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Essential Oil from Pinus Koraiensis Pinecones Inhibits Gastric Cancer Cells Via the HIPPO/YAP Signaling Pathway

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Essential Oil from Pinus Koraiensis Pinecones Inhibits Gastric Cancer Cells Via the HIPPO/YAP Signaling Pathway molecules Article Essential Oil from Pinus Koraiensis Pinecones Inhibits Gastric Cancer Cells via the HIPPO/YAP Signaling Pathway Yandong Zhang 1, Chao Xin 1, Junqiang Qiu 2 and Zhenyu Wang 1,* 1 Department of Food Science and Engineering, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150090, China; [email protected] (Y.Z.); [email protected] (C.X.) 2 Department of Inorganic Chemistry and Analytical Chemistry, School of Pharmacy, Hainan Medical University, Haikou 570100, China; [email protected] * Correspondence: [email protected] Received: 17 September 2019; Accepted: 22 October 2019; Published: 25 October 2019 Abstract: Pinecone is a traditional folk herb, which has been used in China for many years. In this paper, the essential oil from Pinus koraiensis pinecones (PEO) was obtained by hydrodistillation and 41 compounds were identified by gas chromatography–mass spectrometry (GC-MS), mainly including α-Pinene (40.91%), Limonene (24.82%), and β-Pinene (7.04%). The purpose of this study was to investigate the anti-tumor activity of PEO on MGC-803 cells and its mechanism. Anti-tumor experiments in vitro showed PEO could significantly inhibit the proliferation and migration of MGC-803 cells, and it also could arrest the cell cycle in the G2/M phase, decrease the mitochondrial membrane potential, and induce apoptosis. Finally, the effects of PEO on genes expression on MGC-803 cells were analyzed by RNA sequencing, and results showed that after treatment with PEO, 100 genes were up-regulated, and 57 genes were down-regulated. According to the KEGG pathway and GSEA, FAT4, STK3, LATS2, YAP1, and AJUBA were down-regulated, which were related to HIPPO signaling pathway. Real-time PCR and western blot further confirmed the results of RNA sequencing. These results indicated that PEO may exert anti-tumor activity via the HIPPO/YAP signaling pathway. The anti-tumor mechanism of this oil can be further studied, which is important for the development of anti-tumor drugs. Keywords: pinecone; essential oil; gastric cancer; RNA sequencing; HIPPO/YAP signaling pathway 1. Introduction Gastric cancer is one of the most common malignancies, with about one million new cases diagnosed each year, ranking third in cancer-related deaths worldwide [1]. Gastric cancer is often diagnosed at a later stage because of the lack of early symptoms, and the frightening is the poor prognosis and limited treatment options [2]. At present, surgical resection, radiotherapy, and chemotherapy are the main methods for the treatment of gastric cancer [3]. Although chemotherapy is a more effective treatment for potential metastatic and metastatic gastric cancer, the currently applied chemotherapy drugs for gastric cancer are difficult to satisfy [4]. In order to improve the side effects and drug resistance of traditional chemotherapy drugs, more and more researches have focused on the development of natural anti-tumor drugs, including natural polyphenols [5], polysaccharides [6], and terpenoids [7]. Essential oils (EOs) have been paid more and more attention due to their unique properties, which play an important role in natural products [8,9]. It has been reported that EOs have many physiological activities, such as antioxidant [10], antibacterial [11], anti-inflammatory [12], anti-tumor [13] and anti-insect [14]. Pinus koraiensis is a traditional plant belonging to genus of Pinus in the family of Pinaceae [15]. Pinecone is an important organ of Pinus koraiensis, with the effects of antitussive, Molecules 2019, 24, 3851; doi:10.3390/molecules24213851 www.mdpi.com/journal/molecules MoleculesMolecules2019 2019, 24,, 24 3851, x FOR 2 of2 of19 18 of Pinaceae [15]. Pinecone is an important organ of Pinus koraiensis, with the effects of antitussive, expectorantexpectorant and and anti-inflammatory, anti-inflammatory, andand itit isis richrich inin essentialessential oil. In In addition, addition, PinusPinus koraiensis koraiensis pineconespinecones extracts extracts had had a a varietyvariety of of physiological physiological acti activitiesvities such such as anti-tumor, as anti-tumor, anti-angiogenesis, anti-angiogenesis, and andanti-radiation anti-radiation [16–18]. [16–18 According]. According to reports, to reports, pine pine needle needle essential essential oils oils(from (from four fourPinusPinus genera:genera: P. p. mugomugosubsp. subsp. Mugo, P. nigra subsp. NigraNigra, ,P.P. sylvestris, sylvestris, andand P.P. peuce peuce) )had had good good antimicrobial antimicrobial and and insect insect larvicidallarvicidal activities activities [19 [19].]. Earlier, Earlier, the the pine pine needle needle extract extract ofof Cedrus deodara waswas also also proven proven to to be be a good a good naturalnatural antioxidant antioxidant [20 [20].]. However, However, there are are few few reports reports about about PinusPinus koraiensis koraiensis pineconespinecones essential essential oil oil(PEO). (PEO). Therefore, Therefore, it itwas was useful useful for for the the developmen developmentt of natural of natural anti-tumor anti-tumor drugs drugsto explore to explore the anti- the anti-tumortumor activity activity of ofPEO. PEO. HIPPOHIPPO signaling signaling pathway pathway is ais highly a highly conserved conserved cellular cellular signal signal transduction transduction pathway, pathway, which which was firstwas found first in foundDrosophila in Drosophila[21]. HIPPO [21]. pathwayHIPPO pathway controls controls organ size organ and size cell diandfferentiation cell differentiation by regulating by regulating cell proliferation and apoptosis, which is of great significance for the development of cell proliferation and apoptosis, which is of great significance for the development of tumors [22]. tumors [22]. The core of this pathway is a kinase cascade composed of STK3/4-LATS1/2-YAP/TAZ The core of this pathway is a kinase cascade composed of STK3/4-LATS1/2-YAP/TAZ [23]. YAP/TAZ is [23]. YAP/TAZ is the main effector of HIPPO pathway, and dephosphorylated YAP/TAZ promotes the main effector of HIPPO pathway, and dephosphorylated YAP/TAZ promotes the transcription the transcription of genes regulating proliferation and migration after entering the nucleus [21]. It has of genes regulating proliferation and migration after entering the nucleus [21]. It has been reported been reported that YAP is highly expressed in various tumors, including gastric cancer [24]. More that YAP is highly expressed in various tumors, including gastric cancer [24]. More and more studies and more studies have proved that YAP can promote the proliferation and metastasis of tumors, havewhich proved is extremely that YAP unfavorable can promote to the the prognosis proliferation of canc ander patients metastasis [25,26]. of tumors, Therefore, which it is meaningful is extremely unfavorableto study tothe the potential prognosis mechanism of cancer patientsof HIPPO/YAP [25,26]. Therefore,signaling itpathway is meaningful in the to proliferation study the potential and mechanismapoptosis ofof HIPPOcancer /cells.YAP signaling pathway in the proliferation and apoptosis of cancer cells. In thisIn this study, study, we we identified identified the the components components ofof PEOPEO andand analyzedanalyzed its its therapeutic therapeutic potential potential and and mechanismmechanism on on MGC-803 MGC-803 cells cells based based on on RNA RNA sequencingsequencing and bioinformatics analysis, analysis, which which was was necessarynecessary for for the the development development of of anti-tumor anti-tumor drugs. drugs. 2. Results2. Results 2.1.2.1. Chemical Chemical Composition Composition of of Essential Essential Oil Oil GC-MSGC-MS is is the the most most commonly commonly used used methodmethod forfor thethe analysis of of essential essential oils oils components components at at presentpresent [27 [27,28].,28]. The The yellow yellow PEOPEO withwith characteristic odor odor was was obtained obtained by byhydrodistillation hydrodistillation and 41 and 41 components were were identified identified by by GC-MS, GC-MS, accounting accounting 95.73% 95.73% of ofthe the total total oil oil (Table (Table 1),1), and and the the main main componentscomponents were were respectively respectivelyα -Pineneα-Pinene (40.91%), (40.91%), LimoneneLimonene (24.82%), and and ββ-Pinene-Pinene (7.04%). (7.04%). It Itwas was similarsimilar to to the the chemical chemical composition composition ofof oilsoils fromfrom freshfresh needles with with different different pinus pinus species species [19,29]. [19,29 ]. FigureFigure1 shows 1 shows sensitive sensitive signals signals in in the the total total ion ion currency currency (TIC) (TIC) profile.profile. The sequence sequence numbers numbers of of the the peakspeaks in Figurein Figure1 were 1 were consistent consistent with with those those of of the the compounds compounds in in TableTable1 .1. Molecules 2019, 24, x; doi: FOR Figure 1. The total ion currency (TIC) profile of PEO.www.mdpi.com/journal/molecules Molecules 2019, 24, 3851 3 of 18 Table 1. GC-MS analysis results of PEO components. No. Compounds RI a RI b % 1 Santene 882 884 0.06 2 Tricyclene 918 919 0.11 3 Bicyclo [3.1.0]hexane, 4-methyl-1-(1-methylethyl)-didehydro deriv. 926 — 0.11 4 α-Pinene 934 934 40.91 5 Camphene 944 945 1.08 6 Cosmene 968 — 0.07 7 β-Pinene 973 972 7.04 8 β-Myrcene 992 992 2.04 9 3-Carene 1008 1007 4.12 10 Limonene 1030 1031 24.82 11 Bicyclo [3.1.0]hexane, 6-isopropylidene-1-methyl- 1088 — 0.24 12 L-trans-Pinocarveol 1138 1140 0.58 13 L-camphor 1142 1142 0.35
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