α-Pinene inhibits the growth and induces the apoptosis of cervical cancer cells through regulating miR-34a/Bcl-2 signaling axis

Purpose α-pinene was a chemical compound which was extracted from pine needles oil, and it exerted effects on various diseases. However, the effect of α-pinene on cervical cancer had not been reported. The goal of this study was to explore the anti-tumor role of α-pinene. Methods Methyl thiazolyl tetrazolium (MTT) method was used to detect cytotoxicity of α-pinene. Flow cytometry was used to quantify the cell cycle and apoptosis. TUNEL staining was also performed for the revalidation of apoptosis. QRT-PCR and western blot was implemented to detect the expression levels of apoptosis genes and miR-34a-5p. Tumor-bearing nude mouse models was adopted to assess the antitumor action of α-pinene in vivo. Results The results displayed α-pinene restrained proliferation of Hela cells in G1 phase and induced Hela cell apoptosis, which was related to up-regulating expressions of Bax, Bid, Caspase-9, Caspase-3, miR-34a-5p and down-regulating the expression of Bcl-2. Afterwards, α-Pinene could regulate miR-34a-5p/Bcl-2 pathway. Furthermore, α-pinene treatment also induced apoptosis in xenografts tumor models. The uorescence intensity of Bax, Bid, Caspase-9, Caspase-3 increased and uorescence intensity of Bcl-2 decreased. Conclusions Our research demonstrated α-pinene could restrain the development of cervical cancer growth, and it might be an effective chemical compound for therapy of cervical cancer.


Introduction
Cervical cancer (CC) was a prevalent malignant disease for women all around the world [1]. The incidence and mortality of CC was increasing in developing countries [2]. In China, the ve years survival rate of CC patients was only about 36.9% [3]. Chemotherapy is an effective treatment measure to prolong the life length of patients after they had accepted surgical operation [4]. However, chemotherapy resistance seriously affects the effectiveness of treatment and leads to recurrence and metastasis of CC patients [5].
Because of this reason it is important to explore the new and effective chemotherapeutic drugs for CC therapeutic program.
α-pinene was a bicyclic monoterpene compound which was extracted from turpentine. Recently, lots of medicinal values of α-pinene were found in treatment of various diseases, including sleep, ammatory and cancer [6][7][8]. It had been found that α-pinene had anti-cancer effect on lung cancer by promoting apoptosis in lung cancer cells [9]. There was another study which showed that α-pinene inhibited prostate cancer growth in a xenografts model [10]. However, the potential effects of α-pinene and its treatment relative mechanisms in CC were not investigated. So we proceeded biological activity experiments to explore the effect of α-pinene on CC.
It was well known that microRNAs were conserved small non coding RNAs and they could regulate the transcription and translation process of the target genes in different types of cancers [11,12]. Many studies have proved miR-34a could restrict growth and migration of various malignant tumor cells such as osteosarcoma, lung cancer and prostate cancer by controlling c-Met, CDK6 and LEF1 gene [13][14][15], And miR-34a was also reported as a novel molecular biomarker associated with cervical cancer [16,17]. And it was clear that miR-34a acted as a tumor suppressor gene by downregulating Bcl-2 [18]. The synthetic function of miR-34a provided a new way to analyze the mechanism of α-pinene functions.
In our study, we evaluated the therapeutic effect of α-pinene in cervical cancer using Hela cell lines and tumor bearing nude mice models. And anti-cancer mechanism of α-pinene was explained basing on the miR-34a/Bcl-2-mediated regulation functions.
Materials And Methods 2.1. α-pinene purity and structural con rmation Pine pneumatizing powder of the pulverized screen is soaked for a certain period of time, and the water vapor distillation method extracts the pine needle oil in the pine needle, and the fractionation is separated from the extracting high purity α-pinene. Analysis of product purity and structures by gas chromatography instrument, mass spectrometer.

Cell culture
Hela cell line, the human cervical carcinoma cell, was acquired from Suyan Biotechnology Co.

MTT assay
The α-pinene dilution was dissolve with dissolved in the DMSO. The proliferation of Hela cells was detected by MTT assay after they were treated with α-pinene. The Hela cells were inoculated into 96 well plate by adjusting cell accounts to about 4×10 3 cells/well with 100μL.Different concentration of α-pinene (10,20,40,80,160, 320, 640μmol/L,) was administered to each well after the cells adhered to the wall completely and each group contained six parallel wells. 5-FU was used as positive control group (40μmol/L). The control group was cultured in an equal volume of serum RPMI-1640 culture medium containing cells. After 24hours, the supernatant was discarded, and the medium containing 10μL of 5 mg/mL MTT reagent (Meilun Biotechnology Co., Dalian, Liaoning, China) was put into well and incubated in dark at 37 °C for 4 h. After removal of the MTT, cells were treated with 100μL DMSO, and shaken on an oscillator for 15minutes.OD was measured at 490nm using Automated Microplate Reader (Sunrise, Tecan, Switzerland) .Cell survival rate = (OD value of drug group-OD value of blank group)/ OD value of control group -OD value of blank group)×100%. Each experiment was repeated three times. The IC 50 value of α-pinene in Hela cells was calculated with the help of Graphpad Prism 8.0 software.

Cell cycle analysis
1×10 6 Hela cells were inoculated into 6-well plate for each well. When the cultured cell con uency reached at about 60%, the drug was administered into cultured cells. According to IC50 calculated by MTT data, α-pinene groups were designed to three different concentration groups: low concentration group (25μmol/L), medium concentration group (50μmol/L) and high concentration group (100μmol/L).
After incubation for 24 hours, the cells were collected and mixed with 70% alcohol at 4 ℃ for at least 4 hours. According to protocol provided in cell cycle kit, the cells were labeled with 50μg/mL PI staining at 37 ℃ for 30 minutes. Then the samples were measured by Flow Cytometry. The test data was analyzed by ModFit software.

Cell apoptosis proportion detection
The apoptosis of Hela cells treated with α-pinene was assessed by Annexin V-FITC/PI apoptosis detection kit (BestBio, Biotechnology Co., Shanghai, China),. After they were incubated with α-pinene for 24 h, the Hela cells were gathered and cleaned by PBS. Cells were mixed with 5μL Annexin V-FITC dyesolution and 5μL PI dye solution away from light for 10min, the condition of cell apoptosis was tested by ow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

TUNEL staining
Cell apoptosis was also gauged by TUNEL staining kit (Beyotime, Shanghai, China). Hela cells were inoculated into climbing lms and incubated with different concentration of α-pinene for 24h. Then the cells were xed by 1mL 4% paraformaldehyde for 30 minutes. Thereafter, the cells were dipped into PBS containing 0.3% TritonX-100. Then the cells of climbing ake were incubated with 50μL of TUNEL detection solution in dark for 60 minutes at 37 ℃. The uorescence pictures were observed and captured by uorescence microscope (Olympus, Tokyo, Japan), then the uorescence density value was analyzed by Image J software.

qRT-PCR
The cells were handled according to the previous concentration and action time of α-pinene. Total RNA was isolated from the cells of each group by use of Trizol agent (Invitrogen, USA). According to the operating manual of the reverse transcription kit primescript ™ RT Master Mix reagent Kit (TaKaRa Bio, Beijing, China), the acquired RNA was reverse transcripted to cDNA. TB Green Premix Ex TaqII kit (TaKaRa Bio, Beijing, China) was used to measure relative gene cDNA ampli cation. The primers were devised by NCBI website and then synthesized by bioengineering Co. (Shanghai, China). The upstream and downstream primer sequences of Bcl-2, Bax, Bid, Caspase-9, caspase-3 and GAPDH are shown in Table 1.

Western-blot
Proteins were extracted from Hela cells of every group through RIPA liquid containing 1% PMSF Beyotime Biotechnology Co., Ltd., Shanghai, China. After the protein concentration was measured by BCA method (Beckman Coulter, Brea, CA, USA). Each denatured protein specimen was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene di uoride (PVDF) membrane (Millipore, Watford, UK). Then PVDF membrane was blocked by 5% non-fat milk for 1h. According to the operation manual of the primary antibody, the diluted primary antibody (GADPH, Bcl-2, Bax, Bid, Caspase 9, Caspase 3 (Proteintech Group Inc., Wuhan, China) was added to the membrane bearing proteins at 4℃ overnight. After it was washed by TBST, the PVDF membrane was incubated with goat anti-rabbit IgG secondary antibodies (1:5000, Proteintech Group Inc., Wuhan, China) for 1h. At last, the membrane was soaked in ECL light-emitting liquid (Meilun Biotechnology Co., Dalian, China) and then checked by Image Lab™ Software.
According to operation rules of transient transfection, Lipofectamine TM 3000 (Invitrogen, Carlsbad, CA, USA) and Opti-MEMTM (Gibco-BRL, Los Angeles, CA, USA) were added to help miR-34a-5p compounds affect cells. The experiment divided into four groups: negative control group (unrelated sequence transfection), miR-34a-5p mimics group, miR-34a-5p inhibitor group, miR-34a-5p mimics+50umol/L αpinene group). After 24 hours, total RNA was extracted for qPCR detection. Bcl-2 was looked as the target gene of miR-34a-5p, so combining with the results of previous apoptosis experiments, the expression of Bcl-2 was observed by western blot. Finally, the gray value of the stripe is calculated by ImageJ software, and then the statistical calculation was carried out. Immunostaining was performed on deparafnized tissue sections. Each sample was incubated with about 40μL transdermal solution at room temperature for 25 minutes. Subsequently, 40μL TUMEL reaction solution was added to the sample for 60 minutes at 37℃. Finally, the uorescence photograph was captured by the uorescence microscope. Furthermore, apoptotic genes detected in our previous study were detected in tumor tissues by immuno uorescence. 40μL goat serum was used to block each tissue section at 37 ℃ for 10 minutes. After that, every primary antibody were diluted to 1:1000-1:10000 and mixed with 0.2% Triton-100x, and added to each tissue overnight at 4℃. The uorescent second antibody (dylight594, dylight488, Proteintech Group Inc., Wuhan, China) was diluted 1:100 and added to the tissue in dark for 3 minutes, and immediately washed with PBST. After the appropriate amount of anti uorescence quenching liquid was covered on the tissue, the uorescence image was observed by the uorescence microscope (Olympus, Tokyo, Japan).

Statistical analysis
Graphpad prism 8.0 software was applied to draw the histogram and analyze the results. All of the data's were expressed as mean ± SD. Student-t test was used to analyze the comparison between the two samples, and one-way ANOVA was used to compare the multiple copies. The difference was signi cant when p < 0.05.

Product purity and structure are con rmed
This experiment uses gas chromatography and mass spectrometry to analyze the product purity and structure. As shown in (Fig. 1A,) the normalized measured purity product is 91.0% (at 7.297minutes).The sample mass spectrum similarity to the reference spectrum retrieved by the National Institute of Standards and Technology (National Institute of Standards and Technology,NIST) spectrum library is 97%, as shown in (Fig. 1 B-C) The mass spectrogram (B). mass spectrogram of product (C). mass spectrogram of α-pinene which was retrieved from NIST database.

α-Pinene controlled the proliferation of Hela cells
The MTT experiment showed that α-Pinene had obvious inhibition on Hela cells at different concentration gradient. And IC 50 value for α-Pinene was (46.49±0.78μmol/L at 24h,) (Fig. 2). Therefore, 24 hours was selected as the administration time, and 25μmol/L, 50μmol/L and100μmol/L were considered as the low, medium and high concentration gradient groups.

α-Pinene made Hela cells to remain at G1 phase
The cycle distribution in Hela cells was detected by ow cytometry following 24h treatment with α-Pinene at concentrations of 25μmol/L,50μmol/L and100μmol/L. Compared with negative control (Fig. 3A), the proportion of cells in G1 phase increased while the proportion of cells in S phase decreased with increase of concentrations for α-Pinene (Fig. 3B-E). This result displayed that α-Pinene could restrain the proliferation of Hela cells through making the cells to stay at the G1 stage and decreasing the percentage of cells in S stage.
We measured the apoptosis in Hela cells after treatment with α-pinene using AnnexinV-FITC/PI staining and TUNEL uorescent staining. Treatment with α-pinene increased the proportions of apoptotic Hela cells (Fig. 4A). TUNEL results also showed that in comparison with the control group, the uorescence intensity of the α-pinene treatment group was signi cantly enhanced (Fig. 4B). These outcomes demonstrated that α-pinene treatment could lead to apoptosis of Hela cells. In order to further con rm if α-Pinene could in uence expression change of apoptosis related genes, the operations of qPCR and western blot were conducted to detect expression levels of ve genes (Bcl-2, Bax, Bid, Caspase 9, Caspase 3). In comparison with the control group, the outcomes demonstrated that the expression of Bcl-2 decreased, while the expressions of Bax, Bid, Caspase 9, Caspase 3 increased (Fig. 5).

α-pinene inhibited the growth of xenotransplanted tumors
Anti-tumor effect of α-pinene in vivo was evaluated by xenotransplanted tumor model of Hela cells. To test phenomenon of cell apoptosis, we proceeded TUNEL staining in tumor tissue. The uorescence intensity of 5-FU group and α-pinene group was stronger than that of control group (Fig. 7A). TUNEL staining showed that α-pinene could lead to apoptosis activation of tumor cells in vivo. The results concurred with the results of cell experiments in vitro. The proteins which were associated with cell apoptosis were detected through immuno uorescence method in transplanted tumor tissues. DAPI dye was used to dye the nucleus, and the expressed protein was stained green or red uorescence.
Fluorescence analysis showed exhibited lower expression of Bcl-2 in both 5-FU treatment group and αpinene treatment group than the control group. In contrast, the winder expression of Bax, Bid, Caspase 9, Caspase 3 exhibited in 5-FU treatment group and α-pinene treatment group (Fig. 7B-F). The results of in vivo experiments were consistent with those of in vitro. These data further proved that α-pinene can induce apoptosis of HeLa cells by promoting cell apoptosis.

Discussion
Cervical cancer was one of the most prevelant gynecological malignancies, 85% cervical cancer cases appeared in developing countries [19]. Chemotherapy was the common treatment method for cervical cancer. Nowadays, more and more chemosynthetic antitumor drugs had severe toxicity and drug resistance [20]. So lots of natural anticancer drugs including Vincristin, Paclitaxel, Camptothecin were abstracted from plants and displayed effective anti-tumor activity [21][22][23]. α-pinene was separated and distilled from pine needles. In our previous studies, we found α-pinene inhibit the growth of hepatocellular cancer cells [24]. α-pinene also could showed the ability of promoting the apotosis of prostate carcinoma cells [10]. In order to verify pinene was a broad spectrum anti-cancer drug, we evaluated the function of αpinene in Hela cells, a cervical cell line.
As a result, cytotoxicity test in vivo showed that with the improvement of drug concentration, the growth of Hela cells and formed tumors were inhibited. We deeply found that α-pinene could restrain growth of Hela cells depending on affecting the distribution of cell cycle and regulating cell apoptosis.There were increased proportions of cells in G1 phase of the cell cycle after being treated with α-pinene, which suggested that α-pinene could make Hela cells to remain in G1 phase and affect the synthesis of DNA.
Apoptosis played an important role in controlling tumor progress, but apoptosis was often inhibited in cancer, so activating apoptosis was the main target of drug treatment in cancer. In this study, AnnexinV-FITC/PI staining and TUNEL uorescent staining indicated α-pinene promoted cell apoptosis in a concentration dependent manner. And all the results of qPCR data, the western-blot and immuno uorescence staining in tumor tissues showed that the expression of Bax, Bid, caspase-9 and caspase-3 were up-regulated and the expression of Bcl-2 was down regulated after α-pinene administration. The expression change of apoptosis related proteins can fully explain the effect of αpinene induced apoptosis through mitochondrial mediated endogenous apoptosis pathway. In this way, the content of intracellular Bid in cytoplasm increased, which promoted the expression of Bax on mitochondrial membrane and repressed the expression of Bcl-2. Finally, Caspase-9 and Caspase-3 was activated, which always leaded to apoptosis.
The abnormal expression of microRNAs in tumor tissues was closely related to tumor proliferation, apoptosis and metastasis. In the development of cervical cancer, miR-34a-5p could also hold up progress of cancer cells [17]. MiR-34a-5p was looked as one of the prognostic factors of cervical cancer patients [16]. We con rmed that α-Pinene increase expression of miR-34a-5p, and α-Pinene plus miR-34a-5p mimics could obviously enhance expression of miR-34a-5p. The result revealed α-Pinene could regulate miR-34a-5p. Bcl-2 was an important anti-apoptotic protein, which was proved to being regulated by miR-34a-5p in cancers [25]. In our experiments, when miR-34a-5p mimics was transfected the Hela cells, the expression standard of Bcl-2 reduced. On contrary, miR-34a-5p inhibitor could raise the expression standard of Bcl-2. These results proved that the controlling relationship between miR-34a-5p and Bcl-2. In addition, we also found that after α-pinene treatment, the expression of Bcl-2 was downregulated. Meanwhile, our experimental information showed that in comparison with miR-34a-5p transfection group lonely, the expression of Bcl-2 protein was declined obviously after being transfected with miR-34a-5p plus α-pinene. We could speculate α-pinene induced Hela cell apoptosis by controlling miR-34a-5p/Bcl-2 pathway.
In conclusion, we found that α-pinene could inhibit the development of Hela cells both in vitro and in vivo. α-pinene played an anti-tumor role by causing cell cycle to stagnate in G1 phase and promoting apoptosis in Hela cells, along with up-regulating expression of Bax, Bid, Caspase-9, Caspase-3 and downregulating expression of Bcl-2. Furthermore, α-pinene could down-regulate the expression level of Bcl-2 by regulating miR-34a-5p. These results con rmed α-pinene could exert anti-tumor e cacy in Hela cells, which provide theoretically strong for treating cervical cancer with natural plant extracting compound.

Declarations
Author Contributions

Con icts of Interest
All authors have no con ict of interest in the publication of this manuscript. Figure 1 (A). gas chromatography of product.

Figure 2
Page 14/18 Survival rate of Hela cells was detected from 24h, after treatment with α-pinene using the MTT array.