is essential to explore potential curative agents for CC to create new therapeutic tactics. Here, we investigated the effect of α-Pinene on F I G U R E 4 Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. (a) HeLa cells were treated with α-Pinene for 24 h and stained with one step TUNEL apoptosis kit. The image was taken by immunofluorescence microscopy.
(b) The level of fluorescence intensity in HeLa cells was analyzed by the Image J software. All experiments have been performed in triplicate and data were expressed as mean ± standard deviation. Compared with control group, **p < .01.
CC and its basic mechanistic aspects. Our study revealed that α-Pinene increased CC apoptosis by regulating the miR-34a-5p and Bcl-2 mediated apoptosis pathway.
α-Pinene is a type of monoterpene that has been previously studied in several cancers. α-Pinene is effective in the treatment of experimental metastatic melanoma, where it reduces tumor nodules in the lungs Matsuo et al. (2011). The combination of α-Pinene and β-Pinene can reduce serious side effects by reducing the dose of paclitaxel without affecting its effect on non-small cell lung cancer Z. Zhang et al. (2015). α-Pinene, the main constituent of the essential oil, has been reported that stimulates the immune system and induce apoptosis in melanoma cancer (Hakkim et al., 2019). Our previous reports have indicated that α-Pinene inhibited the propagation of HepG2 cells (Chen, Liu, et al., 2015). It was also shown that α-Pinene Numerous studies indicated that noncoding RNAs could act as a crucial part of the pathogenesis of multifarious cancers, including CC (Boija et al., 2021;Pan et al., 2021). For instance, miR-34a-5p mediated CC growth, migration, and other malignant behaviors through the regulation of CDC25A (Jiang & Cheng, 2021). Meanwhile, Wang et al. (2021) suggested that the lncRNA MEF2C-AS1-miR-592-RSPO1 axis was a potential pathway for CC treatment.
Currently, miR-34a-5p has been shown to have important biological functions in tumors where it is often upregulated. It has been found that the expression of miR-34a is relatively lower in tumor tissues than in normal tissues B. Li et al. (2010). miR-34a-5p can also exert oncogenic effects by inhibiting the proliferation, activity, and invasion of cancer cells (Lv et al., 2019). Our study ( Figure 6a) showed the expression of miR-34a-5p in HeLa cells was increased after 24 h of α-Pinene administration compared to the control group by qPCR, which suggested that α-Pinene may promote HeLa cells apoptosis through miR-34a-5p. In other words, exploring the function of miR-34a-5p in the regulatory control of HeLa cells will F I G U R E 6 α-Pinene upregulated miR-34a-5p expression in HeLa cells leading to downregulation of target molecule Bcl-2 expression. (a) The influence of α-Pinene on the expression of miR-34a-5p in HeLa cells using quantitative real-time polymerase chain reaction (qRT-PCR). (b) The expression of miR-34a-5p was determined after miR-34a-5p mimics and inhibitor transfection by qRT-PCR. (c) The effect of miR-34a-5p mimics and inhibitors on Bcl-2 gene expression is determined by qRT-PCR. (d, e) The expression level of the Bcl-2 protein was detected by applying Western blot analysis. All data were shown as the mean ± standard deviation from three independent experiments. Significant differences were indicated by *p < .05; **p < .01; and ****p < .001, as compared with the control group.
T A B L E 2 Growth inhibition of transplanted tumor tissues in nude mice by α-Pinene contribute to the development of effective drugs for CC treatment.
Intriguingly, miR-34a-5p had previously been demonstrated to downregulate Bcl-2 (Qu et al., 2021). In this study, we used bioinformatics to confirm that Bcl-2 is the downstream target gene of miR-34a-5p. In cell transfection experiments, on one hand, the expression of miR-34a-5p was downregulated upon transfection of miR-34a-5p inhibitor, and correspondingly, the expression of Bcl-2 increased. On the other hand, intracellular transfection of miR-34a-5p mimics resulted in upregulation of miR-34a-5p expression and downregulation of Bcl-2 expression. Based on the results mentioned above, Bcl-2 is a target molecule of miR-34a-5p, and α-Pinene induced apoptosis in HeLa cells via the miR-34a-5p/Bcl-2 pathway.
To summarize, α-Pinene was a natural extract that is characterized by its low price, abundant sources, and small adverse effects.
Our study demonstrated that α-Pinene induced cell cycle arrest and apoptosis in CC, which provided the theoretical foundation for treating CC with natural plant extract. Our finding highlighted the requirement of an apoptotic pathway to achieve induced cell death, a high concordance between observed in vitro and in vivo after treatment with α-Pinene.

| CONCLUSION
Collectively, the current study provided evidence that α-Pinene induced CC apoptosis through activation of the miR-34a-5p/Bcl-2dependent pathway both in vitro and in vivo. Accordingly, α-Pinene may be an effective therapy for CC. Nevertheless, the pharmacokinetics of α-Pinene in CC remains unclear so far, more specific work and in-depth discussion employing additional cellular and in vivo animal models or clinical experiments will be necessary to validate the potential of α-Pinene as a useful drug for CC treatment.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Data available on request from the author. Data supporting the findings of this study are available from the corresponding author upon reasonable request. (b-f) Protein levels of Bcl, Bax, Bid, caspase-9, and caspase-3 were determined in immunofluorescence staining. Magnification: ×100. The results were presented as mean ± standard deviation, n = 3. Significant differences were indicated as **p < .01, compared with the control group.