Carbonyl cyanide 3-chlorophenylhydrazone promotes of mitophagy in gastric cancer cells MKN1 and MKN45 via PINK1/parkin pathway

Carbonyl cyanide 3-chlorophenylhydrazone promotes of mitophagy in gastric cancer cells MKN1 and MKN45 via PINK1/parkin pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Carbonyl cyanide 3-chlorophenylhydrazone promotes of mitophagy in gastric cancer cells MKN1 and MKN45 via PINK1/parkin pathway Jun Yao, Sun-yuan LV, Jia-jie Xia, Chen-nan Xu, Xiao Ying, Jing-jing Guo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4099767/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract BACKGROUND Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), as a common mitochondrial uncoupling agent, inducer of mitochondrial autophagy, has been used in nervous system and digestive system diseases. Mitochondrial autophagy is rarely reported in gastric cancer (GC). In this study, we used CCCP to treat MKN1 and MKN45 gastric cancer cells. METHODS We studied its effects on cell viability, migration and invasion, mitochondrial membrane potential level, apoptosis, cell cycle and mitophagy of GC were determined respectively by CCK8, Wound-Healing, Transwell, JC-1 dye, Western blot analysis and Flow cytometry assays. CCCP drugs and saline were injected intravenously, respectively, to clarify the effects of the drugs on HGC27 xenograft tumours in nude mice. RESULTS CCCP significantly inhibited the activity of MKN1 and MKN45 cells in a dose-dependent manner, and inhibited cell proliferation, migration and invasion. CCCP induced MKN1 and MKN45 cell apoptosis and arrested cell cycle in G1 phase. Furthermore, CCCP inhibited the expression of CyclinD1, CDK4 and CDK6, thus inhibited the cell cycle, and promoted the decline of mitochondrial membrane potential, which induced cells to enter PINK1 / parkin pathway mitophagy. Intravenous CCCP drugs significantly inhibited nude mouse HGC27 xenograft tumour Growth. CONCLUSIONS In general, CCCP, can regulate gastric cancer cells by inducing mitophagy, and may be a potential therapy for gastric cancer. Mitophagy Mitochondrial Membrane Potential Gastric Cancer mitophagy Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Gastric cancer is one of the most common malignant tumors in the world, which is a serious threat to human health. There are about 1 million new cases every year, and in 2018 alone, 784,000 people died of gastric cancer worldwide [ 1 ]. With the development of metagenomics sequencing, targeted therapy and immunotherapy, the prognosis of gastric cancer has been significantly improved. However, there is still a lack of effective treatment for advanced gastric cancer, especially for implanted metastatic gastric cancer. Therefore, it is of great significance to find new drugs for the treatment of gastric cancer. Autophagy is a catabolic pathway that can lead to the degradation of proteins and organelles [ 2 ]. Mitochondria, as organelles of cell power source, play an important role in cell metabolism, energy production, REDOX homeostasis and cell apoptosis [ 3 ]. Excessive mitochondrial autophagy results in loss of normal mitochondrial function, loss of energy sources and eventual death of the cell. However, mitochondrial autophagy can also promote cell survival by eliminating damaged mitochondria, thus better adapting to aggressive conditions. Mitochondrial autophagy pathway usually involves PINK1/Parkin, BNIP3/NIX/FUNDC1 and p62/SQSTM1 signaling pathways [ 3 ]. It has been reported that PINK1/PARK2 gene is associated with juvenile Parkinson's disease and acts as a tumor suppressor in cancer [ 4 ]. Carbonyl cyanide 3-chlorobenzene (CCCP) is a typical oxidative phosphoric acid uncoupling agent, which is usually used as a mitochondrial uncoupling agent and photosynthetic inhibitor [5]. The mechanism of CCCP is to uncouple the proton concentration gradient formed by the electron carrier under the normal activity of the electron transport chain, which mainly affects the protein synthesis in mitochondria. CCCP acts as an inducer of mitochondrial autophagy in the treatment of Alzheimer's disease, avian influenza virus, alcoholic liver disease and other diseases [4,6,7]. At present, it has been confirmed that the combination of CCCP and TRAIL can induce apoptosis of gastric cancer cells, but the mechanism of action remains unclear. In this study, we hypothesized that CCCP might lead to mitochondrial function loss by inducing autophagy in human gastric cancer cells, and explored its mechanism. 2. Materials and methods 2.1 Cell culture Human gastric cancer cells MKN1 and MKN45 were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences. Cells were cultured using 1640 medium containing 10%FBS (Excell), (hyclone). Culture bottles were incubated at 37℃ in 5%CO 2 incubator. 2.2 Drugs and antibodies CCCP purchased from Sigma-Aldrich. Anti-LC3II (abcam), anti-PINK1 (immunoway), Caspase-3, cyclinD1, CDK4, CDK6 were purchased from abcam. 2.3 Cell Viability Assay 100 uL medium containing 5000 cells was added to each well of 96-well plate (NEST, Shanghai, China). After one day of culture, the number of cells reached 80–90%. CCCP was added at the concentrations of 2.5, 5, 10, 20, 30, 40, and 50µg/ml, respectively. CCK8 (10µM) reagent (GLPBIO, Shanghai, China) was added 24h and 48h after addition, respectively, and cells were cultured for 2 h. The fractional luminosity was measured at 480 mm using an enzyme marker. The change of cell activity value under concentration gradient was measured, and IC50 of gastric cancer cells under CCCP was calculated to be 10µg/ml. 2.4 Wound Healing Assay Cells (1*10 5 cells/mL) were inoculated into 6-well plates (NEST, Shanghai, China). When the cells were overgrown, CCCP was added into 6-well plates at concentrations of 0, 2.5, 5, and 10 µg/ml, respectively. After the cells filled the whole hole, a certain distance was marked and photographed, and 0 h and 48 h were recorded to calculate the change of the distance. 2.5 Transwell Assay The cells were treated with 10 µg/ml of CCCP for 24 h and digested with pancreatic enzymes. The spare cells (3*10 4 ) were added to the upper chamber of 70 uL serum-free 1640 medium transwell. 600uL 1640 medium containing 20%FBS was added to transwell subchamber. After incubation for 24 h, the transwell chamber was fixed with 4% paraformaldehyde and stained with 1% crystal violet. Cells migrating to the lower surface of the superior compartment membrane were photographed under a microscope (NIKON, Japan). The average number of cells in the three random Wells was calculated. 2.6 EdU cell proliferation Assay Inoculate cells into each well of the 6-well plate. After being adherent cells, respectively, in accordance with the concentration of 0, 2.5, 5, and 10 µg/ml CCCP drugs in 6 orifice plate, and then according to the EdU kit (from Beyotime, Shanghai, China) cells, Images were obtained using a fluorescence microscope (NIKON, Japan). 2.7 Mitochondrial membrane potential was detected by JC-1 staining Cells were collected and stained with JC-1 (Applygen, Beijing, China) according to the given regimen, then detected by flow cytometry (ACEA NovoCyte 3130, USA). The data were processed by FlowJo (10.8.1) and the red/green fluorescence ratio was calculated. 2.8 Western blot analysis Protein was extracted from CCCP drugs with concentrations of 0, 2.5, 5, 10 µg/ml, respectively. The protein was isolated using 10% SDS-PAGE and transferred to a PVDF (millipore, USA) membrane. After being closed in the sealing solution (Beyotime Shanghai, China) for 30min, the film was added to the primary antibody and incubated at 4℃. After the second day, the film was taken out and added to the secondary antibody and incubated at room temperature for 2 h. ECL enhanced fluorescence reagent (Biosharp, Shanghai, China) was exposed under Tanon-5200 machine (Beijing, China) and photographed . 2.9 Flow cytometry Cells (1*10 5 cells/mL) were inoculated into 6-well plates. When the cells were overgrown, CCCP was added into 6-well plates at different concentrations and cultured for 24 h. Apoptosis was analyzed, cells digested with trypsin without EDTA were washed twice with PBS and re-suspended (including the broken cells in the original medium), followed by Annexin V-FITC/PI staining (MultiSciences, Zhejiang, China) with staining agent, and incubated in the dark for 15 min before flow cytometer analysis. The cell cycle was analyzed, and the cells collected were analyzed by flow cytometry according to the cycle kit (Biosharp, Shanghai, China). 2.10 In vivo antitumor growth effect of drugs In vivo anticancer activity was evaluated in mice bearing subcutaneous HGC27 tumor. The treatment schedule started when the tumor volume reached approximate 50 mm 3 . The mice were randomly divided into two groups (n = 5) and treated with CCCP (10µg/kg). via tail vein injection on the day 0 and 6. And the mice treated with 0.9% NaCl solution was used as control. Tumor volume was measured via serial caliper measurement every other 3 days and estimated using the formula: Volume = 0.5 × length × (width) 2 . On the day 12, the animals were sacrificed by cervical dislocation, and the tumor mass was harvested, weighted and photographed. 2.11 Statistical Analysis Three independent experiments were conducted for all studies and all assay conditions were established in duplicate or triplicate. Student's t-test was used to analyze the data, and the difference was statistically significant when p < 0.05. 3. Results 3.1 CCCP inhibited gastric cancer cell activity Firstly, we used CCK8 method to measure the effect of CCCP on the viability of MKN1 and MKN45 gastric cancer cells under different concentration gradients. The results showed that CCCP inhibited MKN1 and MKN45 gastric cancer cells in a dose-dependent manner. Among them, MKN45 cells were more resistant than MKN1 cells (Figure 1A). For further experiments, the GraphPad Prism 8 software was used to calculate the 50% inhibition concentration (IC50) of CCCP on MKN1 cells and MKN45 cells as 10 μg/ml and 8 μg/ml, respectively (Figure1B). Next, we analyzed the effect of CCCP on the proliferation of gastric cancer cells by EDU assay. The results showed that higher concentrations of CCCP drugs showed stronger inhibitory effects than those at lower concentrations (Figure1C, D). figure1. Effects of CCCP compounds on cell viability and clonal ability of MKN1 and MKN45 cells. A. Cell viability of MKN1 and MKN45 cells treated with different concentrations of CCCP compounds for 24 hours. B. According to the results in Figure 1, the IC50 was determined by software (granphad prism8), and the IC50 of the CCCP compound for MKN1 cells was determined to be 10 μg/ml, and the MKN45 was 8 μg/ml. C. D. Images of the effect of different concentrations of CCCP compounds on the colony-forming ability of MKN1 and MKN45 cells. Data are presented as mean ± SD of three independent experiments (*p<0.05 **p<0.01, compared to control group) 3.2 CCCP inhibited the migration and invasion of gastric cancer cells According to the previous experimental results, we determined the action concentration of CCCP. Then, we found through scratch test that the migration ability of MKN1 and MKN45 cells gradually decreased with the increase of concentration (Figure 2A). In addition, transwell experiment found that under different concentrations of CCCP, the invasion level was positively correlated with the level of CCCP (Figure 2B-C). figure2. CCCP compounds for MKN1 and MKN45 cell scratch experiments and in migration and invasion abilities. Images obtained using microscope magnification (40x) at 0 and 48 hours when CCCP compounds acted on M1 and MKN45 cells at different concentrations, The image obtained by A was obtained using Adobe Photoshop 2020 software to calculate the wound distance at 48 hours, and the ratio of its value to the 0 hour distance was obtained. B. C. M1 and MKN45 cells were treated with different concentrations of CCCP compounds, and images of invasion and migration cells were observed at 0 and 24 hours using microscope magnification (100x). B. After calculating the number of cells using imageJ software, the ratio was obtained compared with the control group. Data are presented as mean ± SD of three independent experiments (*p<0.05 **p<0.01, compared to control group) 3.3 CCCP promotes apoptosis of gastric cancer cells It has been reported in previous studies that human gastric cancer cells SNU-638 incubated with CCCP in combination with TRAIL can significantly enhance apoptosis compared with TRAIL alone. And the CCCP itself is the uncoupling agent. Therefore, we verified whether CCCP can also induce apoptosis of MKN1 and MKN45 gastric cancer cells. Flow cytometry showed that CCCP could induce apoptosis of MKN1 and MKN45 cells (Figure 3A-B). We then used western blotting to assess Caspase-3 protein expression levels. The results showed that the expression level of Caspase-3 protein was significantly increased after exposure (Figure3 C-D), and CCCP could promote the apoptosis of gastric cancer cells. figure3.CCCP compounds induce apoptosis in MKN1 and MKN45 cells. A, B. MKN1 and MKN45 cells were treated with different concentrations of CCCP drugs, stained with V-FITC/PI, and determined by flow cytometry, and their apoptosis rates were calculated. C, D. The expression levels of caspase-3 were detected by western blotting. The gray value was calculated using imageJ through the image of panel C, and the ratio of the expression level of β-Actin protein. Data are presented as mean ± SD of three independent experiments (*p<0.05 **p<0.01, compared to control group) 3.4 CCCP blocked the G0/G1 phase of gastric cancer cells We previously confirmed that CCCP can promote the apoptosis of gastric cancer cells. cyclin-D1, CDK4 and CDK6 are cell cyclin-related proteins. Western blot analysis showed that the expression levels of cyclin-D1, CDK4 and CDK6 proteins were significantly reduced (Figure 4C-D). In addition, the cell cycle distribution was analyzed by flow cytometry. The results showed that the number of G0/G1 stage gastric cancer cells significantly increased with the increase of CCCP concentration (Figure 4A-B). figure4. CCCP compounds inhibit the cell cycle of MKN1 and MKN45 cells. A. MKN1 and MKN45 cells were treated with different concentrations of CCCP drugs, and the effect on cell cycle was evaluated by flow cytometry after PI staining; B. The percentage of cells in each stage of the cell cycle. C, D. MKN1 and MKN45 cells were treated with different concentrations of CCCP drugs, and the expression levels of cyclinD1, CDK4 and CDK6 were detected by western blotting; The gray value was calculated using imageJ through the image of panel C, and the ratio of the expression level of β-Actin protein. Data are presented as mean ± SD of three independent experiments (*p<0.05 **p<0.01, compared to control group) 3.5 CCCP induced mitochondrial dysfunction and promoted mitochondrial autophagy in gastric cancer cells. As a mitochondrial uncoupling agent, CCCP can induce mitochondrial membrane depolarization and lead to autophagy. We detected mitochondrial membrane potential in MKN1 and MKN45 cells after CCCP treatment. The results showed that MKN1 and MKN45 cells treated with CCCP showed significantly decreased membrane potential compared with the control group (Figure 5A-B). As a marker protein of mitochondrial autophagy, PINK1 specifically targets damaged mitochondria to promote the progress of mitochondrial autophagy [8]. Western blot results showed that the protein levels of LC3-II and PINK1 were significantly increased (Figure 5C-D). This suggests that CCCP can induce mitochondrial autophagy by activating PINK1/Parkin signaling pathway. figure5. Effects of CCCP compounds on mitochondrial membrane potential of MKN1 and MKN45 cells. A. B. MKN1 and MKN45 cells were treated with CCCP at a concentration of 0 and 10 ug/ml, and after 24 hours of JC-1 staining, flow cytometry was performed and the red/green ratio was calculated. C. D. The expression levels of PINK1 and LC3IIB were detected by western blotting. The gray value was calculated using imageJ through the image of panel C, and the ratio of the expression level of β-Actin protein. Data are presented as mean ± SD of three independent experiments (*p<0.05 **p<0.01, compared to control group) 3.6 In vivo therapeutic efficacy We found that CCCP drug significantly inhibited the growth of tumours in mice by using a certain concentration of CCCP drug compared with saline-treated control group (Figure 6). After 6 days of injecting the drug, the average tumour volume of mice was 274.02 mm3, which was smaller than the control group 497.49 mm3, a reduction of about 44.86%, and after 12 days of CCCP administration for the pair of mice the tumour volume was reduced by about 65.69%. In terms of the weight of the tumour, after 12 injections of the drug, the average weight of the tumour in the CCCP-administered group was about 786 mg, compared with 2979 mg in the control group, a reduction in weight of about 73.61%. In conclusion, combined with the previous effects of CCCP drug to inhibit the growth of gastric cancer cells and promote apoptosis, it is known that CCCP drug in mice can inhibit the enlargement of tumour volume in mice, and has certain therapeutic effects. Figure6.Anti-tumor growth effects of CCCP drug and salineon nude mice bearing subcutaneous HGC27 tumours (CCCP dose of 10 μg/kg). The injection was repeated every 6 days for 1 time. A. Tumour morphology and size at the endpoint of the experiment (day 12). B. Measurement of tumour volume. C. Tumour weight at the experimental endpoint (day 12). Data are given as mean ± SD (n = 5). 4. Discussion Gastric cancer has gradually become one of the cancers with the highest mortality rate in the world, with about 784,000 people dying from it every year [ 1 ]. At present, conventional chemotherapy is mainly used in the treatment of gastric cancer, but the median survival time is less than 1 year [9]. No matter the subsequent immune checkpoint blocking for patients with advanced gastric cancer [10.11] or the use of fibroblast growth receptor-2 pathway inhibitors [12,13,14], the treatment for patients with gastric cancer is still unsatisfactory. We found that its application in mitochondrial autophagy is not only related to cardiovascular and liver diseases and neurodegenerative diseases, but also to tumorgenesis and tumor progression of multiple tumors [14,15,16], which has great potential in the treatment of cancer. We set out to explore the possible pathways regulating mitochondrial autophagy in gastric cancer cells. To seek new treatment options for gastric cancer. Firstly, CCCP can dissolve in the inner mitochondrial membrane (MIM) and selectively increase the permeability of the mitochondrial matrix, which then leads to the release of protons in the matrix, and finally leads to the change of MIM potential (DWm) and transmembrane PH (DpH) of the inner mitochondrial membrane [17]. We measured the membrane potential after CCCP treatment by flow cytometry, and found that the treated mitochondria significantly decreased the membrane potential level compared with the control group, indicating that CCCP can also reduce the mitochondrial membrane potential of gastric cancer cells. As an important organelle for energy metabolism and homeostasis of cells [18]. When mitochondrial autophagy is shown to affect the phenotype of gastric cancer cells, we confirmed that the decline in cell viability, proliferation and invasion ability of gastric cancer cells induced by CCCP at different concentration gradients is positively correlated with CCCP concentration. In order to address the change in the inhibitory ability of CCCP on the proliferation of gastric cancer cells, we found that CCCP blocked the G1 to S phase of gastric cancer cells by flow cytometry. In order to explore the expression level of cyclin-D1, CDK4 and CDK6, as key proteins in the transition from G1 phase to S phase of cell division cycle [19], western blotting experiments were used to find that the expression level of cyclin gradually decreased with the increase of concentration. Mitochondrial autophagy has been reported to play a dual role in cancer cells depending on the cell type. On the one hand, pro-apoptotic mitochondrial clearance mediated by mitochondrial autophagy may have cell protective effects [20]. On the other hand, excessive mitochondrial autophagy may induce cancer cell death [21]. Cleaved caspase-3 is one of the markers of apoptosis, and in this study, we found that CCCP promoted caspase-3 protein expression levels. In mitochondrial autophagy, PINK1-mediated PRKN-dependent mitochondrial autophagy is the main pathway [22,23]. It has been confirmed that PINK1 deletion in mice can lead to the spontaneous development of hepatocellular carcinoma [24], while Parkin, as the downstream of PINK1, is down-regulated or absent in human glioblastoma [25], melanoma [26], breast cancer [27], etc. In addition, it has been reported that mitochondrial autophagy controlled by PINK1/Parkin pathway affects many mitochondrial physiological processes [28,29]. In this study, western blotting showed that compared with the control group, the treated cells showed higher expression of PINK1, and directly interacted with LC3 and its receptor in the pathway of PINK1-mediated PRKN-dependent mitochondrial autophagy [30]. In conclusion, this study provides more directions and means for the treatment of gastric cancer. 5. Conclusion Our aim was to investigate whether CCCP can induce mitochondrial autophagy in gastric cancer and to identify one of the pathways of mitochondrial autophagy in gastric cancer cells. We found that CCCP not only inhibited cell viability, proliferation, migration and invasion of gastric cancer cells, but also induced apoptosis and blocked cell cycle, it is dose dependent. However, from the known regulation of mitochondrial autophagy in other cancers, mitochondrial autophagy has dual effects on different cells, and the existence of differences is still unclear. At present, CCCP can not only induce mitochondrial autophagy, but also promote cell apoptosis. So, the potential of CCCP in the treatment of gastric cancer and even other cancers remains to be explored. Declarations Availability of data and materials The datasets used and/or analysed during the present are available from the corresponding author on reasonable request. Authors ’ contributions Jun Yao conducted the experiments, analyzed data, and drafted the manuscript; Jia-jie Xia and Sun-yuan Lv has contributed to the experiment and manuscript preparation; Chen-nan Xu contributed to the revision of the manuscript; Jing-jing Guo and Xiao Ying participated in the design of the research and experiment; All authors read and agree to the final manuscript. Ethics approval and consent to participate Not applicable. Manuscript Statement: This study follows the guidelines set by our institution and national regulations for the ethical care and use of laboratory animals, as stated in the manuscript. All animal experiments were conducted with ethical approval and aimed to minimize discomfort and ensure animal welfare. We are committed to upholding these standards and continually improving our practices for the welfare of animals involved in our research. References Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R. L.; Torre, L. 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T.; Bressac-de-Paillerets, B.; Tsalamlal, A.; Kumar, R.; Klebe, S.; Grandchamp, B.; Andrieu-Abadie, N.; Thomas, L.; Brice, A.; Dumaz, N.; Soufir, N., PARKIN Inactivation Links Parkinson's Disease to Melanoma. J Natl Cancer Inst 2016, 108 (3). Letessier, A.; Garrido-Urbani, S.; Ginestier, C.; Fournier, G.; Esterni, B.; Monville, F.; Adelaide, J.; Geneix, J.; Xerri, L.; Dubreuil, P.; Viens, P.; Charafe-Jauffret, E.; Jacquemier, J.; Birnbaum, D.; Lopez, M.; Chaffanet, M., Correlated break at PARK2/FRA6E and loss of AF-6/Afadin protein expression are associated with poor outcome in breast cancer. Oncogene 2007, 26 (2), 298-307. Pickles, S.; Vigie, P.; Youle, R. J., Mitophagy and Quality Control Mechanisms in Mitochondrial Maintenance. Curr Biol 2018, 28 (4), R170-R185. Harper, J. W.; Ordureau, A.; Heo, J. M., Building and decoding ubiquitin chains for mitophagy. Nat Rev Mol Cell Biol 2018, 19 (2), 93-108. Yoo, S. M.; Jung, Y. K., A Molecular Approach to Mitophagy and Mitochondrial Dynamics. Mol Cells 2018, 41 (1), 18-26. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted 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 Our Team In Review Editorial Policies Advisory Board 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-4099767","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":279802088,"identity":"8118bad9-0c04-4616-b292-b8efe9ac5d54","order_by":0,"name":"Jun Yao","email":"","orcid":"","institution":"The Central Hospital Affiliated to Shaoxing University, Shaoxing Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Yao","suffix":""},{"id":279802089,"identity":"4cbbcd45-8772-4036-80aa-2971b529967e","order_by":1,"name":"Sun-yuan LV","email":"","orcid":"","institution":"The Chengdu Fifth People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sun-yuan","middleName":"","lastName":"LV","suffix":""},{"id":279802090,"identity":"7b20fc87-95da-4c06-b82a-4e457fabb5c7","order_by":2,"name":"Jia-jie Xia","email":"","orcid":"","institution":"The Central Hospital Affiliated to Shaoxing University, Shaoxing Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jia-jie","middleName":"","lastName":"Xia","suffix":""},{"id":279802091,"identity":"08b30b65-529c-468b-988a-45155bdc8716","order_by":3,"name":"Chen-nan Xu","email":"","orcid":"","institution":"The Central Hospital Affiliated to Shaoxing University, Shaoxing Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chen-nan","middleName":"","lastName":"Xu","suffix":""},{"id":279802092,"identity":"ba37c859-5d81-409c-800e-021db1f5b10e","order_by":4,"name":"Xiao Ying","email":"","orcid":"","institution":"The First People's Hospital Yongkang","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Ying","suffix":""},{"id":279802093,"identity":"7ead9fed-657d-452c-b2ae-9d67dc6903b9","order_by":5,"name":"Jing-jing Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYJACgwQGBmY2KEeOjb39AGlajPl4ziSQZmXiPAkHA7wq5COSDxQ83FHLzifdfoGZt+1wepsEQwLDj4ptOLUY3khLMEg8c5yZTeZMAUhLbpt04wHGnjO3cWuZkWNgkNh2jJlNIif9d+6227ltMgcSmBnbiNOSwAzUks4mkWCAV4u8BFhLDVBL+gGQlgSCWgx4ngH90nYAZAsD899//w3bgIF8EJ9f5NuTjxn+bKtLlp+R/oBxxpk0efn29oMPflTgseUAAxswGg4nMzDwIKLjAE71IFsaGJgfMDDU2TEwsD/Ap3AUjIJRMApGMAAA2lhWg6PActcAAAAASUVORK5CYII=","orcid":"","institution":"The Central Hospital Affiliated to Shaoxing University, Shaoxing Central Hospital","correspondingAuthor":true,"prefix":"","firstName":"Jing-jing","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2024-03-14 10:26:54","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4099767/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4099767/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52926567,"identity":"23e0d7f7-cc61-441c-9bd7-a7572cb335f9","added_by":"auto","created_at":"2024-03-18 18:36:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1092984,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of CCCP compounds on cell viability and clonal ability of MKN1 and MKN45 cells.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Cell viability of MKN1 and MKN45 cells treated with different concentrations of CCCP compounds for 24 hours. \u003cstrong\u003e(B)\u003c/strong\u003e According to the results in Figure 1, the IC50 was determined by software (granphad prism8), and the IC50 of the CCCP compound for MKN1 cells was determined to be 10 μg/ml, and the MKN45 was 8 μg/ml. \u003cstrong\u003e(C,D)\u003c/strong\u003e Images of the effect of different concentrations of CCCP compounds on the colony-forming ability of MKN1 and MKN45 cells. Data are presented as mean ± SD of three independent experiments (*p\u0026lt;0.05 **p\u0026lt;0.01, compared to control group)\u003c/p\u003e","description":"","filename":"Figure.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4099767/v1/40263a19771f7babcc3b40db.png"},{"id":52926568,"identity":"09282ffb-3e55-4cf2-8f68-a9941c1304ee","added_by":"auto","created_at":"2024-03-18 18:36:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3035426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCCP compounds for MKN1 and MKN45 cell scratch experiments and in migration and invasion abilities.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Images obtained using microscope magnification (40x) at 0 and 48 hours when CCCP compounds acted on M1 and MKN45 cells at different concentrations, The image obtained by A was obtained using Adobe Photoshop 2020 software to calculate the wound distance at 48 hours, and the ratio of its value to the 0 hour distance was obtained. \u003cstrong\u003e(B,C)\u003c/strong\u003e M1 and MKN45 cells were treated with different concentrations of CCCP compounds, and images of invasion and migration cells were observed at 0 and 24 hours using microscope magnification (100x). B. After calculating the number of cells using imageJ software, the ratio was obtained compared with the control group. Data are presented as mean ± SD of three independent experiments (*p\u0026lt;0.05 **p\u0026lt;0.01, compared to control group)\u003c/p\u003e","description":"","filename":"Figure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4099767/v1/4bc483353531029a757634a1.png"},{"id":52926569,"identity":"e2909622-a3e6-49b9-b67f-27bb307a598a","added_by":"auto","created_at":"2024-03-18 18:36:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":572479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCCP compounds induce apoptosis in MKN1 and MKN45 cells. (A,B)\u003c/strong\u003eMKN1 and MKN45 cells were treated with different concentrations of CCCP drugs, stained with V-FITC/PI, and determined by flow cytometry, and their apoptosis rates were calculated. \u003cstrong\u003e(C,D)\u003c/strong\u003e The expression levels of caspase-3 were detected by western blotting. The gray value was calculated using imageJ through the image of panel C, and the ratio of the expression level of β-Actin protein. Data are presented as mean ± SD of three independent experiments (*p\u0026lt;0.05 **p\u0026lt;0.01, compared to control group)\u003c/p\u003e","description":"","filename":"Figure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4099767/v1/29920f0293005e44c6a00c9f.png"},{"id":52927082,"identity":"12edd2c4-ec30-4444-bda9-b039f866a607","added_by":"auto","created_at":"2024-03-18 18:44:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":863025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCCP compounds inhibit the cell cycle of MKN1 and MKN45 cells. (A)\u003c/strong\u003e MKN1 and MKN45 cells were treated with different concentrations of CCCP drugs, and the effect on cell cycle was evaluated by flow cytometry after PI staining; \u003cstrong\u003e(B)\u003c/strong\u003e The percentage of cells in each stage of the cell cycle. \u003cstrong\u003e(C,D)\u003c/strong\u003e MKN1 and MKN45 cells were treated with different concentrations of CCCP drugs, and the expression levels of cyclinD1, CDK4 and CDK6 were detected by western blotting; The gray value was calculated using imageJ through the image of panel C, and the ratio of the expression level of β-Actin protein. Data are presented as mean ± SD of three independent experiments (*p\u0026lt;0.05 **p\u0026lt;0.01, compared to control group)\u003c/p\u003e","description":"","filename":"Figure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4099767/v1/0bc8d03ff3ab2e638e82e651.png"},{"id":52926571,"identity":"efd3e681-3c58-4939-8b43-a44f39625c73","added_by":"auto","created_at":"2024-03-18 18:36:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":496920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of CCCP compounds on mitochondrial membrane potential of MKN1 and MKN45 cells. (A,B)\u003c/strong\u003e MKN1 and MKN45 cells were treated with CCCP at a concentration of 0 and 10 ug/ml, and after 24 hours of JC-1 staining, flow cytometry was performed and the red/green ratio was calculated. \u003cstrong\u003e(C,D)\u003c/strong\u003e The expression levels of PINK1 and LC3IIB were detected by western blotting. The gray value was calculated using imageJ through the image of panel C, and the ratio of the expression level of β-Actin protein. Data are presented as mean ± SD of three independent experiments (*p\u0026lt;0.05 **p\u0026lt;0.01, compared to control group)\u003c/p\u003e","description":"","filename":"Figure.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4099767/v1/6ade8685804cbee5868acb0a.png"},{"id":52926572,"identity":"ec7ef723-5021-4202-8083-dccf862d191a","added_by":"auto","created_at":"2024-03-18 18:36:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":321211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-tumor growth effects of CCCP drug and salineon nude mice bearing subcutaneous HGC27 tumours (CCCP dose of 10 μg/kg). The injection was repeated every 6 days for 1 time.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Tumour morphology and size at the endpoint of the experiment (day 12). \u003cstrong\u003e(B)\u003c/strong\u003eMeasurement of tumour volume. \u003cstrong\u003e(C)\u003c/strong\u003e Tumour weight at the experimental endpoint (day 12). Data are given as mean ± SD (n = 5).\u003c/p\u003e","description":"","filename":"Figure.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4099767/v1/5dec93bd16a9122d1e20c9d5.png"},{"id":52984357,"identity":"26221f47-6b4f-44dd-83ec-4e162ff4f390","added_by":"auto","created_at":"2024-03-19 10:54:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3519373,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4099767/v1/1ba68487-2ce7-4681-acc1-2806917f6c9c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Carbonyl cyanide 3-chlorophenylhydrazone promotes of mitophagy in gastric cancer cells MKN1 and MKN45 via PINK1/parkin pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGastric cancer is one of the most common malignant tumors in the world, which is a serious threat to human health. There are about 1\u0026nbsp;million new cases every year, and in 2018 alone, 784,000 people died of gastric cancer worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. With the development of metagenomics sequencing, targeted therapy and immunotherapy, the prognosis of gastric cancer has been significantly improved. However, there is still a lack of effective treatment for advanced gastric cancer, especially for implanted metastatic gastric cancer. Therefore, it is of great significance to find new drugs for the treatment of gastric cancer.\u003c/p\u003e \u003cp\u003eAutophagy is a catabolic pathway that can lead to the degradation of proteins and organelles [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Mitochondria, as organelles of cell power source, play an important role in cell metabolism, energy production, REDOX homeostasis and cell apoptosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Excessive mitochondrial autophagy results in loss of normal mitochondrial function, loss of energy sources and eventual death of the cell. However, mitochondrial autophagy can also promote cell survival by eliminating damaged mitochondria, thus better adapting to aggressive conditions. Mitochondrial autophagy pathway usually involves PINK1/Parkin, BNIP3/NIX/FUNDC1 and p62/SQSTM1 signaling pathways [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It has been reported that PINK1/PARK2 gene is associated with juvenile Parkinson's disease and acts as a tumor suppressor in cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCarbonyl cyanide 3-chlorobenzene (CCCP) is a typical oxidative phosphoric acid uncoupling agent, which is usually used as a mitochondrial uncoupling agent and photosynthetic inhibitor [5]. The mechanism of CCCP is to uncouple the proton concentration gradient formed by the electron carrier under the normal activity of the electron transport chain, which mainly affects the protein synthesis in mitochondria. CCCP acts as an inducer of mitochondrial autophagy in the treatment of Alzheimer's disease, avian influenza virus, alcoholic liver disease and other diseases [4,6,7]. At present, it has been confirmed that the combination of CCCP and TRAIL can induce apoptosis of gastric cancer cells, but the mechanism of action remains unclear. In this study, we hypothesized that CCCP might lead to mitochondrial function loss by inducing autophagy in human gastric cancer cells, and explored its mechanism.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell culture\u003c/h2\u003e \u003cp\u003eHuman gastric cancer cells MKN1 and MKN45 were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences. Cells were cultured using 1640 medium containing 10%FBS (Excell), (hyclone). Culture bottles were incubated at 37℃ in 5%CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Drugs and antibodies\u003c/h2\u003e \u003cp\u003eCCCP purchased from Sigma-Aldrich. Anti-LC3II (abcam), anti-PINK1 (immunoway), Caspase-3, cyclinD1, CDK4, CDK6 were purchased from abcam.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell Viability Assay\u003c/h2\u003e \u003cp\u003e100 uL medium containing 5000 cells was added to each well of 96-well plate (NEST, Shanghai, China). After one day of culture, the number of cells reached 80\u0026ndash;90%. CCCP was added at the concentrations of 2.5, 5, 10, 20, 30, 40, and 50\u0026micro;g/ml, respectively. CCK8 (10\u0026micro;M) reagent (GLPBIO, Shanghai, China) was added 24h and 48h after addition, respectively, and cells were cultured for 2 h. The fractional luminosity was measured at 480 mm using an enzyme marker. The change of cell activity value under concentration gradient was measured, and IC50 of gastric cancer cells under CCCP was calculated to be 10\u0026micro;g/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Wound Healing Assay\u003c/h2\u003e \u003cp\u003eCells (1*10\u003csup\u003e5\u003c/sup\u003e cells/mL) were inoculated into 6-well plates (NEST, Shanghai, China). When the cells were overgrown, CCCP was added into 6-well plates at concentrations of 0, 2.5, 5, and 10 \u0026micro;g/ml, respectively. After the cells filled the whole hole, a certain distance was marked and photographed, and 0 h and 48 h were recorded to calculate the change of the distance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Transwell Assay\u003c/h2\u003e \u003cp\u003eThe cells were treated with 10 \u0026micro;g/ml of CCCP for 24 h and digested with pancreatic enzymes. The spare cells (3*10\u003csup\u003e4\u003c/sup\u003e) were added to the upper chamber of 70 uL serum-free 1640 medium transwell. 600uL 1640 medium containing 20%FBS was added to transwell subchamber. After incubation for 24 h, the transwell chamber was fixed with 4% paraformaldehyde and stained with 1% crystal violet. Cells migrating to the lower surface of the superior compartment membrane were photographed under a microscope (NIKON, Japan). The average number of cells in the three random Wells was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 EdU cell proliferation Assay\u003c/h2\u003e \u003cp\u003eInoculate cells into each well of the 6-well plate. After being adherent cells, respectively, in accordance with the concentration of 0, 2.5, 5, and 10 \u0026micro;g/ml CCCP drugs in 6 orifice plate, and then according to the EdU kit (from Beyotime, Shanghai, China) cells, Images were obtained using a fluorescence microscope (NIKON, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Mitochondrial membrane potential was detected by JC-1 staining\u003c/h2\u003e \u003cp\u003e Cells were collected and stained with JC-1 (Applygen, Beijing, China) according to the given regimen, then detected by flow cytometry (ACEA NovoCyte 3130, USA). The data were processed by FlowJo (10.8.1) and the red/green fluorescence ratio was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Western blot analysis\u003c/h2\u003e \u003cp\u003eProtein was extracted from CCCP drugs with concentrations of 0, 2.5, 5, 10 \u0026micro;g/ml, respectively. The protein was isolated using 10% SDS-PAGE and transferred to a PVDF (millipore, USA) membrane. After being closed in the sealing solution (Beyotime Shanghai, China) for 30min, the film was added to the primary antibody and incubated at 4℃. After the second day, the film was taken out and added to the secondary antibody and incubated at room temperature for 2 h. ECL enhanced fluorescence reagent (Biosharp, Shanghai, China) was exposed under Tanon-5200 machine (Beijing, China) and photographed .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Flow cytometry\u003c/h2\u003e \u003cp\u003eCells (1*10\u003csup\u003e5\u003c/sup\u003e cells/mL) were inoculated into 6-well plates. When the cells were overgrown, CCCP was added into 6-well plates at different concentrations and cultured for 24 h. Apoptosis was analyzed, cells digested with trypsin without EDTA were washed twice with PBS and re-suspended (including the broken cells in the original medium), followed by Annexin V-FITC/PI staining (MultiSciences, Zhejiang, China) with staining agent, and incubated in the dark for 15 min before flow cytometer analysis. The cell cycle was analyzed, and the cells collected were analyzed by flow cytometry according to the cycle kit (Biosharp, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 In vivo antitumor growth effect of drugs\u003c/h2\u003e \u003cp\u003eIn vivo anticancer activity was evaluated in mice bearing subcutaneous HGC27 tumor. The treatment schedule started when the tumor volume reached approximate 50 mm\u003csup\u003e3\u003c/sup\u003e. The mice were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;5) and treated with CCCP (10\u0026micro;g/kg). via tail vein injection on the day 0 and 6. And the mice treated with 0.9% NaCl solution was used as control. Tumor volume was measured via serial caliper measurement every other 3 days and estimated using the formula: Volume\u0026thinsp;=\u0026thinsp;0.5 \u0026times; length \u0026times; (width)\u003csup\u003e2\u003c/sup\u003e. On the day 12, the animals were sacrificed by cervical dislocation, and the tumor mass was harvested, weighted and photographed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical Analysis\u003c/h2\u003e \u003cp\u003eThree independent experiments were conducted for all studies and all assay conditions were established in duplicate or triplicate. Student's t-test was used to analyze the data, and the difference was statistically significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 CCCP inhibited gastric cancer cell activity\u003c/p\u003e\n\u003cp\u003eFirstly, we used CCK8 method to measure the effect of CCCP on the viability of MKN1 and MKN45 gastric cancer cells under different concentration gradients. The results showed that CCCP inhibited MKN1 and MKN45 gastric cancer cells in a dose-dependent manner. Among them, MKN45 cells were more resistant than MKN1 cells (Figure 1A). For further experiments, the GraphPad Prism 8 software was used to calculate the 50% inhibition concentration (IC50) of CCCP on MKN1 cells and MKN45 cells as 10 \u0026mu;g/ml and 8 \u0026mu;g/ml, respectively (Figure1B). Next, we analyzed the effect of CCCP on the proliferation of gastric cancer cells by EDU assay. The results showed that higher concentrations of CCCP drugs showed stronger inhibitory effects than those at lower concentrations (Figure1C, D).\u003c/p\u003e\n\u003cp\u003efigure1. Effects of CCCP compounds on cell viability and clonal ability of MKN1 and MKN45 cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA. Cell viability of MKN1 and MKN45 cells treated with different concentrations of CCCP compounds for 24 hours. B. According to the results in Figure 1, the IC50 was determined by software (granphad prism8), and the IC50 of the CCCP compound for MKN1 cells was determined to be 10 \u0026mu;g/ml, and the MKN45 was 8 \u0026mu;g/ml. C. D. Images of the effect of different concentrations of CCCP compounds on the colony-forming ability of MKN1 and MKN45 cells. Data are presented as mean \u0026plusmn; SD of three independent experiments (*p\u0026lt;0.05 **p\u0026lt;0.01, compared to control group)\u003c/p\u003e\n\u003cp\u003e3.2 CCCP inhibited the migration and invasion of gastric cancer cells\u003c/p\u003e\n\u003cp\u003eAccording to the previous experimental results, we determined the action concentration of CCCP. Then, we found through scratch test that the migration ability of MKN1 and MKN45 cells gradually decreased with the increase of concentration (Figure 2A). In addition, transwell experiment found that under different concentrations of CCCP, the invasion level was positively correlated with the level of CCCP (Figure 2B-C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003efigure2. CCCP compounds for MKN1 and MKN45 cell scratch experiments and in migration and invasion abilities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImages obtained using microscope magnification (40x) at 0 and 48 hours when CCCP compounds acted on M1 and MKN45 cells at different concentrations, The image obtained by A was obtained using Adobe Photoshop 2020 software to calculate the wound distance at 48 hours, and the ratio of its value to the 0 hour distance was obtained. B. C. M1 and MKN45 cells were treated with different concentrations of CCCP compounds, and images of invasion and migration cells were observed at 0 and 24 hours using microscope magnification (100x). B. After calculating the number of cells using imageJ software, the ratio was obtained compared with the control group. Data are presented as mean \u0026plusmn; SD of three independent experiments (*p\u0026lt;0.05 **p\u0026lt;0.01, compared to control group)\u003c/p\u003e\n\u003cp\u003e3.3 CCCP promotes apoptosis of gastric cancer cells\u003c/p\u003e\n\u003cp\u003eIt has been reported in previous studies that human gastric cancer cells SNU-638 incubated with CCCP in combination with TRAIL can significantly enhance apoptosis compared with TRAIL alone. And the CCCP itself is the uncoupling agent. Therefore, we verified whether CCCP can also induce apoptosis of MKN1 and MKN45 gastric cancer cells. Flow cytometry showed that CCCP could induce apoptosis of MKN1 and MKN45 cells (Figure 3A-B). We then used western blotting to assess Caspase-3 protein expression levels. The results showed that the expression level of Caspase-3 protein was significantly increased after exposure (Figure3 C-D), and CCCP could promote the apoptosis of gastric cancer cells.\u003c/p\u003e\n\u003cp\u003efigure3.CCCP compounds induce apoptosis in MKN1 and MKN45 cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA, B. MKN1 and MKN45 cells were treated with different concentrations of CCCP drugs, stained with V-FITC/PI, and determined by flow cytometry, and their apoptosis rates were calculated. C, D. The expression levels of caspase-3 were detected by western blotting. The gray value was calculated using imageJ through the image of panel C, and the ratio of the expression level of \u0026beta;-Actin protein. Data are presented as mean \u0026plusmn; SD of three independent experiments (*p\u0026lt;0.05 **p\u0026lt;0.01, compared to control group)\u003c/p\u003e\n\u003cp\u003e3.4 CCCP blocked the G0/G1 phase of gastric cancer cells\u003c/p\u003e\n\u003cp\u003eWe previously confirmed that CCCP can promote the apoptosis of gastric cancer cells. cyclin-D1, CDK4 and CDK6 are cell cyclin-related proteins. Western blot analysis showed that the expression levels of cyclin-D1, CDK4 and CDK6 proteins were significantly reduced (Figure 4C-D). In addition, the cell cycle distribution was analyzed by flow cytometry. The results showed that the number of G0/G1 stage gastric cancer cells significantly increased with the increase of CCCP concentration (Figure 4A-B).\u003c/p\u003e\n\u003cp\u003efigure4. CCCP compounds inhibit the cell cycle of MKN1 and MKN45 cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; A. MKN1 and MKN45 cells were treated with different concentrations of CCCP drugs, and the effect on cell cycle was evaluated by flow cytometry after PI staining; B. The percentage of cells in each stage of the cell cycle. C, D. MKN1 and MKN45 cells were treated with different concentrations of CCCP drugs, and the expression levels of cyclinD1, CDK4 and CDK6 were detected by western blotting; The gray value was calculated using imageJ through the image of panel C, and the ratio of the expression level of \u0026beta;-Actin protein. Data are presented as mean \u0026plusmn; SD of three independent experiments (*p\u0026lt;0.05 **p\u0026lt;0.01, compared to control group)\u003c/p\u003e\n\u003cp\u003e3.5 CCCP induced mitochondrial dysfunction and promoted mitochondrial autophagy in gastric cancer cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs a mitochondrial uncoupling agent, CCCP can induce mitochondrial membrane depolarization and lead to autophagy. We detected mitochondrial membrane potential in MKN1 and MKN45 cells after CCCP treatment.\u003c/p\u003e\n\u003cp\u003eThe results showed that MKN1 and MKN45 cells treated with CCCP showed significantly decreased membrane potential compared with the control group (Figure 5A-B). As a marker protein of mitochondrial autophagy, PINK1 specifically targets damaged mitochondria to promote the progress of mitochondrial autophagy [8]. Western blot results showed that the protein levels of LC3-II and PINK1 were significantly increased (Figure 5C-D). This suggests that CCCP can induce mitochondrial autophagy by activating PINK1/Parkin signaling pathway.\u003c/p\u003e\n\u003cp\u003efigure5. Effects of CCCP compounds on mitochondrial membrane potential of MKN1 and MKN45 cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA. B. MKN1 and MKN45 cells were treated with CCCP at a concentration of 0 and 10 ug/ml, and after 24 hours of JC-1 staining, flow cytometry was performed and the red/green ratio was calculated. C. D. The expression levels of PINK1 and LC3IIB were detected by western blotting. The gray value was calculated using imageJ through the image of panel C, and the ratio of the expression level of \u0026beta;-Actin protein. Data are presented as mean \u0026plusmn; SD of three independent experiments (*p\u0026lt;0.05 **p\u0026lt;0.01, compared to control group)\u003c/p\u003e\n\u003cp\u003e3.6 In vivo therapeutic efficacy\u003c/p\u003e\n\u003cp\u003eWe found that CCCP drug significantly inhibited the growth of tumours in mice by using a certain concentration of CCCP drug compared with saline-treated control group (Figure 6). After 6 days of injecting the drug, the average tumour volume of mice was 274.02 mm3, which was smaller than the control group 497.49 mm3, a reduction of about 44.86%, and after 12 days of CCCP administration for the pair of mice the tumour volume was reduced by about 65.69%. In terms of the weight of the tumour, after 12 injections of the drug, the average weight of the tumour in the CCCP-administered group was about 786 mg, compared with 2979 mg in the control group, a reduction in weight of about 73.61%. In conclusion, combined with the previous effects of CCCP drug to inhibit the growth of gastric cancer cells and promote apoptosis, it is known that CCCP drug in mice can inhibit the enlargement of tumour volume in mice, and has certain therapeutic effects.\u003c/p\u003e\n\u003cp\u003eFigure6.Anti-tumor growth effects of CCCP drug and salineon nude mice bearing subcutaneous HGC27 tumours (CCCP dose of 10 \u0026mu;g/kg). The injection was repeated every 6 days for 1 time.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA. Tumour morphology and size at the endpoint of the experiment (day 12). B. Measurement of tumour volume. C. Tumour weight at the experimental endpoint (day 12). Data are given as mean \u0026plusmn; SD (n = 5).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eGastric cancer has gradually become one of the cancers with the highest mortality rate in the world, with about 784,000 people dying from it every year [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. At present, conventional chemotherapy is mainly used in the treatment of gastric cancer, but the median survival time is less than 1 year [9]. No matter the subsequent immune checkpoint blocking for patients with advanced gastric cancer [10.11] or the use of fibroblast growth receptor-2 pathway inhibitors [12,13,14], the treatment for patients with gastric cancer is still unsatisfactory. We found that its application in mitochondrial autophagy is not only related to cardiovascular and liver diseases and neurodegenerative diseases, but also to tumorgenesis and tumor progression of multiple tumors [14,15,16], which has great potential in the treatment of cancer. We set out to explore the possible pathways regulating mitochondrial autophagy in gastric cancer cells. To seek new treatment options for gastric cancer.\u003c/p\u003e \u003cp\u003eFirstly, CCCP can dissolve in the inner mitochondrial membrane (MIM) and selectively increase the permeability of the mitochondrial matrix, which then leads to the release of protons in the matrix, and finally leads to the change of MIM potential (DWm) and transmembrane PH (DpH) of the inner mitochondrial membrane [17]. We measured the membrane potential after CCCP treatment by flow cytometry, and found that the treated mitochondria significantly decreased the membrane potential level compared with the control group, indicating that CCCP can also reduce the mitochondrial membrane potential of gastric cancer cells. As an important organelle for energy metabolism and homeostasis of cells [18]. When mitochondrial autophagy is shown to affect the phenotype of gastric cancer cells, we confirmed that the decline in cell viability, proliferation and invasion ability of gastric cancer cells induced by CCCP at different concentration gradients is positively correlated with CCCP concentration. In order to address the change in the inhibitory ability of CCCP on the proliferation of gastric cancer cells, we found that CCCP blocked the G1 to S phase of gastric cancer cells by flow cytometry. In order to explore the expression level of cyclin-D1, CDK4 and CDK6, as key proteins in the transition from G1 phase to S phase of cell division cycle [19], western blotting experiments were used to find that the expression level of cyclin gradually decreased with the increase of concentration. Mitochondrial autophagy has been reported to play a dual role in cancer cells depending on the cell type. On the one hand, pro-apoptotic mitochondrial clearance mediated by mitochondrial autophagy may have cell protective effects [20]. On the other hand, excessive mitochondrial autophagy may induce cancer cell death [21]. Cleaved caspase-3 is one of the markers of apoptosis, and in this study, we found that CCCP promoted caspase-3 protein expression levels. In mitochondrial autophagy, PINK1-mediated PRKN-dependent mitochondrial autophagy is the main pathway [22,23]. It has been confirmed that PINK1 deletion in mice can lead to the spontaneous development of hepatocellular carcinoma [24], while Parkin, as the downstream of PINK1, is down-regulated or absent in human glioblastoma [25], melanoma [26], breast cancer [27], etc.\u003c/p\u003e \u003cp\u003eIn addition, it has been reported that mitochondrial autophagy controlled by PINK1/Parkin pathway affects many mitochondrial physiological processes [28,29].\u003c/p\u003e \u003cp\u003eIn this study, western blotting showed that compared with the control group, the treated cells showed higher expression of PINK1, and directly interacted with LC3 and its receptor in the pathway of PINK1-mediated PRKN-dependent mitochondrial autophagy [30]. In conclusion, this study provides more directions and means for the treatment of gastric cancer.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur aim was to investigate whether CCCP can induce mitochondrial autophagy in gastric cancer and to identify one of the pathways of mitochondrial autophagy in gastric cancer cells. We found that CCCP not only inhibited cell viability, proliferation, migration and invasion of gastric cancer cells, but also induced apoptosis and blocked cell cycle, it is dose dependent. However, from the known regulation of mitochondrial autophagy in other cancers, mitochondrial autophagy has dual effects on different cells, and the existence of differences is still unclear. At present, CCCP can not only induce mitochondrial autophagy, but also promote cell apoptosis. So, the potential of CCCP in the treatment of gastric cancer and even other cancers remains to be explored.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the present are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJun Yao conducted the experiments, analyzed data, and drafted the manuscript; Jia-jie Xia and Sun-yuan Lv has contributed to the experiment and manuscript preparation; Chen-nan Xu contributed to the revision of the manuscript; Jing-jing Guo and Xiao Ying participated in the design of the research and experiment; All authors read and agree to the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eManuscript Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study follows the guidelines set by our institution and national regulations for the ethical care and use of laboratory animals, as stated in the manuscript. All animal experiments were conducted with ethical approval and aimed to minimize discomfort and ensure animal welfare. We are committed to upholding these standards and continually improving our practices for the welfare of animals involved in our research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray, F.; Ferlay, J.;\u0026nbsp; Soerjomataram, I.;\u0026nbsp; Siegel, R. L.;\u0026nbsp; Torre, L. 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K., A Molecular Approach to Mitophagy and Mitochondrial Dynamics. \u003cem\u003eMol Cells \u003c/em\u003e\u003cstrong\u003e2018,\u003c/strong\u003e \u003cem\u003e41\u003c/em\u003e (1), 18-26.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"Mitophagy, Mitochondrial Membrane Potential, Gastric Cancer, mitophagy, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-4099767/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4099767/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBACKGROUND\u003c/h2\u003e \u003cp\u003eCarbonyl cyanide 3-chlorophenylhydrazone (CCCP), as a common mitochondrial uncoupling agent, inducer of mitochondrial autophagy, has been used in nervous system and digestive system diseases. Mitochondrial autophagy is rarely reported in gastric cancer (GC). In this study, we used CCCP to treat MKN1 and MKN45 gastric cancer cells.\u003c/p\u003e\u003ch2\u003eMETHODS\u003c/h2\u003e \u003cp\u003eWe studied its effects on cell viability, migration and invasion, mitochondrial membrane potential level, apoptosis, cell cycle and mitophagy of GC were determined respectively by CCK8, Wound-Healing, Transwell, JC-1 dye, Western blot analysis and Flow cytometry assays. CCCP drugs and saline were injected intravenously, respectively, to clarify the effects of the drugs on HGC27 xenograft tumours in nude mice.\u003c/p\u003e\u003ch2\u003eRESULTS\u003c/h2\u003e \u003cp\u003eCCCP significantly inhibited the activity of MKN1 and MKN45 cells in a dose-dependent manner, and inhibited cell proliferation, migration and invasion. CCCP induced MKN1 and MKN45 cell apoptosis and arrested cell cycle in G1 phase. Furthermore, CCCP inhibited the expression of CyclinD1, CDK4 and CDK6, thus inhibited the cell cycle, and promoted the decline of mitochondrial membrane potential, which induced cells to enter PINK1 / parkin pathway mitophagy. Intravenous CCCP drugs significantly inhibited nude mouse HGC27 xenograft tumour Growth.\u003c/p\u003e\u003ch2\u003eCONCLUSIONS\u003c/h2\u003e \u003cp\u003eIn general, CCCP, can regulate gastric cancer cells by inducing mitophagy, and may be a potential therapy for gastric cancer.\u003c/p\u003e","manuscriptTitle":"Carbonyl cyanide 3-chlorophenylhydrazone promotes of mitophagy in gastric cancer cells MKN1 and MKN45 via PINK1/parkin pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-18 18:36:53","doi":"10.21203/rs.3.rs-4099767/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":"88a46bfb-a10e-45f6-af29-57b45b0c1afc","owner":[],"postedDate":"March 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-19T10:46:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-18 18:36:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4099767","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4099767","identity":"rs-4099767","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}