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

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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 anti-tumor 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 fluorescence intensity of Bax, Bid, Caspase-9, Caspase-3 increased and fluorescence 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. Drug Discovery, Design, & Development α-Pinene Cervical cancer Hela cells Apoptosis MicroRNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 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 five 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, flammatory and cancer [6-8]. 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 [13-15], 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 confirmation 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. 2.2 . Cell culture Hela cell line, the human cervical carcinoma cell, was acquired from Suyan Biotechnology Co. (Guangzhou, Guangdong, China). The cells were cultured in RPMI-1640 medium (Gibco-BRL, Los Angeles, CA, USA), mixed with 10% fetal bovine serum (Gibco-BRL, Los Angeles, CA, USA) and 1% streptomycin/-penicillin (Solarbio Corp., Beijing, China). The cells were applied to constant-temperature incubator with 5% CO2 at 37°C. 2.3 . 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. 2.4. Cell cycle analysis 1×10 6 Hela cells were inoculated into 6-well plate for each well. When the cultured cell confluency 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. 2.5. 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 flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) . 2.6. TUNEL staining Cell apoptosis was also gauged by TUNEL staining kit (Beyotime, Shanghai, China). Hela cells were inoculated into climbing films and incubated with different concentration of α-pinene for 24h. Then the cells were fixed by 1mL 4% paraformaldehyde for 30 minutes. Thereafter, the cells were dipped into PBS containing 0.3% TritonX-100. Then the cells of climbing flake were incubated with 50μL of TUNEL detection solution in dark for 60 minutes at 37 ℃. The fluorescence pictures were observed and captured by fluorescence microscope (Olympus, Tokyo, Japan), then the fluorescence density value was analyzed by Image J software. 2.7 . 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 amplification. 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. The two-step procedure was: 95 ℃, 5s (thermal denaturation); 60 ℃, 30s (primer annealing), The Cycle is repeated for 40 times. As for microRNA detection, PrimeScript™ RT reagent Kit (TaKaRa Bio, Beijing, China). Total RNA was extracted 24 hours after medicine administration. MiR-34a-5p was reverse transcripted by cervical loop method according to PrimeScript™ RT reagent Kit rules (TaKaRa Bio, Beijing, China). The reverse transcription primer sequences were 5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACACAACCAG-3’, primers of miR-34a-5p and U6 were also shown in Table 1 . Expression levels of miR-34a-5p in Hela cells were measured by qPCR. The relative quantity was calculated by means of 2 −ΔΔCt method. Table 1 Primer sequences Gene name Up sequence Down sequence GADPH 5’-ACAACTTTGGTATCGTGGAAGG-3’ 5’-GCCATCACGCCACAGTTTC-3’ Bid 5’-CCACACCGTGGTCTTTCCA-3’ 5’-GCACATCATTGCCAGTGCTC-3’ Bcl-2 5’-TGGTGATGTGAGTCTGGGCT-3’ 5’-CTGATTTTATTTCGCCGGCTCC-3’ Bax 5’-TCACTGAAGCGACTGATGTCC-3’ 5’-CTTCCAGATGGTGAGTGAGGC-3’ Caspase-3 5’-TGCTATTGTGAGGCGGTTGT-3’ 5’-CGCTTCCATGTATGATGGTTCC-3’ Caspase-9 miR-34a-5p U6 5’-GCTCAGACCAGAGATTCGCA-3’ 5’-TCGGCAGGTGGCAGTGTCTTAG-3’ 5’-CTCGCTTCGGCAGCACA-3’ 5’-CTTTCTGCTCGACATCACCAA-3’ 5’-TATCCAGTGCGTGTCGTG-3’ 5’-AACGCTTCACGAATTTGCGT-3’ 2. 8 . 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 difluoride (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. 2.9. miR-34a-5p mimics and inhibitor transfection MiR-34a-5p mimics (5’-UGGCAGUGUCUUAGCUGGUUGU-3’) and miR-34a-5p inhibitor (5’-ACAACCAGCUAAGACACUGCCA-3’) were obtained from RiboBio, (Guangzhou, Guangdong, China). 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 α -pinene 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. 2.10 . Animal experiments Thirty female BALB/C nude mice were obtained from Guangdong Medical Experimental Animal Center. The nude mice were raised in SPF environment. Then 1×107/200μL Hela cells were injected at the right back of nude mice. When the volumes of tumors rose to 100mm3, the mice with tumors were divided into three groups: control group (PBS group), 5-FU group (15μmol/L) and α -pinene group (60μmol/L). The mice of each group were intraperitoneal injected 200μL corresponding drug every two days for 14 days. 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 fluorescence photograph was captured by the fluorescence microscope. Furthermore, apoptotic genes detected in our previous study were detected in tumor tissues by immunofluorescence. 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 fluorescent 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 fluorescence quenching liquid was covered on the tissue, the fluorescence image was observed by the fluorescence microscope (Olympus, Tokyo, Japan). 2.11 . 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 significant when p < 0.05. Results 3.1. Product purity and structure are confirmed 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. 3.2 . 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. 3.3 . α-Pinene made Hela cells to remain at G1 phase The cycle distribution in Hela cells was detected by flow 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 fluorescent 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 fluorescence intensity of the α -pinene treatment group was significantly enhanced (Fig. 4B). These outcomes demonstrated that α -pinene treatment could lead to apoptosis of Hela cells. In order to further confirm if α -Pinene could influence expression change of apoptosis related genes, the operations of qPCR and western blot were conducted to detect expression levels of five 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). 3.5. α-Pinene regulated m iR-34a-5p/Bcl-2 pathway We further explored the molecular mechanism that α -Pinene induced apoptosis of Hela cells. MicroRNA were important for regulating expression change of apoptosis related genes. Bcl-2 was always considered as the target gene of miR-34a-5p. So we detected the expression of miR-34a-5p following treatment with α -Pinene. The data displayed that miR-34a-5p was high-expressed after Hela cells was treated by α -Pinene (Fig. 6A). After that,we tried to certify α -Pinene could regulate miR-34a-5p/Bcl-2 pathway to induce cells apoptosis. Transfection assay showed that compared with negative group, miR-34a-5p mimics could decrease expression of miR-34a-5p, while miR-34a-5p inhibitor increased the expression of miR-34a-5p, and α -Pinene plus miR-34a-5p mimics could obviously enhance expression of miR-34a-5p (Fig.6B). Meanwhile, compared with negative group, miR-34a-5p mimics could decrease expression of Bcl-2, while miR-34a-5p inhibitor increased the expression of Bcl-2. miR-34a-5p allied with α -Pinene induce obvious down-regulation of Bcl-2 (Fig. 6C, D). In conclusion, α -Pinene affected the expression of miR-34a-5p and Bcl-2, miR-34a-5p could influence expression of Bcl-2, so α -Pinene could regulate miR-34a-5p/Bcl-2 pathway. 3.6. 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 fluorescence 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 immunofluorescence method in transplanted tumor tissues. DAPI dye was used to dye the nucleus, and the expressed protein was stained green or red fluorescence. Fluorescence analysis showed exhibited lower expression of Bcl-2 in both 5-FU treatment group and α -pinene 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 [21-23]. 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 α -pinene 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 fluorescent staining indicated α-pinene promoted cell apoptosis in a concentration dependent manner. And all the results of qPCR data, the western-blot and immunofluorescence 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 α -pinene 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 confirmed 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 down-regulated. 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 down-regulating expression of Bcl-2. Furthermore, α -pinene could down-regulate the expression level of Bcl-2 by regulating miR-34a-5p. These results confirmed α -pinene could exert anti-tumor efficacy in Hela cells, which provide theoretically strong for treating cervical cancer with natural plant extracting compound. Declarations Author Contributions Huang Xiaosu: Conceptualization Methodology, Data curation, Writing-Original draft preparation, Writing- Reviewing and Editing Yan pei: Methodology, Data curation, Writing-Original draft preparation Zhou chang: Conceptualization, Software . Xu qiuxiang: Visualization. Li ming: Software, Validation. Ye lianbao: Supervision. Ding wenqing: Validation. Chen weiqiang: Funding acquisition. Conflicts of Interest All authors have no conflict of interest in the publication of this manuscript. Acknowledgments This project was financially supported by Special Areas of New Generation of Information Technology of Guangdong Provincial Department of Education: (grant nos.2020ZDZX3026). Youth Innovative Talent Project of Guangdong Provincial Department of Education (grant nos. 201810573041). Special fund for economic and technological development of Longgang District, Shenzhen (grant nos. LGKCYLWS2020103） Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Experimental Animal Ethics Review Committee of Guangdong Pharmaceutical University (gdpulac2019074) References 1. 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Cao, W., Wei, W., Zhan, Z., Xie, D., Xie, Y., and Xiao, Q.(2018) Regulation of drug resistance and metastasis of gastric cancer cells via the microRNA647-ANK2 axis.Int J Mol Med 41, 1958-1966 22. Abu Samaan, T. M., Samec, M., Liskova, A., Kubatka, P., and Büsselberg, D(2019) aclitaxel's Mechanistic and Clinical Effects on Breast Cancer.Biomolecules9, 789 23. Su, P., Yang, Y., Wang, G., Chen, X., and Ju, Y(2018) Curcumin attenuates resistance to irinotecan via induction of apoptosis of cancer stem cells in chemoresistant colon cancer cells. Int J Oncol 53, 1343-1353 24. Xu, Q., Li, M., Yang, M., Yang, J., Xie, J., Lu, X., Wang, F., and Chen, W (2018) α-pinene regulates miR-221 and induces G(2)/M phase cell cycle arrest in human hepatocellular carcinoma cells.Biosci Rep38 ,6 25. Feng, S. D., Mao, Z., Liu, C., Nie, Y. S., Sun, B., Guo, M., and Su, C, Onco (2017) Simultaneous overexpression of miR-126 and miR-34a induces a superior antitumor efficacy in pancreatic adenocarcinoma. Onco Targets Ther,10, 5591-5604 Supplementary Files BCL2.tif BID.tif Bax.tif CASPASE3.tif CASPASE9.tif GraphicalAbstract.pdf gapdh.tif Cite Share Download PDF Status: Published Journal Publication published 07 Sep, 2022 Read the published version in Drug Development Research → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-789119","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":44425619,"identity":"e651d94e-5872-4eee-90d9-803cb340c0a9","order_by":0,"name":"Huang Xiao-Su","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huang","middleName":"","lastName":"Xiao-Su","suffix":""},{"id":44425620,"identity":"df46496b-175e-404a-85c7-84e44b3b4085","order_by":1,"name":"Yan Pei","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Pei","suffix":""},{"id":44425621,"identity":"f7fa9b8c-af77-492b-9f72-3ef555b70b8b","order_by":2,"name":"Zhou Chang","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhou","middleName":"","lastName":"Chang","suffix":""},{"id":44425622,"identity":"6dc6eaad-7e44-44d4-91f8-e22c37f9d8fd","order_by":3,"name":"Xu Qiuxiang","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Qiuxiang","suffix":""},{"id":44425623,"identity":"ba5cd509-33ae-479e-a78f-e287028c03a4","order_by":4,"name":"Li Ming","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Ming","suffix":""},{"id":44425624,"identity":"f626885a-ad86-444e-a8e8-4f5916f6a7d8","order_by":5,"name":"Ye Lianbao","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Lianbao","suffix":""},{"id":44425625,"identity":"eb156eeb-8aa9-4545-9cf0-011c96e84ee0","order_by":6,"name":"Ding Wenqing","email":"","orcid":"","institution":"Shenzhen Children's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ding","middleName":"","lastName":"Wenqing","suffix":""},{"id":44425626,"identity":"68c11dcd-7ba5-4e76-b569-190fe7f6a0c8","order_by":7,"name":"Chen Wei-Qiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYHACxgcJFf/l2NibDxCthdngwRlmYz6eYwlEa2GTfNjGnDhPIkeBOPX8M5I3SCScYUtvY8hhYPhRsY2wFokbaQUGCRU8uW0MZw8w9py5TViLgXSOQULCGYncNsa+BGbGNiK1HEhsM0hnY+YxIFqLYUNiW0ICGxuxWiTuPytmSDhzwLCNhy3hIFF+4e85vP3nj4oD8vLzHx988KOCCC0gt8FZB4hSj6JlFIyCUTAKRgFWAABAmjx56H0XEgAAAABJRU5ErkJggg==","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Wei-Qiang","suffix":""}],"badges":[],"createdAt":"2021-08-07 01:18:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-789119/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-789119/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1002/ddr.21994","type":"published","date":"2022-09-08T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":12261749,"identity":"419151f8-8b28-4355-acd4-dd1eedddd8f3","added_by":"auto","created_at":"2021-08-09 19:45:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29748,"visible":true,"origin":"","legend":"(A). gas chromatography of product. ","description":"","filename":"f1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/cac0c3ce5c181ad5d40cef07.jpg"},{"id":12261747,"identity":"e6e5d444-3c86-4ecf-86f5-136b00a09456","added_by":"auto","created_at":"2021-08-09 19:45:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":32453,"visible":true,"origin":"","legend":"Survival rate of Hela cells was detected from 24h, after treatment with α-pinene using the MTT array.","description":"","filename":"f2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/927ab23608ce249bfae89203.jpg"},{"id":12261746,"identity":"6595df0c-75aa-4724-a59d-67e0a3489c12","added_by":"auto","created_at":"2021-08-09 19:45:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":33475,"visible":true,"origin":"","legend":"Cell cycle detection revealed an increase in G0/G1 phase of HeLa cells. A the negative control group; B 5-Fu control group; C α–pinene low concentration group; D α–pinene medium concentration group; E α–pinene high concentration group. F Statistical analysis graph of Cell cycle distribution. In comparison with the negative control group, * P \u003c 0.05; ** P \u003c 0.01.\n3.4. α-Pinene induced apoptosis of Hela cells \n","description":"","filename":"f3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/a97b529f974c6b896f408ec7.jpg"},{"id":12261834,"identity":"1aa7d0f4-79c1-4481-8d72-37fb6d56fcc9","added_by":"auto","created_at":"2021-08-09 19:48:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39653,"visible":true,"origin":"","legend":"α–pinene promoted the apoptosis of Hela cells. (A) Apoptotic cells were checked by Annexin V-FITC/PI apoptosis detection kit after treatment with α–pinene for 24h. a: the negative control group; b: 5-Fu control group; c: α–pinene low concentration group; d: α–pinene medium concentration group; e: α–pinene high concentration group. In comparison with the negative control group, *P\u003c 0.05; **P \u003c 0.01. (B) Apoptotic cells were also stained with TUNEL test kit, and cells with green fluorescence were observed by fluorescence microscopy. *P \u003c 0.05, **P \u003c 0.01. ","description":"","filename":"f4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/df91f34d7aa91de6e600106d.jpg"},{"id":12261758,"identity":"06031dec-01bd-462a-9efc-6e6074533cbb","added_by":"auto","created_at":"2021-08-09 19:45:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":48589,"visible":true,"origin":"","legend":"α–pinene influenced the expression of apoptosis related genes in Hela cells. The effect of α–pinene on expression of apoptosis related genes were detected by qRT-PCR (A) and Western blot (B) in Hela cells. a: the negative control group; b: 5-Fu control group; c: α–pinene low concentration group; d: α–pinene medium concentration group; e: α–pinene high concentration group. In comparison with the negative control group, *P \u003c 0.05, **P \u003c 0.01.","description":"","filename":"f5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/45c8a9e703ff3f4d31e3709a.jpg"},{"id":12261833,"identity":"67f61946-1562-4328-99b7-de4625c0f49e","added_by":"auto","created_at":"2021-08-09 19:48:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":52644,"visible":true,"origin":"","legend":"α–Pinene regulatediR-34a and Bcl-2 expression. (A) The influnece of α-pinene on the expression of miR-34a-5p in Hela cells was evaluated using qRT-PCR. (B) The expression of miR-34a-5p was determined after miR-34a-5p mimics and inhibitor transfection. (C) The effect of miR-34a-5p mimics and inhibitor on Bcl-2 gene expression determined by qRT-PCR. (D) Protein expression of Bcl-2 was determined after miR-34a-5p mimics (b), miR-34a-5p mimics with α-pinene (c) and miR-34a-5p inhibitor transfection (d). In comparison with the negative control group (a), **P \u003c 0.01, ****P\u003c0.001.","description":"","filename":"f6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/ef5a16c4f207d1c9cddcf9af.jpg"},{"id":12261745,"identity":"82b982f1-20f9-4ea8-8b65-0389dacebdf7","added_by":"auto","created_at":"2021-08-09 19:45:17","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":58960,"visible":true,"origin":"","legend":"α–pinene promoted the apoptosis of Hela cells in vivo. (A) TUNEL staining was applied to test the affect of α–pinene on apoptosis of tumor cells. green: TUNEL- positive cells; blue; DAPI. (B-F) The expression of Bcl-2, Bax, Bid, Caspase 9, Caspase 3 protein was detected by immunofluorescence staining after α–pinene treatment. green: Bcl-2, Bid, Caspase 3; red: Bax, Caspase 9; blue: DAPI. (original magnification ×100)","description":"","filename":"f7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/b60c7a1ffda827584f6f36e2.jpg"},{"id":26261965,"identity":"85e0a415-da14-41ab-b7fe-33dfc638caf4","added_by":"auto","created_at":"2022-09-09 14:45:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":500753,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/f66397de-d266-4ad0-8c34-2cfb22e94e31.pdf"},{"id":12261748,"identity":"eaefa6c3-909c-4123-aa52-dc09230e80a2","added_by":"auto","created_at":"2021-08-09 19:45:17","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":397332,"visible":true,"origin":"","legend":"","description":"","filename":"BCL2.tif","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/2473c1f01ed5ecc949834e03.tif"},{"id":12261756,"identity":"7ffffa7d-65c0-40f7-bf15-5a05b5605047","added_by":"auto","created_at":"2021-08-09 19:45:18","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":241540,"visible":true,"origin":"","legend":"","description":"","filename":"BID.tif","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/402de52bf4d1669e89ac6b58.tif"},{"id":12261751,"identity":"12749ca8-bba9-4e44-8e99-11d93339a183","added_by":"auto","created_at":"2021-08-09 19:45:17","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":100760,"visible":true,"origin":"","legend":"","description":"","filename":"Bax.tif","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/9d60688556062961f9e3be91.tif"},{"id":12261752,"identity":"7450744d-1c33-4c19-9422-07d464617f64","added_by":"auto","created_at":"2021-08-09 19:45:17","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":106090,"visible":true,"origin":"","legend":"","description":"","filename":"CASPASE3.tif","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/8ef696a85e9fb6bd1de3e513.tif"},{"id":12261832,"identity":"a7ee62d0-686c-482d-9a8a-a80882228aac","added_by":"auto","created_at":"2021-08-09 19:48:17","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":106140,"visible":true,"origin":"","legend":"","description":"","filename":"CASPASE9.tif","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/7494872edd4c169772b44c4f.tif"},{"id":12261835,"identity":"975f6089-1445-495c-a846-acd66211297b","added_by":"auto","created_at":"2021-08-09 19:48:18","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2037152,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/d09cb2def67ff4555d74697c.pdf"},{"id":12261754,"identity":"1c67ad25-c071-4690-ab0e-d50a974df8ef","added_by":"auto","created_at":"2021-08-09 19:45:17","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":67408,"visible":true,"origin":"","legend":"","description":"","filename":"gapdh.tif","url":"https://assets-eu.researchsquare.com/files/rs-789119/v1/fc8ed1c183920ef180a8b727.tif"}],"financialInterests":"","formattedTitle":"α-Pinene inhibits the growth and induces the apoptosis of cervical cancer cells through regulating miR-34a/Bcl-2 signaling axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCervical 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 five years survival rate of CC patients was only about 36.9%\u0026nbsp;[3]. Chemotherapy is an\u0026nbsp;effective treatment measure to prolong the life length of patients after they had accepted\u0026nbsp;surgical operation [4].\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eHowever,\u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene was a bicyclic monoterpene compound which was extracted from turpentine. Recently, lots of medicinal values of \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene were found in treatment of various diseases, including sleep, flammatory and cancer [6-8]. It had been found that \u0026alpha;-pinene had anti-cancer effect on lung cancer by promoting apoptosis in lung cancer cells [9]. There was another study which showed that \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene inhibited prostate cancer growth in a xenografts model [10]. However, the potential effects of \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene and its treatment relative mechanisms in CC were not investigated. So we proceeded biological activity experiments to explore the effect of \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene on CC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt was well known that\u0026nbsp;microRNAs were conserved small non coding RNAs and they\u0026nbsp;could\u0026nbsp;regulate the transcription and translation process of the target genes in different types of cancers\u0026nbsp;[11,12].\u0026nbsp;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\u0026nbsp;[13-15],\u0026nbsp;And miR-34a was also reported as a novel molecular biomarker associated with cervical cancer [16,17]. And\u0026nbsp;it was clear that miR-34a acted as a tumor suppressor gene by downregulating Bcl-2[18].\u0026nbsp;The synthetic function of miR-34a provided a new way to analyze the mechanism of \u0026alpha;-pinene functions.\u003c/p\u003e\n\u003cp\u003eIn our study, we evaluated the therapeutic effect of \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene in cervical cancer using Hela cell lines and tumor bearing nude mice models. And anti-cancer mechanism of \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene was explained basing on the miR-34a/Bcl-2-mediated regulation functions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cem\u003e2.1.\u0026nbsp;\u003c/em\u003e\u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene\u0026nbsp;purity and structural confirmation\u003c/p\u003e\n\u003cp\u003ePine 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\u0026nbsp;\u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene.\u0026nbsp;Analysis of product purity and structures by gas chromatography instrument, mass spectrometer.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;Cell culture\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHela cell line, the human cervical carcinoma cell, was acquired from Suyan Biotechnology Co. (Guangzhou, Guangdong, China). The cells were cultured in RPMI-1640 medium (Gibco-BRL, Los Angeles, CA, USA), mixed with 10% fetal bovine serum (Gibco-BRL, Los Angeles, CA, USA) and 1% streptomycin/-penicillin (Solarbio Corp., Beijing, China). The cells were applied to constant-temperature incubator with 5% CO2 at 37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.3\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003eMTT assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe\u003cem\u003e\u0026nbsp;\u0026alpha;\u003c/em\u003e-pinene dilution was dissolve with\u0026nbsp;dissolved in the DMSO.\u0026nbsp;The proliferation of Hela cells was detected by MTT assay after they were treated with \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene. The Hela cells were inoculated into 96 well plate by adjusting cell accounts to about 4\u0026times;10\u003csup\u003e3\u003c/sup\u003ecells/well with 100\u0026mu;L.Different concentration of \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene (10, 20, 40, 80, 160, 320, 640\u0026mu;mol/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 (40\u0026mu;mol/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 10\u0026mu;L of 5 mg/mL MTT reagent (Meilun Biotechnology Co., Dalian, Liaoning, China) was put into well and incubated in dark at 37\u0026nbsp;\u0026deg;C for 4 h. After removal of the MTT, cells were treated with 100\u0026mu;L 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)\u0026times;100%. Each experiment was repeated three times. The IC\u003csub\u003e50\u003c/sub\u003e value of \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene in Hela cells was calculated with the help of Graphpad Prism 8.0 software.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eCell cycle analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e1\u0026times;10\u003csup\u003e6\u003c/sup\u003e Hela cells were inoculated into 6-well plate for each well. When the cultured cell confluency reached at about 60%, the drug was administered into cultured cells. According to IC50 calculated by MTT data, \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene groups were designed to three different concentration groups: low concentration group (25\u0026mu;mol/L), medium concentration group (50\u0026mu;mol/L) and high concentration group (100\u0026mu;mol/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 \u0026nbsp; cell cycle kit, the cells were labeled with 50\u0026mu;g/mL PI staining at 37\u0026nbsp;℃\u0026nbsp;for 30 minutes. Then the samples were measured by Flow Cytometry. The test data was analyzed by ModFit software.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.5.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eCell apoptosis proportion detection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe apoptosis of Hela cells treated with\u0026nbsp;\u0026alpha;-pinene was assessed by Annexin V-FITC/PI apoptosis detection kit (BestBio, Biotechnology Co., Shanghai, China),. After they were incubated with \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene for 24 h, the Hela cells were gathered and cleaned by PBS. Cells were mixed with 5\u0026mu;L Annexin V-FITC dyesolution and 5\u0026mu;L PI dye solution away from light for 10min, the condition of cell apoptosis was tested by flow cytometry\u0026nbsp;\u003cem\u003e(BD\u003c/em\u003e \u003cem\u003eBiosciences,\u003c/em\u003e Franklin Lakes, NJ,\u0026nbsp;\u003cem\u003eUSA)\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.6.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eTUNEL staining\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCell apoptosis was also gauged by TUNEL staining kit\u0026nbsp;(Beyotime, Shanghai, China). Hela cells were inoculated into climbing films and incubated with different concentration of \u0026alpha;-pinene for 24h. Then the cells were fixed by 1mL 4% paraformaldehyde for 30 minutes. Thereafter, the cells were dipped into PBS containing 0.3% TritonX-100. Then the cells\u0026nbsp;of climbing flake\u0026nbsp;were incubated with 50\u0026mu;L of TUNEL detection solution in dark for 60 minutes at 37\u0026nbsp;℃. The fluorescence pictures were observed and captured by fluorescence microscope (Olympus, Tokyo, Japan), then the fluorescence density value was analyzed by Image J software.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.7\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;qRT-PCR\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were handled according to the previous concentration and action time of \u0026alpha;-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 \u0026trade; RT Master Mix reagent Kit (TaKaRa Bio, Beijing, China), the acquired RNA was reverse transcripted to cDNA.\u0026nbsp;TB Green Premix Ex TaqII kit\u0026nbsp;(TaKaRa Bio, Beijing, China) was used to measure relative gene cDNA amplification. 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. The two-step procedure was: 95\u0026nbsp;℃, 5s (thermal denaturation); 60\u0026nbsp;℃, 30s (primer annealing), The Cycle is repeated for 40 times. As for microRNA detection,\u0026nbsp;PrimeScript\u0026trade; RT reagent Kit\u0026nbsp;(TaKaRa Bio, Beijing, China). Total RNA was extracted 24 hours after medicine administration. MiR-34a-5p was reverse transcripted by cervical loop method according to PrimeScript\u0026trade; RT reagent Kit rules\u0026nbsp;(TaKaRa Bio, Beijing, China). The reverse transcription primer sequences were 5\u0026rsquo;-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACACAACCAG-3\u0026rsquo;, primers of miR-34a-5p and U6 were also shown in \u003cstrong\u003eTable 1\u003c/strong\u003e. Expression levels of miR-34a-5p in Hela cells were measured by qPCR.\u0026nbsp;The relative quantity was calculated by means of 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrimer sequences\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"604\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.417218543046358%\"\u003e\n \u003cp\u003eGene name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"45.36423841059602%\"\u003e\n \u003cp\u003eUp\u003c/p\u003e\n \u003cp\u003esequence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"42.21854304635762%\"\u003e\n \u003cp\u003eDown\u0026nbsp;\u003c/p\u003e\n \u003cp\u003esequence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.417218543046358%\"\u003e\n \u003cp\u003eGADPH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"45.36423841059602%\"\u003e\n \u003cp\u003e5\u0026rsquo;-ACAACTTTGGTATCGTGGAAGG-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"42.21854304635762%\"\u003e\n \u003cp\u003e5\u0026rsquo;-GCCATCACGCCACAGTTTC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.417218543046358%\"\u003e\n \u003cp\u003eBid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"45.36423841059602%\"\u003e\n \u003cp\u003e5\u0026rsquo;-CCACACCGTGGTCTTTCCA-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"42.21854304635762%\"\u003e\n \u003cp\u003e5\u0026rsquo;-GCACATCATTGCCAGTGCTC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.417218543046358%\"\u003e\n \u003cp\u003eBcl-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"45.36423841059602%\"\u003e\n \u003cp\u003e5\u0026rsquo;-TGGTGATGTGAGTCTGGGCT-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"42.21854304635762%\"\u003e\n \u003cp\u003e5\u0026rsquo;-CTGATTTTATTTCGCCGGCTCC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.417218543046358%\"\u003e\n \u003cp\u003eBax\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"45.36423841059602%\"\u003e\n \u003cp\u003e5\u0026rsquo;-TCACTGAAGCGACTGATGTCC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"42.21854304635762%\"\u003e\n \u003cp\u003e5\u0026rsquo;-CTTCCAGATGGTGAGTGAGGC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.417218543046358%\"\u003e\n \u003cp\u003eCaspase-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"45.36423841059602%\"\u003e\n \u003cp\u003e5\u0026rsquo;-TGCTATTGTGAGGCGGTTGT-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"42.21854304635762%\"\u003e\n \u003cp\u003e\u0026nbsp;5\u0026rsquo;-CGCTTCCATGTATGATGGTTCC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.417218543046358%\"\u003e\n \u003cp\u003eCaspase-9\u003c/p\u003e\n \u003cp\u003emiR-34a-5p\u003c/p\u003e\n \u003cp\u003eU6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"45.36423841059602%\"\u003e\n \u003cp\u003e5\u0026rsquo;-GCTCAGACCAGAGATTCGCA-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003e5\u0026rsquo;-TCGGCAGGTGGCAGTGTCTTAG-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003e5\u0026rsquo;-CTCGCTTCGGCAGCACA-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"42.21854304635762%\"\u003e\n \u003cp\u003e5\u0026rsquo;-CTTTCTGCTCGACATCACCAA-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003e5\u0026rsquo;-TATCCAGTGCGTGTCGTG-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003e5\u0026rsquo;-AACGCTTCACGAATTTGCGT-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e2.\u003c/em\u003e\u003cem\u003e8\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;Western-blot\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eProteins 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 difluoride (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)\u0026nbsp;was added to the membrane bearing proteins at 4℃\u0026nbsp;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\u0026nbsp;Biotechnology Co., Dalian, China) and then checked by Image Lab\u0026trade; Software.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.9. miR-34a-5p mimics and inhibitor transfection\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMiR-34a-5p mimics (5\u0026rsquo;-UGGCAGUGUCUUAGCUGGUUGU-3\u0026rsquo;) and miR-34a-5p inhibitor (5\u0026rsquo;-ACAACCAGCUAAGACACUGCCA-3\u0026rsquo;) were obtained from RiboBio, (Guangzhou, Guangdong, China). According to operation rules of transient transfection, Lipofectamine\u003csup\u003eTM\u003c/sup\u003e 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 \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene 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.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.10\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;Animal experiments\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThirty female BALB/C nude mice were obtained from Guangdong Medical Experimental Animal Center. The nude mice were raised in SPF environment. Then 1\u0026times;107/200\u0026mu;L Hela cells were injected at the right back of nude mice. When the volumes of tumors rose to 100mm3, the mice with tumors were divided into three groups: control group (PBS group), 5-FU group (15\u0026mu;mol/L) and \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene group (60\u0026mu;mol/L). The mice of each group were intraperitoneal injected 200\u0026mu;L corresponding drug every two days for 14 days. Immunostaining was performed on deparafnized tissue sections. Each sample was incubated with about 40\u0026mu;L transdermal solution at room temperature for 25 minutes. Subsequently, 40\u0026mu;L TUMEL reaction solution was added to the sample for 60 minutes at 37℃. Finally, the fluorescence photograph was captured by the fluorescence microscope. Furthermore, apoptotic genes detected in our previous study were detected in tumor tissues by immunofluorescence. 40\u0026mu;L goat serum was used to block each tissue section at 37\u0026nbsp;℃\u0026nbsp;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 fluorescent 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 fluorescence quenching liquid was covered on the tissue, the fluorescence image was observed by the fluorescence microscope (Olympus, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.11\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003eStatistical analysis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGraphpad prism 8.0 software was applied to draw the histogram and analyze the results. All of the data\u0026rsquo;s were expressed as mean \u0026plusmn; 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.\u0026nbsp;The difference was significant\u0026nbsp;when \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003e3.1. Product purity and structure are confirmed\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis 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)\u003c/p\u003e\n\u003cp\u003eThe mass spectrogram (B). mass spectrogram of product (C). mass spectrogram of\u0026nbsp;\u0026alpha;-pinene which was retrieved from NIST database.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003e\u0026alpha;-Pinene controlled the proliferation of Hela cells\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe MTT experiment showed that \u003cem\u003e\u0026alpha;-\u003c/em\u003ePinene\u003cem\u003e\u0026nbsp;\u003c/em\u003ehad obvious inhibition on Hela cells at different concentration gradient. And\u0026nbsp;IC\u003csub\u003e50\u003c/sub\u003e value for \u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene was (46.49\u0026plusmn;0.78\u0026mu;mol/L at 24h,) (Fig. 2). Therefore, 24 hours was selected as the administration time, and 25\u0026mu;mol/L, 50\u0026mu;mol/L and100\u0026mu;mol/L were considered as the low, medium and high concentration gradient groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003e\u0026alpha;-Pinene made Hela cells to remain at G1 phase\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe cycle distribution in Hela cells was detected by flow cytometry following 24h treatment with \u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene at concentrations of 25\u0026mu;mol/L,50\u0026mu;mol/L and100\u0026mu;mol/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 \u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene (Fig. 3B-E). This result displayed that\u003cem\u003e\u0026nbsp;\u0026alpha;\u003c/em\u003e-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.\u003c/p\u003e\n\u003cp\u003eWe measured the apoptosis in Hela cells after treatment with \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene using AnnexinV-FITC/PI staining and TUNEL fluorescent staining. Treatment with \u003cem\u003e\u0026alpha;-\u003c/em\u003epinene increased the proportions of apoptotic Hela cells (Fig. 4A). TUNEL results also showed that in comparison with the control group, the fluorescence intensity of the\u003cem\u003e\u0026nbsp;\u0026alpha;\u003c/em\u003e-pinene treatment group was significantly enhanced (Fig. 4B). These outcomes demonstrated that\u003cem\u003e\u0026nbsp;\u0026alpha;\u003c/em\u003e-pinene treatment could lead to apoptosis of Hela cells.\u0026nbsp;In order to further confirm if\u003cem\u003e\u0026nbsp;\u0026alpha;\u003c/em\u003e-Pinene could influence expression change of apoptosis related genes, the operations of qPCR and western blot were conducted to detect expression levels of five 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).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.5.\u0026nbsp;\u0026alpha;-Pinene regulated\u0026nbsp;\u003c/em\u003e\u003cem\u003em\u003c/em\u003e\u003cem\u003eiR-34a-5p/Bcl-2 pathway\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe further explored the molecular mechanism that \u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene induced apoptosis of Hela cells. MicroRNA were important for regulating expression change of apoptosis related genes. Bcl-2 was always considered as the target gene of miR-34a-5p. So we detected the expression of miR-34a-5p following treatment with \u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene. The data displayed that miR-34a-5p was high-expressed after Hela cells was treated by \u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene (Fig. 6A). After that,we tried to certify \u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene could regulate miR-34a-5p/Bcl-2 pathway to induce cells apoptosis. Transfection assay showed that compared with negative group, miR-34a-5p mimics could decrease expression of miR-34a-5p, while miR-34a-5p inhibitor increased the expression of miR-34a-5p, and \u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene plus miR-34a-5p mimics could obviously enhance expression of miR-34a-5p (Fig.6B). Meanwhile, compared with negative group, miR-34a-5p mimics could decrease expression of Bcl-2, while miR-34a-5p inhibitor increased the expression of Bcl-2. miR-34a-5p allied with \u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene induce obvious down-regulation of Bcl-2 (Fig. 6C, D). In conclusion, \u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene affected the expression of miR-34a-5p and Bcl-2, miR-34a-5p could influence expression of Bcl-2, so \u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene could regulate miR-34a-5p/Bcl-2 pathway.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.6.\u0026nbsp;\u0026alpha;-pinene inhibited the growth of xenotransplanted tumors\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAnti-tumor effect of \u0026alpha;-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 fluorescence intensity of 5-FU group and \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene group was stronger than that of control group (Fig. 7A). TUNEL staining showed that \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene could lead to\u0026nbsp;apoptosis\u0026nbsp;activation of tumor cells in\u003cem\u003e\u0026nbsp;vivo\u003c/em\u003e. The results concurred with the results of cell experiments in \u003cem\u003evitro\u003c/em\u003e. The proteins which were associated with cell apoptosis were detected through immunofluorescence method in transplanted tumor tissues. DAPI dye was used to dye the nucleus, and the expressed protein was stained green or red fluorescence. Fluorescence analysis showed exhibited lower expression of Bcl-2 in both 5-FU treatment group and \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene 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 \u0026alpha;-pinene treatment group (Fig. 7B-F). The results of \u003cem\u003ein vivo\u003c/em\u003e experiments were consistent with those of \u003cem\u003ein vitro\u003c/em\u003e. These data further proved that \u0026alpha;-pinene can induce apoptosis of HeLa cells by promoting cell apoptosis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCervical cancer was one of the most prevelant gynecological malignancies, 85% cervical cancer cases appeared in developing countries\u0026nbsp;[19]. Chemotherapy was the common treatment method for cervical cancer. Nowadays, more and more chemosynthetic antitumor drugs had severe toxicity and drug resistance\u0026nbsp;[20]. So lots of natural anticancer drugs including Vincristin,\u0026nbsp;Paclitaxel, Camptothecin\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewere abstracted from plants and displayed effective anti-tumor activity\u0026nbsp;[21-23]. \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene was separated and distilled from pine needles. In our previous studies, we found \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene inhibit the growth of\u0026nbsp;hepatocellular cancer cells\u0026nbsp;[24].\u0026nbsp;\u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene also could showed the ability of promoting the apotosis of\u0026nbsp;prostate carcinoma cells\u0026nbsp;[10]. In order to verify pinene was a broad spectrum anti-cancer drug, we evaluated the function of \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene in Hela cells, a cervical cell line.\u003c/p\u003e\n\u003cp\u003eAs a result, cytotoxicity test \u003cem\u003ein vivo\u003c/em\u003e showed that with the improvement of drug concentration, the growth of Hela cells and formed tumors were inhibited. We deeply found that \u003cem\u003e\u0026alpha;\u003c/em\u003e-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\u0026nbsp;after being treated with \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene, which suggested that \u003cem\u003e\u0026alpha;-\u003c/em\u003epinene 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,\u0026nbsp;AnnexinV-FITC/PI staining and\u0026nbsp;TUNEL\u0026nbsp;fluorescent staining\u0026nbsp;indicated \u0026alpha;-pinene promoted cell apoptosis\u0026nbsp;in a concentration dependent manner.\u0026nbsp;And all the results of qPCR data, the western-blot and immunofluorescence 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 \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene administration.\u0026nbsp;The expression change of apoptosis related proteins can fully explain the effect of \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene 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.\u003c/p\u003e\n\u003cp\u003eThe abnormal expression of microRNAs in tumor tissues was closely related to tumor proliferation, apoptosis and metastasis.\u0026nbsp;In the development of cervical cancer, miR-34a-5p could also hold up progress of cancer cells\u0026nbsp;[17]. MiR-34a-5p was looked as one of the prognostic factors of cervical cancer patients\u0026nbsp;[16]. We confirmed that\u0026nbsp;\u0026alpha;-Pinene increase expression of miR-34a-5p, and\u0026nbsp;\u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene plus miR-34a-5p mimics could obviously enhance expression of miR-34a-5p.\u0026nbsp;The result revealed\u0026nbsp;\u003cem\u003e\u0026alpha;\u003c/em\u003e-Pinene\u0026nbsp;could regulate\u0026nbsp;miR-34a-5p.\u0026nbsp;Bcl-2 was an important anti-apoptotic protein, which was proved to being regulated by miR-34a-5p in cancers\u0026nbsp;[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\u0026nbsp;\u0026alpha;-pinene\u0026nbsp;treatment, the expression of Bcl-2 was down-regulated. 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 \u0026alpha;-pinene. We could speculate \u0026alpha;-pinene induced Hela cell apoptosis by controlling miR-34a-5p/Bcl-2 pathway.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we found that \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene could inhibit the development of Hela cells both\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e and\u003cem\u003e\u0026nbsp;in vivo.\u003c/em\u003e \u003cem\u003e\u0026alpha;\u003c/em\u003e-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 down-regulating expression of Bcl-2. Furthermore, \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene could down-regulate the expression level of Bcl-2 by regulating miR-34a-5p. These results confirmed \u003cem\u003e\u0026alpha;\u003c/em\u003e-pinene could exert anti-tumor efficacy in Hela cells, which provide theoretically strong for treating cervical cancer with natural plant extracting compound.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuang Xiaosu:\u003c/strong\u003e Conceptualization Methodology, Data curation, Writing-Original draft preparation, Writing- Reviewing and Editing\u003cstrong\u003e\u0026nbsp;Yan pei:\u003c/strong\u003e Methodology, Data curation, Writing-Original draft preparation\u0026nbsp;\u003cstrong\u003eZhou chang:\u003c/strong\u003e Conceptualization, Software\u003cstrong\u003e. Xu qiuxiang:\u0026nbsp;\u003c/strong\u003eVisualization. \u003cstrong\u003eLi ming:\u0026nbsp;\u003c/strong\u003eSoftware, Validation. \u003cstrong\u003eYe lianbao:\u003c/strong\u003e Supervision.\u003cstrong\u003e\u0026nbsp;Ding wenqing:\u003c/strong\u003eValidation. \u003cstrong\u003eChen weiqiang:\u003c/strong\u003e Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have no conflict of interest in the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was financially supported by Special Areas of New Generation of Information Technology of Guangdong Provincial Department of Education: (grant nos.2020ZDZX3026). Youth Innovative Talent Project of Guangdong Provincial Department of Education (grant nos. 201810573041). Special fund for economic and technological development of Longgang District, Shenzhen (grant nos. LGKCYLWS2020103）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Experimental Animal Ethics Review Committee of Guangdong Pharmaceutical University (gdpulac2019074)\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1. Arbyn, M., Weiderpass, E., Bruni, L., de Sanjos\u0026eacute;, S., Saraiya, M., Ferlay, J., and Bray, F (2020)\u0026nbsp;Estimates of incidence and mortality of cervical cancer in 2018: a worldwide analysis.\u0026nbsp;Lancet Glob Health 8, e191-e203\u003c/p\u003e\n\u003cp\u003e2. \u0026nbsp;Canfell, K., Kim, J. J., Brisson, M., Keane, A., Simms, K. T., Caruana, M. , Burger EA., Martin D., Nguyen DTN., \u0026nbsp;B\u0026eacute;nard \u0026Eacute; (2020)\u0026nbsp;Mortality impact of achieving WHO cervical cancer elimination targets: a comparative modelling analysis in 78 low-income and lower-middle-income countries.\u0026nbsp;Lancet 395, 591-603\u003c/p\u003e\n\u003cp\u003e3. Hui Z, D. W., Ming L, Zhongqiu L(2019)\u0026nbsp;FIGO 2018 gynecological cancer report: Interpretation of cervical cancer guidelines.\u0026nbsp;Journal of Practical Gynecology and obstetric 35(1):99-107\u003c/p\u003e\n\u003cp\u003e4. Hosaka, M., Watari, H., Kato, T., Odagiri, T., Konno, Y., Endo, D., Mitamura, T., Kikawa, S., Suzuki, Y., Sakuragi N (2012 )\u0026nbsp;Clinical efficacy of paclitaxel/cisplatin as an adjuvantchemotherapy for patients with cervical cancer who underwent radical hysterectomy and systematic lymphadenectomy.\u0026nbsp;J Surg Oncol 105, 612-616\u003c/p\u003e\n\u003cp\u003e5. Jung, J., Kim, S., An, H. T., and Ko, J (2020)\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/31584624/\"\u003e\u0026alpha;-Actinin-4 regulates cancer stem cell properties and chemoresistance in cervical cancer.\u0026nbsp;\u003c/a\u003eCarcinogenesis .2020,41, 940-949\u003c/p\u003e\n\u003cp\u003e6. \u0026nbsp;Yang, H., Woo, J., Pae, A. N., Um, M. Y., Cho, N. C., Park, K. D., Yoon, M., Kim, J., Lee, C. J., and Cho, S (2016)\u0026nbsp;\u0026alpha;-Pinene, a major constituent of pine tree oils, enhances non-rapid eye movement sleep in mice through GABAA-benzodiazepine receptors.\u0026nbsp;Mol Pharmacol 90, 530-539\u003c/p\u003e\n\u003cp\u003e7. \u0026nbsp;Li, X. J., Yang, Y. J., Li, Y. S., Zhang, W. K., and Tang, H. B, J\u0026nbsp;）(2016)\u0026nbsp;\u0026alpha;-Pinene, linalool, and 1-octanol contribute to the topical anti-inflammatory and analgesic activities of frankincense by inhibiting COX-2.Ethnopharmacol,2016.179, 22-26\u003c/p\u003e\n\u003cp\u003e8. Matsuo, A. L., Figueiredo, C. R., Arruda, D. C., Pereira, F. V., Scutti, J. A., Massaoka, M. H., Travassos, L. R., Sartorelli, P., and Lago, J. H, (2011)\u0026nbsp;\u0026alpha;-Pinene isolated from Schinus terebinthifolius Raddi (Anacardiaceae) induces apoptosis and confers antimetastatic protection in a melanoma model.Biochem Biophys Res Commun. \u0026nbsp;411, 449-454\u003c/p\u003e\n\u003cp\u003e9. Zhang, Z., Guo, S., Liu, X., and Gao, X( 2015)\u0026nbsp;Synergistic antitumor effect of \u0026alpha;-pinene and \u0026beta;-pinene with paclitaxel against non-small-cell lung carcinoma (NSCLC)\u0026nbsp;Drug Res (Stuttg) 65, 214-218\u003c/p\u003e\n\u003cp\u003e10.Zhao, Y., Chen, R., Wang, Y., and Yang, Y(2018)\u0026nbsp;\u0026alpha;-Pinene inhibits human prostate cancer growth in a mouse Xenograft Model.Chemotherapy. 2018,63, 1-7\u003c/p\u003e\n\u003cp\u003e11.Iorio, M. V., and Croce, C. M(2012) microRNA involvement in human cancer. Carcinogenesis 33, 1126-1133\u003c/p\u003e\n\u003cp\u003e12. Calin, G. A., and Croce, C. M, (2006)\u0026nbsp;MicroRNA-cancer connection: the beginning of a new tale.Cancer Res 66, 7390-7394\u003c/p\u003e\n\u003cp\u003e13. Sun, Z., Zhang, T., and Chen, B(2019)\u0026nbsp;Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes proliferation and metastasis of osteosarcoma cells by targeting c-Met and SOX4 via miR-34a/c-5p and miR-449a/b.Med Sci Monit25, 1410-1422\u003c/p\u003e\n\u003cp\u003e14. Hong, J. H., Roh, K. S., Suh, S. S., Lee, S., Sung, S. W., Park, J. K., Byun, J. H., and Kang, J ,（2015）The expression of microRNA-34a is inversely correlated with c-MET and CDK6 and has a prognostic significance in lung adenocarcinoma patients.Tumour Biol. 2015,36, 9327-9337\u003c/p\u003e\n\u003cp\u003e15. Liang, J., Li, Y., Daniels, G., Sfanos, K., De Marzo, A., Wei, J., Li, X., Chen, W., Wang, J., Zhong, X., et al,\u0026nbsp;（2015）LEF1 targeting EMT in prostate cancer invasion is regulated by miR-34a.Mol Cancer Res 13, 681-688\u003c/p\u003e\n\u003cp\u003e16. Pardini, B., De Maria, D., Francavilla, A., Di Gaetano, C., Ronco, G., and Naccarati, A\u0026nbsp;（2018）MicroRNAs as markers of progression in cervical cancer: a systematic review.BMC Cancer .2018,18, 696\u003c/p\u003e\n\u003cp\u003e17. Pang, R. T., Leung, C. O., Ye, T. M., Liu, W., Chiu, P. C., Lam, K. K., Lee, K. F., and Yeung, W. S,(2010)MicroRNA-34a suppresses invasion through downregulation of Notch1 and Jagged1 in cervical carcinoma and choriocarcinoma cells.Carcinogenesis31, 1037-1044\u003c/p\u003e\n\u003cp\u003e18. Wang, X., Xie, Y., and Wang, J (2018)Overexpression of MicroRNA-34a-5p inhibits proliferation and promotes apoptosis of human cervical cancer cells by downregulation of Bcl-2.\u0026nbsp;Oncol Res.26, 977-985\u003c/p\u003e\n\u003cp\u003e19. Jemal, A., Bray, F., Center, M. M., Ferlay, J., Ward, E., and Forman, D, CA (2011).\u0026nbsp;Global cancer statistics.Cancer J Clin 61, 69-90\u003c/p\u003e\n\u003cp\u003e20. Chen, J., Solomides, C., Parekh, H., Simpkins, F., and Simpkins, H (2015)\u0026nbsp;Cisplatin resistance in human cervical, ovarian and lung cancer cells.Cancer Chemother Pharmacol .2015,75, 1217-1227\u003c/p\u003e\n\u003cp\u003e21. Cao, W., Wei, W., Zhan, Z., Xie, D., Xie, Y., and Xiao, Q.(2018)\u0026nbsp;Regulation of drug resistance and metastasis of gastric cancer cells via the microRNA647-ANK2 axis.Int J Mol Med 41, 1958-1966\u003c/p\u003e\n\u003cp\u003e22. Abu Samaan, T. M., Samec, M., Liskova, A., Kubatka, P., and B\u0026uuml;sselberg, D(2019) \u0026nbsp;aclitaxel\u0026apos;s Mechanistic and Clinical Effects on Breast Cancer.Biomolecules9, 789\u003c/p\u003e\n\u003cp\u003e23. \u0026nbsp;Su, P., Yang, Y., Wang, G., Chen, X., and Ju, Y(2018)\u0026nbsp;Curcumin attenuates resistance to irinotecan via induction of apoptosis of cancer stem cells in chemoresistant colon cancer cells.\u0026nbsp;Int J Oncol 53, 1343-1353\u003c/p\u003e\n\u003cp\u003e24. Xu, Q., Li, M., Yang, M., Yang, J., Xie, J., Lu, X., Wang, F., and Chen, W (2018)\u0026nbsp;\u0026alpha;-pinene regulates miR-221 and induces G(2)/M phase cell cycle arrest in human hepatocellular carcinoma cells.Biosci Rep38 ,6\u003c/p\u003e\n\u003cp\u003e25. Feng, S. D., Mao, Z., Liu, C., Nie, Y. S., Sun, B., Guo, M., and Su, C, Onco (2017)\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/29200874/\"\u003eSimultaneous overexpression of miR-126 and miR-34a induces a superior antitumor efficacy in pancreatic adenocarcinoma.\u0026nbsp;\u003c/a\u003eOnco Targets Ther,10, 5591-5604\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"α-Pinene, Cervical cancer, Hela cells, Apoptosis, MicroRNA","lastPublishedDoi":"10.21203/rs.3.rs-789119/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-789119/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u0026nbsp;\u003c/strong\u003e\u003cem\u003eα-\u003c/em\u003epinene was a chemical compound which was extracted from pine needles oil, and it\u0026nbsp;exerted effects\u0026nbsp;on various diseases. However, the effect of\u0026nbsp;\u003cem\u003eα\u003c/em\u003e-pinene on cervical cancer had not been reported. The goal of this study was to explore the anti-tumor role of\u0026nbsp;\u003cem\u003eα-\u003c/em\u003epinene.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u0026nbsp;\u003c/strong\u003eMethyl thiazolyl tetrazolium (MTT)\u0026nbsp;method was used to detect cytotoxicity of\u0026nbsp;\u003cem\u003eα\u003c/em\u003e-pinene.\u0026nbsp;Flow cytometry was used to quantify the cell cycle and apoptosis.\u0026nbsp;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 anti-tumor action of\u0026nbsp;\u003cem\u003eα\u003c/em\u003e-pinene\u0026nbsp;\u003cem\u003ein vivo\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u0026nbsp;\u003c/strong\u003eThe results displayed\u0026nbsp;\u003cem\u003eα\u003c/em\u003e-pinene\u0026nbsp;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,\u0026nbsp;\u003cem\u003eα\u003c/em\u003e-pinene treatment also induced apoptosis in xenografts tumor models. The fluorescence intensity of Bax, Bid, Caspase-9, Caspase-3 increased and fluorescence intensity of Bcl-2 decreased.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u0026nbsp;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.\u003c/p\u003e","manuscriptTitle":"α-Pinene inhibits the growth and induces the apoptosis of cervical cancer cells through regulating miR-34a/Bcl-2 signaling axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-08-09 19:45:15","doi":"10.21203/rs.3.rs-789119/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9f29fe4c-fee8-49ee-8430-05bcf9b65b43","owner":[],"postedDate":"August 9th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":6332060,"name":"Drug Discovery, Design, \u0026 Development"}],"tags":[],"updatedAt":"2022-09-09T14:45:15+00:00","versionOfRecord":{"articleIdentity":"rs-789119","link":"https://doi.org/10.1002/ddr.21994","journal":{"identity":"drug-development-research","isVorOnly":true,"title":"Drug Development Research"},"publishedOn":"2022-09-08 00:00:00","publishedOnDateReadable":"September 8th, 2022"},"versionCreatedAt":"2021-08-09 19:45:15","video":"","vorDoi":"10.1002/ddr.21994","vorDoiUrl":"https://doi.org/10.1002/ddr.21994","workflowStages":[]},"version":"v1","identity":"rs-789119","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-789119","identity":"rs-789119","version":["v1"]},"buildId":"DfRiq_wiF3Ryq-tyrab2w","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}