Baicalein improves cisplatin resistance in gastric cancer through OMA1/OPA1 axis mediated mitochondrial dynamics

Baicalein improves cisplatin resistance in gastric cancer through OMA1/OPA1 axis mediated mitochondrial dynamics | 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 Baicalein improves cisplatin resistance in gastric cancer through OMA1/OPA1 axis mediated mitochondrial dynamics chunmei Yu, chunxia Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8726309/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Resistance to chemotherapy represents a major obstacle to the successful treatment of gastric cancer (GC). This study aimed to investigate the role and underlying mechanism of baicalein in cisplatin resistance in gastric cancer through the OMA1/OPA1 axis.Baicalein treatment significantly increased mitochondrial fission and ROS levels in AGS cells, leading to reduced ATP production and consequently enhancing cisplatin-induced cytotoxicity in gastric cancer cells.Intriguingly,OMA1 expression was most significantly increased upon combined treatment with baicalein and cisplatin in AGS cells.In contrast,OPA1 expression was significantly reduced.Mechanistically, we define a signaling axis linking OMA1 to OPA1 and mitochondrial fission in Gastric cancer. Moreover, overexpression of OMA1 in AGS/DDP cells also significantly increased mitochondrial fission and ROS levels, decreased OPA1 protein expression and mitochondrial membrane potential, and reduced ATP production.Consistently, the cell viability and cell apoptosis assay showed that OMA1 overexpression synergistically enhancing cisplatin sensitivity in AGS/DDP cells.Taken together, all data in this study indicate that baicalein improves cisplatin resistance in gastric cancer through OMA1/OPA1 axis mediated mitochondrial dynamics,and repurpose baicalein as a potential agent to inhibit cisplatin resistance in GC.Furthermore,improve our knowledge about how OMA1 regulates mitochondria and provide justification for combining OMA1 overexpression with chemotherapy in GC. Baicalein Gastric cancer Cisplatin resistance Mitochondrial dynamics OMA1 OPA1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Gastric cancer is a fatal disease with low survival rate worldwide. It is reported that there are over one million new cases every year, and gastric cancer is the fifth largest diagnosed malignant tumor in the world [ 1 ] . Chemotherapy is an important means for the treatment of gastric cancer, but drug resistance often leads to treatment failure, which has become the main clinical problem of poor prognosis in patients with gastric cancer [ 2 ] . Therefore, re-sensitizing gastric cancer cells to chemotherapy and investigating the mechanism of drug resistance is of clinical significance. Mitochondria are highly dynamic organelles with constantly division, elongation and fusion to each other, forming networks or fragments [ 3 ] .Mitochondrial fusion generates a tubular mitochondrial network that counteracts metabolic stress, maintains cellular integrity, and provides protection against cell death [ 4 ] .In contrast, mitochondrial fission produces numerous small and fragmented mitochondria, a process linked to cellular stress or apoptotic signaling [ 5 ] . In our previous studies, it was found that mitochondrial dynamics participate in cisplatin resistance in ovarian cancer, and the level of mitochondrial fusion was higher in cisplatin resistant ovarian cancer cells [ 6 ] .Excessive mitochondrial fragmentation is linked to platinum sensitivity, whereas chemoresistant cells exhibit a more fused mitochondrial network.These suggest that mitochondrial fusion plays a key role in the acquisition of chemotherapy resistance. Mitochondrial dynamics regulate mitochondrial function through a balance of fusion and fission. The fusion process, mediated by Mitofusion 1/2 (Mfn1/2 )and Optic atrophy 1(OPA1), facilitates the exchange of mitochondrial contents and repairs damaged mtDNA, thereby maintaining oxidative phosphorylation efficiency and ATP synthesis capacity. Conversely, mitochondrial fission, driven by Dynamin-related protein 1 (Drp1) [ 7 ] .Mitochondrial fusion enhances the efficiency of ATP synthesis and increases mitochondrial stability [ 8 ] .However, excessive fission induced by apoptotic/stress signals produces too much reactive oxygen species (ROS), a prerequisite for apoptosis [ 9 ] .Enhancing mitochondrial fission may be a promising strategy to induce apoptosis in gastric cancer cells. Stress-sensitive OMA1 mediates proteolytic cleavage of L-OPA1 into S-OPA1, which disrupts mitochondrial fusion and causes network fragmentation [ 10 ] . Mitochondrial protease OMA1 has been shown to be involved in tumor progression [ 11 ] , but its role in promoting or inhibiting tumor growth has not been determined.Some studies have found that OMA1 activation increases the sensitivity of ovarian cancer to cisplatin both in vivo and in vitro. This effect may be mediated by the cleavage of OPA1, which disrupts the coordination between mitochondrial inner and outer membranes, ultimately leading to ovarian cancer cell death. Conversely, following OMA1 knockout, OPA1 levels increase and apoptosis is attenuated [ 12 ] . Baicalein is a natural product with antioxidant, antiviral, anti-tumor and other biological activities [ 13 ] . Studies have suggested that it plays an important role in the treatment of cancer [ 14 ] . When combined with existing chemotherapy drugs, it can improve the therapeutic effect and reduce the toxicity of chemotherapy drugs [ 15 ] . The pathway and mechanism of baicalein's anti-tumor and improvement of chemotherapy resistance are still in the exploration stage. In lung cancer cells, baicalein can reduce mitochondrial membrane potential (MMP) and decrease the level of mitochondrial dynamic protein OPA1 [ 16 ] . Whether baicalein plays a role in drug resistance of gastric cancer cells through OMA1-mediated mitochondrial dynamics remains unclear. The purpose of this study was to investigate the effect of baicalin on cisplatin resistance in AGS gastric cancer, and whether the dynamic mitochondrial function is mediated by affecting the OMA1/OPA1 axis, so as to provide new ideas and basis for the solution of chemotherapy resistance in gastric cancer.Here, we investigate the consequence of altered OMA1 expression on mitochondrial dynamics, ATP,ROS and therapeutic resistance in gastric cancer. Materials and Methods 1. Cell culture Human gastric cancer AGS cells and cisplatin-resistant AGS/DDP cells were purchased from the Jinyuan Biotechnology(Shanghai, China). AGS and AGS/DDP cells were cultured in RPMI 1640 (BI, Israel) and supplemented with 1% penicillin/streptomycin (Solarbio, China) and 10% fetal bovine serum (FBS, BI, Israel) at 37°C with 5% CO 2 in a humidified atmosphere. 2. Cell viability assay Cells were collected through pancreatic enzyme digestion and spread on 96-well cell culture plates with 5*103 cells/holes in complete medium (plates and cell numbers were selected according to specific experiments). The cells were incubated in a 37℃ temperature box overnight according to the cell adhesion conditions, and the treatment could be started after the cell adhesion was complete. Discard the old medium, add the drug-containing medium for treatment, and culture for 48h. Discard the supernatant, add 10%CCK-8(#CA1210,Solarbio, Beijing,China)detection reagent, culture for 1 ~ 2h, and then test with enzyme marker. 3. Annexin V-FITC/PI apoptosis assay After the cells were treated according to the experimental requirements, the culture supernatant was discarded, washed with PBS((Solarbio, China) for 2 to 3 times, digested with pancreatic enzyme for 2 to 3 min, the reaction was terminated with cell culture medium, and cells were collected by centrifugation at 500 ×g for 5 min. Apoptosis was detected using Annexin V-FITC/PI kit(#E-CK-A211,Solarbio, Beijing,China). The cells were washed with pre-cooled PBS solution for 2–3 times, centrifuged at 500 ×g for 5 min, and the supernatant was discarded and the cells were collected. Add 100µL pre-cooled Binding Buffer to the suspension cells, add 5µL An‑nexin V-FITC and 5µL PI solution, gently mix, avoid light at room temperature for 15 min, add 400µL pre-cooled binding buffer and mix well. The proportion of cells expressing Annexin V-FITC fluorescence was measured by flow cytometry, which was the apoptosis rate.Cells were analyzed using a flow cytometer equipped with a laser emitting at 488 nm and an optical filter FL1 (530/30 nm). 4.Intracellular ROS Level Assay After treatment, the cells were digested and collected by pancreatic enzymes, centrifuged at 500×g for 5 min, and the supernatant was discarded. DCFH-DA (#MA0219,Dalian Meilun Biotechnology Co., Ltd.,Dalian,China)was diluted in serum-free medium at 1:1000 to a final concentration of 10µM. After the cells were collected, they were suspended in the DCFH-DA working liquid with an appropriate volume of diluted DCFH-DA, and the cell density was 1.0×10^6 ~ 2.0×10^7/mL (500uL/ well). The cells were incubated in the cell incubator at 37℃ for 20 ~ 30 min in dark light, and mixed upside down every 3–5 min. The probe was fully in contact with the cells, and then the cells were washed with serum-free culture solution 3 times. FITC channel was selected and samples were detected by flow cytometry. 5. Intracellular ATP Level Assay The cells of each group treated with experimental drugs were separated from the culture supernatant, and the culture supernatant was removed to obtain the bottom layer of precipitated cells. The collected cells were added to 300 ~ 500µL cold double steaming water and homogenized in an ice water bath for crushing (part of the cells were taken out for determination of protein concentration), and then the cell suspension was heated in a boiling water bath for 10 minutes, then the cells were mixed and extracted for 1 minute. The ATP levels were detected with commercial ATP assay kit (#A095-1-1, Nanjing Jiancheng Bioengineering Institute,Nanjing,China) following manufacturer’s protocol. 6.Transmission electron microscopy AGS/DDP and AGS cells were seeded at 5×10 5 cells/culture dish (35mm) and treated with indicated reagents. Then cells were collected and fixed by 3% glutaraldehyde at 4°C followed by 1% osmium tetroxide. Next, cells were dehydrated in 30%~100% acetone, embedded with Epox 812 and cut to semithin sections. After stained with methylene bule, the ultrathin sections (60 nm ~ 90 nm) were prepared by EM UC7 (Leica, Germany) and stained with lead citrate and uranyl acetate. The sections were detected by transmission electron microscope (FEI, Ltd, America). 7.Intracellular MMP Level Assay After treating the cells according to the experimental requirements, pancreatic enzyme digestion and collection of cells, according to the JC-1(#E-CK-A301,Wuhan,China) staining kit operation is as follows: The cells to be tested in each group were added with JC-1 dye and incubated for 20 min at room temperature in the dark. After gradient washing with the buffer delivered with the kit, the cells were detected by upflow cytometry. The relative number of mitochondrial membrane potential polarization and depolarization of the cells was analyzed to reflect the changes of cell MMP. 8.Western blot analysis 8.Western blot analysis After administration according to the above experimental groups, the proteins of the cells were extracted, the proteins were analyzed by BCA protein kit, SDS-PAGE glue, sample loading, electrophoresis, and membrane transfer were prepared, sealed with 5% skim milk powder, incubated overnight at 4℃ with primary antibody, incubated overnight at 37℃ with secondary antibody, TBST membrane washing, and then ECL light-producing liquid was added after TBST membrane washing. The protein expressions of MFN1(#13798-1-AP,proteintech), MFN2(#12186-1-AP,proteintech), Drp1( #DF7037,Affinity), OPA1 (#27733-1-AP,proteintech)and OMA1( #DF12435,Affinity) were detected in the gel imaging system, and the gray values of the bands were analyzed using Image J software for statistical analysis. 9.OMA1 overexpression transfection AGS/DDP cells were transfected with OMA1 overexpression plasmid(GeneCreate,Wuhan, China).The cells were collected through pancreatic enzyme digestion, the cells were suspended in the medium, the cells were counted, the cell density was diluted to 3*10 ^ 5 cells /ml, and the 2ml holes were inoculated on the 6-well cell culture plate. The cells were incubated in a 37℃ temperature box overnight according to the cell adhesion conditions. After the cell adhesion was complete, the treatment could be started. The supernatant was discarded and replaced with 1mL basic medium. According to the transfection group, two sterile EP tubes were prepared for each group, 125ul Opti-MEM was added to each tube, 5uL lipofectamine 3000 was added to one tube, 2.5ug plasmid and 5ul lipofectamine 3000 was added to the other tube, and the tubes were gently mixed and left for 5min at room temperature. The above two EP tubes were mixed well, left at room temperature for 15min, the mixed liquid drops were placed into the corresponding holes in the six-well plate, and the cells were put back into the incubator for culture. 4-6h after transfection, 1ml complete medium containing 20% serum was added into the six-well plate. The culture plates were cultured in a cell incubator at 37℃ and 5% CO 2 for 24h, cell precipitate was collected, cleaned once with PBS, and 1mL Tranzol was added. 10.Statistical analysis Using Prism 6 statistical software, measurement data were expressed as mean ± standard deviation (± s), T-test was used for inter-group comparison, and one-way analysis of variance was used for inter-group comparison. All experiments were independently repeated 3 or more times. P < 0.05 was considered statistically significant. Results 1. Baicalein increased cisplatin-induced cell proliferation and apoptosis. Cisplatin is one of the most commonly used chemotherapy drugs for gastric cancer. However, its toxic side effects greatly limit the clinical application of cisplatin. The dose of cisplatin needs to be reduced by combination with some synergist drugs. In order to investigate whether baicalein can enhance the sensitivity of cisplatin chemotherapy. First,AGS cells were treated with baicalein 20µmol/L, cisplatin 2.5µg/mL, baicalein 20µmol/L combined with cisplatin 2.5µg/mL for 48h, and the activity of the cells was detected by CCK8.As shown in Fig. 1 A,DDP treatment inhibited cell viability. While Baicalein had no effect on cell activity, DDP treatment in combination with Baicalein showed greater efficiency in inhibiting cell activity than treatment with DDP alone.Furthermore, cell apoptosis was examined in AGS cells treated with DDP and/or baicalein. Baicalein treatment combination with DDP efficiently promote cisplatin-induced apoptosis in AGS cells(Fig. 1 B,C).All these results indicate that baicalein may increase the sensitivity to cisplatin chemotherapy. 2.Baicalein increased cisplatin-induced mitochondrial fission and ROS level ,while decreased the intracellular ATP level in AGC cells. Disturbance of homeostasis results in ROS accumulation and ATP reduction, leading to mitochondrial damage, which is crucial in cancer progression [ 17 ] . The intracellular ROS levels in AGS cells were measured by detection of the DCFH-DA staining.As shown in Fig. 2 A,B,While Baicalein had no effect on the level of ROS, 2.5µg/l of cisplatin significantly increased the intracellular ROS level in AGS cells. DDP treatment in combination with Baicalein significantly increase cisplatin-induced intracellular ROS production in AGS cells.Mitochondria are the major source for ATP production. Therefore, intracellular ATP level was detected in AGS cells. Our results showed that Fig. 2 C, Baicalein in combination with DDP significantly decreased the intracellular ATP levels in AGC cell.Mitochondrial fusion enhances the efficiency of ATP synthesis and increases mitochondrial stability [ 8 ] . The results of TEM indicated that cisplatin treatment shifted mitochondrial morphology toward smaller, more rounded/oval shapes compared to the control, and this effect was further enhanced when cisplatin was combined with baicalein(Fig. 2 C).All these results indicated that baicalein in combination with DDP impaire mitochondrial function, which might stimulated ROS accumulation and mitochondrial fission ,while prevent ATP generation. 3.Baicalein regulates the expression of mitochondrial dynamics proteins and OMA1 in cisplatin-treated AGC cells. Our previous research results showed that compared with AGS cells, OMA1 mRNA was low expressed in AGS/DDP cells, while OPA1 mRNA was high expressed [ 18 ] .As shown in Fig. 2 D ,decreased mitochondrial fusion was detected in AGS cells upon cisplatin treatment.These suggest that OMA1and mitochondrial dynamics may be related to cisplatin resistance in GC.Thus, the mechanism of mitochondrial dynamics which baicalin reduces cisplatin resistance in AGS cells was further explored.It was found that cisplatin down-regulated the expression of MFN1,MFN2 and OPA1, which was attenuated by cisplatin in combination with baicalein( Fig. 3 A-D). In contrast, the expression of Drp1and OMA1 was significantly upregulated following cisplatin in combination with baicalein treatment, suggesting that mitochondria have undergone division( Fig. 3 E,F). These results suggest that OMA1 and mitochondrial dynamics may be involved in the mechanism baicalein reverses cisplatin resistance in GC cells. 4.Overexpression of OMA1 stimulated cisplatin-induced mitochondrial fission in AGS/DDP cells by regulating OPA1. It is reported that the activation of OMA1 can mediate the hydrolysis of OPA1 and the division of mitochondria [ 19 , 20 ] .To further investigate the underlying mechanism of OMA1 mediated mitochondrial dynamics, the lentiviral plasmid was used to establish OMA1 overexpressed AGS/DDP cells.Western blot results showed that OPA1 expression was significantly decreased following OMA1overexpressed in AGS/DDP cells.After combined with cisplatin, the decrease in OPA1 level was greater than that when cisplatin was used alone,while OMA1 expression were increased(Fig. 4 A-C).Moreover, the results of TEM also indicated that compared with the control(NC), both OMA1 overexpression and cisplatin treatment alone induced a shift in mitochondrial morphology towards smaller, more rounded and oval shapes, and this effect was further enhanced when cisplatin was combined with OMA1 overexpression than cisplatin alone. 5. Overexpression of OMA1 re-sensitized AGS/DDP cells to cisplatin-induced cytotoxicity. To examine the role of OMA1 in cisplatin resistance, AGS/DDP cells were transfected with OMA1 overexpression plasmid or treated with cisplatin, cell viability and apoptosis assay were detected using CCK-8 assay and flow cytometry. As expected, overexpression of OMA1mediated suppression on cisplatin-induced growth inhibition and apoptosis was also significantly increased in AGS/DDP cells (Fig. 5 A-B). In conclusion, all these results highlight the important role of OMA1/OPA1 axis-mediated mitochondrial fission in the development of cisplatin resistance in GC. 6.OMA1/OPA1 axis enhanced cisplatin-induced cytotoxicity by mitochondrial dysfunction. Although the mitochondria are in a constant state of transition between fission and fusion, excessive mitochondrial fission leads to a decrease in metabolic function and an increase in oxidative stress [ 21 ] . As shown in Fig. 6 A,B,E,OMA1 overexpression reduces ATP levels and increases ROS levels in AGS/DDP cells. DDP in combination with OMA1 overexpression showed higher ROS levels and lower ATP levels than DDP treatment alone. ΔψM serves as an indirect indicator of mitochondrial ATP synthesis capability. The shape of mitochondria may directly affect biological energy production. For instance, longer mitochondria are sometimes linked to more efficient ATP production [ 22 ] .We then evaluated the changes in ΔψM in mitochondria that were measured by flow cytometry using JC-1 staining. OMA1 overexpression reduces ΔψM levels in AGS/DDP cells. DDP in combination with OMA1 overexpression showed lower ψM levels than DDP treatment alone.It was also indirectly implied that OMA1overexpression caused damage to mitochondrial function.These results provide additional evidence that OMA1/OPA1 axis causes mitochondrial dysfunction via mitochondrial fragmentation. In conclusion, all these data suggest that OMA1 signaling pathway may be involved in cisplatin-induced mitochondrial dynamics, and that baicalein reduces cisplatin resistance in gastric cancer cells via the OMA1/OPA1 axis. Discussion Gastric cancer represents one of the leading causes of cancer deaths worldwide. Cisplatin (DDP) is a basic chemotherapy drug for gastric cancer (GC). With the increase of DDP drug concentration in clinical treatment, cancer cells gradually became resistant. Therefore,identifying novel molecular targets and drugs capable of overcoming drug resistance is crucial for improving patient prognosis. Baicalein showed anti-tumor effect on gynecological tumor, lung cancer, liver cancer and other tumor cells, and combined with chemotherapy drugs can enhance the effect of chemotherapy. The combination of baicalein and cisplatin has a synergistic effect on cervical cancer cells, which can promote cell apoptosis and inhibit cell vitality, and has the potential to improve the sensitivity of cervical cancer cells to cisplatin [ 23 ] . Similarly, baicalein treatment can increase the sensitivity of ovarian cancer cells to cisplatin and inhibit cell proliferation, metastasis and tumor growth [ 24 ] . A number of studies have found that baicalin is an important candidate drug for the treatment of lung cancer, and its mechanism of action includes regulating cell proliferation, metastasis, apoptosis, autophagy, etc [ 25 ] . In addition, baicalein can induce apoptosis and autophagy of multi-drug resistant liver cancer cells and reverse multi-drug resistance of liver cancer, which may contribute to the treatment of drug-resistant liver cancer [ 26 ] . In our previous study, we found that baicalin inhibited the proliferation and promoted apoptosis of gastric cancer resistant cells AGS/DDP through the mitochondrial pathway [ 18 ] . Here, baicalein combined with cisplatin can significantly increase the proliferation inhibition and induce apoptosis of AGS gastric cancer cells, which is consistent with the results of previous studies showing that baicalein enhances the chemotherapy sensitivity of tumor cells, providing further supporting evidence for the anti-tumor treatment of baicalein, especially in the treatment of gastric cancer. At present, the mechanism of baicalein enhancing tumor chemotherapy sensitivity has not been determined. Studies have shown that baicalein enhances the sensitivity of cervical cancer cells to cisplatin through the Akt pathway [ 23 ] , improves the drug resistance of ovarian cancer by regulating CirSLC7A6 [ 24 ] , and enhances the sensitivity of triple-negative breast cancer MDA-MB-231 cells to adriamycin through autophagy mediated down-regulation of CDK1 [ 27 ] . Studies have also confirmed that baicalein inhibits the glutamine-mtor metabolic pathway, induces apoptosis of lung cancer cells [ 28 ] , and triggers siderptosis of colorectal cancer cells by blocking the JAK2/STAT3/GPX4 axis [ 29 ] . In gastric cancer cells, baicalin can induce apoptosis and autophagy by regulating miR-7/FAK/AKT [ 30 ] , Akt/mTOR and Nrf2/ Keap1 pathways [ 31 ] , thereby enhancing cisplatin sensitivity of gastric cancer cells. Studies have found that microRNA is a key factor in the cisplatin resistance mechanism of gastric cancer [ 32 ] , but interestingly, microRNA may enhance the sensitivity of cisplatin chemotherapy by promoting threadgrain fission [ 33 ] .In recent years, researchers have observed that tumor cells manipulate mitochondrial dynamics to acquire proliferation and survival advantages [ 34 ] . It has been reported that baicalein can resensitize tamoxifen-resistant breast cancer cells by inhibiting hypoxia-inducing factor-1 α, reducing aerobic glycolysis and reversing mitochondrial dysfunction [ 35 ] . When the morphology of mitochondria tends to be prolonged and networked, it is beneficial to maintain the normal function of mitochondria, such as enhancing energy metabolism and reducing the excessive production of reactive oxygen species (ROS). In order to confirm that baicalein can improve the sensitivity of tumor chemotherapy through mitochondrial pathway, cisplatin combined with baicalein was applied to gastric cancer cells, and it was found that mitochondrial division increased,ROS production increased, and ATP decreased, indicating that baicalein affects mitochondrial dynamics and damages mitochondrial function in the process of anti-tumor cells. The morphology and ultrastructure of mitochondria are regulated by mitochondrial shaping proteins,and the dynamic changes of mitochondrial shape can in turn control mitochondrial function [ 36 ] . OPA1, Mfn1, and Mfn2 regulate mitochondrial fusion, and Drp1 is the main protein regulating division [ 37 ] . In lung cancer cells, baicalein activates AMPK by activating phosphorylated adenylate, and then promotes the phosphorylation of mitochondrial fission protein Drp1, inducing mitochondrial division and apoptosis [ 38 ] . We determined proteins related to mitochondrial dynamics and found that mitochondrial fusion protein decreased and fission protein increased in gastric cancer cells, which further confirmed that baicalin reshaps mitochondrial dynamics by affecting mitochondrial regulatory proteins.OPA1 plays a crucial role in the dysregulation of key processes such as apoptosis and cell proliferation in cancer by directly controlling mitochondrial biology [ 39 ] . In unbiased screening of the Venetoclaxresistance gene, it was found that OPA1 was up-regulated, and its deletion restored the sensitivity of cells to Venetoclax [ 40 ] , suggesting that OPA1 may be a key target for overcoming resistance. In this study, OPA1 was significantly down-regulated after baicalein combined with cisplatin treatment of gastric cancer cells, and the sensitivity of gastric cancer cells to cisplatin was enhanced.Thus, we sought to identify upstream regulators of OPA1 to further clarify the precise mechanism through which baicalein sensitizes gastric cancer cells to cisplatin. It is well-known that OMA1 is the protease responsible for cleaving and regulating OPA1.Some studies have found that OMA1 activation increases the sensitivity of ovarian cancer to cisplatin in vivo and in vitro, possibly through the cleavage of OPA1, coordinating the inner and outer membrane of mitochondria, and ultimately inducing ovarian cancer cell death, while the OPA1 increases and apoptosis is reversed after OMA1 knockout [ 41 ] . However, OMA1 can also promote the development of colorectal cancer by enhancing glycolysis and suppressing OXPHOS in colorectal cancer cells [ 42 ] .Therefore, we overexpressed OMA1 in AGS/DDP cells and found that OPA1 expression decreased, and mitochondrial fusion was reduced.OMA1 overexpression treatment was performed on AGS/DPP cells of gastric cancer, and it was found that overexpression of OMA1 combined with cisplatin treatment inhibited proliferation, induced apoptosis, and impaired mitochondrial function of AGS/DPP cells. In conclusion, our results indicate that baicalin may overcome cisplatin resistance in GC cells by OMA1/OPA1 axis-mediated mitochondrial dynamics. In summary, this study found that baicalein plays an important role in the cisplatin resistance of AGS gastric cancer. Baicalein, as a natural product, has the advantages of relatively low toxicity and wide sources, which is expected to become a new choice for adjuvant cisplatin chemotherapy, providing a new theoretical basis and potential treatment strategy for solving drug resistance in gastric cancer. In addition, the combined application of baicalein and cisplatin may reduce the clinical dose of cisplatin, reduce the toxic side effects of cisplatin while ensuring the therapeutic effect, and improve the quality of life of patients. Declarations Author Contribution We declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere.We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.We understand that the Corresponding Author is the sole contact for the Editorial process. She is responsible for communicatine with the other authors about progress, submissions of revisions and final approval ot proofs.All authors as follows：Chunmei Yu (First Author):Conceptualization,Funding Acquisition, Resources,Supervision,Experimental verification;Chunxia Yu: Wrote the main manuscript text,Data Curation,Formal Analysis, Investigation, Software; References Smyth EC, Nilsson M, Grabsch HI et al (2020) Gastric cancer[J] Lancet 396(10251):635–648 Mora-Lagos B, Cartas-Espinel I, Riquelme I et al (2020) Functional and transcriptomic characterization of cisplatin-resistant AGS and MKN-28 gastric cancer cell lines[J]. 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Clin Transl Med 11(11):e577 Quintana-Cabrera R, Scorrano L (2023) Determinants and outcomes of mitochondrial dynamics[J]. Mol Cell 83(6):857–876 Bereiter-Hahn J, Voth M (1994) Dynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria[J]. Microsc Res Tech 27(3):198–219 Deng X, Liu J, Liu L et al (2020) Drp1-mediated mitochondrial fission contributes to baicalein-induced apoptosis and autophagy in lung cancer via activation of AMPK signaling pathway[J]. Int J Biol Sci 16(8):1403–1416 Herkenne S, Scorrano L (2020) OPA1, a new mitochondrial target in cancer therapy[J]. Aging 12(21):20931–20933 Chen X, Glytsou C, Zhou H et al (2019) Targeting Mitochondrial Structure Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment[J]. Cancer Discov 9(7):890–909 Cheng M, Yu H, Kong Q et al (2022) The Mitochondrial PHB2/OMA1/DELE1 Pathway Cooperates with Endoplasmic Reticulum Stress to Facilitate the Response to Chemotherapeutics in Ovarian Cancer[J]. Int J Mol Sci, 23(3) Wu Z, Zuo M, Zeng L et al (2021) OMA1 reprograms metabolism under hypoxia to promote colorectal cancer development[J]. EMBO Rep 22(1):e50827 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 01 Apr, 2026 Reviews received at journal 23 Mar, 2026 Reviews received at journal 16 Mar, 2026 Reviews received at journal 11 Mar, 2026 Reviewers agreed at journal 11 Mar, 2026 Reviewers agreed at journal 10 Mar, 2026 Reviewers agreed at journal 09 Mar, 2026 Reviewers invited by journal 09 Mar, 2026 Editor assigned by journal 09 Feb, 2026 Submission checks completed at journal 07 Feb, 2026 First submitted to journal 28 Jan, 2026 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. 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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-8726309","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":604732124,"identity":"b48bc4d4-04ac-4d54-ae4c-05d564a3f2fc","order_by":0,"name":"chunmei Yu","email":"","orcid":"","institution":"Jiangxi Health Vocational College","correspondingAuthor":false,"prefix":"","firstName":"chunmei","middleName":"","lastName":"Yu","suffix":""},{"id":604732125,"identity":"0d838c71-ce36-4b8f-b79e-9db50eaf5bff","order_by":1,"name":"chunxia Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACNv7mg4//VPxj5udvPkCcFj6JY8kGPGcOsEvOOJZAnBY5hhwzCd62A/wGB3IMiHQYwwEzCQm2O9IGB858vPGGwU5Ot4GQFuaGZAsDnmfGkod7N1vOYUg2NjtA2JaDNxIkmJP5DpzdJs3DcCBxG2EtiQ0SBwyY6xsO5DwjVksyk2RDwmFmgQM5bERqkTjGbMxwII0ZGMjGlnMMiPCLfH//x8eM/2xAUfnwxpsKOzmCWlCABA+RUYOshVQdo2AUjIJRMCIAAAK9Q/OvKudYAAAAAElFTkSuQmCC","orcid":"","institution":"Jiangxi Provincial Institute of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"chunxia","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2026-01-29 02:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8726309/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8726309/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104780795,"identity":"4879cde3-1bc7-4900-9ced-9222a2033f3a","added_by":"auto","created_at":"2026-03-17 07:53:59","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":333124,"visible":true,"origin":"","legend":"\u003cp\u003eBaicalein increased cisplatin-induced cell proliferation and apoptosis.\u003cstrong\u003e(A) \u003c/strong\u003eIt indicated that AGS cells were treated with baicalein 20umol/L, cisplatin 2.5ug/mL, baicalein 20umol/L combined with cisplatin 2.5ug/mL for 48h, and the cell viability was detected by CCK8.\u003cstrong\u003e (B)\u003c/strong\u003e Cell apoptosis was analyzed by Annexin V/PI staining. Bar diagram represents percentage of apoptotic cells. Data were presented as the means ± SD. The experiments were repeated three times independently.\u003csup\u003e##\u003c/sup\u003ep\u0026lt;0.01,\u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001, ***p\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8726309/v1/b670c9414ed412fb41f4fd09.jpeg"},{"id":104522514,"identity":"8d3c37cd-a04d-4cae-a500-841aa0aab87d","added_by":"auto","created_at":"2026-03-12 20:24:47","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":700278,"visible":true,"origin":"","legend":"\u003cp\u003eBaicalein reduces cisplatin resistance of gastric cancer cells by impairing mitochondrial function.\u003cstrong\u003e(A,B)\u003c/strong\u003e It indicated that AGS cells were treated with baicalein 20μmol/L, cisplatin 2.5μg/mL and baicalein 20μmol/L combined with cisplatin 2.5μg/mL for 48h, and the ROS levels of the cells were detected by cytometry.\u003cstrong\u003e(C)\u003c/strong\u003e ATP levels were detected by ATP assay kit. \u003cstrong\u003e(D) \u003c/strong\u003eMitochondrial morphology in AGS cells treated for 48 h with baicalein (20 μM), cisplatin (2.5 μg/mL), or their combination was assessed by transmission electron microscopy (TEM).Circled images at higher magnification are shown in the below panels. Scale bar=1 µm. The experiment was repeated at least three times, and the experimental results were statistically analyzed by one-way analysis of variance, and the histogram was drawn.*p\u0026lt;0.05; **p\u0026lt;0.01.***P\u0026lt;0.001, \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e###\u003c/sup\u003eP\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8726309/v1/a68b06a66767e529e5fbfe93.jpeg"},{"id":104522507,"identity":"c2fb1f6e-c323-4120-91ee-f10453ea7197","added_by":"auto","created_at":"2026-03-12 20:24:46","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":404046,"visible":true,"origin":"","legend":"\u003cp\u003eBaicalein increases cisplatin chemosensitivity through mitochondrial dynamics. \u003cstrong\u003e(A) \u003c/strong\u003eindicated that AGS cells were treated with baicalein 20umol/L, cisplatin 2.5μg/mL and baicalein 20umol/L combined with cisplatin 2.5μg/mL for 48h, respectively. \u003cstrong\u003e(B-F) \u003c/strong\u003eThe expression levels of MFN2, MFN1, OPA1, OMA1 and DRP1 were detected by Western Blot, with GAPDH as the internal reference.One-way analysis of variance was used to analyze the data and draw corresponding statistical charts.\u003csup\u003e * \u003c/sup\u003eP \u0026lt; 0.05, * *P \u0026lt; 0.01,\u003csup\u003e * * * \u003c/sup\u003e\u0026nbsp;P \u0026lt; 0.001;\u003csup\u003e #\u003c/sup\u003e P \u0026lt; 0.05, \u003csup\u003e# # # \u003c/sup\u003eP \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8726309/v1/cecb748a21d7074b4d0c390a.jpeg"},{"id":104522511,"identity":"5cb946c6-0942-474b-a017-192cdc79cd25","added_by":"auto","created_at":"2026-03-12 20:24:46","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":778498,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of OMA1 re-sensitized AGS/DDP cells to cisplatin-induced cytotoxicity.\u003cstrong\u003e(A-C) \u003c/strong\u003eAGS/DDP cells were transfected with OMA1 overexpression plasmid for 4-6h. Then,AGS/DDP cells were treated with OMA1-overexpression plasmid and 2.5 μg/mL cisplatin individually, or co-treated with both for 48 hours.The expression of OPA1 and OMA1 was determined by western blot.One-way analysis of variance was used to analyze the data and draw corresponding statistical charts.\u003cstrong\u003e(D) \u003c/strong\u003eAGS/DDP cells were transfected with OMA1 overexpression plasmid for 4-6h. Then,mitochondrial morphology in AGS/DDP cells, treated with OMA1-overexpression plasmid or 2.5 μg/mL cisplatin individually or in combination for 48 hours, was assessed by transmission electron microscopy (TEM).Circled images at higher magnification are shown in the below panels. Scale bar=1 µm.\u003cstrong\u003e \u003c/strong\u003e* * P \u0026lt; 0.01, * * * P \u0026lt; 0.001; \u003csup\u003e#\u003c/sup\u003e P \u0026lt; 0.05, \u003csup\u003e# # \u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e# # # \u003c/sup\u003eP \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8726309/v1/163148231bd75443ef9bd3e9.jpeg"},{"id":104522508,"identity":"84fbb8f8-d5d0-436a-a1d8-71663eab0020","added_by":"auto","created_at":"2026-03-12 20:24:46","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":368303,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of OMA1 re-sensitized AGS/DDP cells to cisplatin-induced cytotoxicity. \u003cstrong\u003e(A,B) \u003c/strong\u003eAGS/DDP cells were transfected with OMA1 overexpression plasmid for 4-6h. Then,AGS/DDP cells were treated with OMA1 overexpression plasmid and 2.5 μg/mL cisplatin individually, or co-treated with both for 48 hours. Cell apoptosis was analyzed by Annexin V/PI staining. Bar diagram represents percentage of apoptotic cells. Data were presented as the means ± SD. The experiments were repeated three times independently.\u003cstrong\u003e(B) \u003c/strong\u003eAGS/DDP cells were transfected with OMA1 overexpression plasmid for 4-6h. Then,AGS/DDP cells were treated with OMA1 overexpression plasmid and 2.5 μg/mL cisplatin individually, or co-treated with both for 48 hours. Cell viability was detected by CCK8 kit.* * * P \u0026lt; 0.001;\u003csup\u003e # # #\u003c/sup\u003e P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8726309/v1/e559b0cba177f4325b266d92.jpeg"},{"id":104522509,"identity":"710ee45e-994a-4dbb-9542-449b80e60911","added_by":"auto","created_at":"2026-03-12 20:24:46","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":448741,"visible":true,"origin":"","legend":"\u003cp\u003eOMA1/OPA1 axis enhanced cisplatin-induced cytotoxicity by mitochondrial dysfunction.\u003cstrong\u003e(A,B\u003c/strong\u003e) AGS/DDP cells were transfected with OMA1 overexpression plasmid for 4-6h. Then,AGS/DDP cells were treated with OMA1 overexpression plasmid and 2.5 μg/mL cisplatin individually, or co-treated with both for 48 hours,and the ROS levels of the cells were detected by cytometry.One-way analysis of variance was used to analyze the data and draw corresponding statistical charts.\u003cstrong\u003e(C,D) \u003c/strong\u003eAGS/DDP cells were transfected with OMA1 overexpression plasmid for 4-6h. Then,AGS/DDP cells were treated with OMA1 overexpression plasmid and 2.5 μg/mL cisplatin individually, or co-treated with both for 48 hours,and theψM\u0026nbsp; levels of the cells were detected by cytometry.One-way analysis of variance was used to analyze the data and draw corresponding statistical charts. \u003cstrong\u003e(E) \u003c/strong\u003eAGS/DDP cells were transfected with OMA1 overexpression plasmid for 4-6h. Then,AGS/DDP cells were treated with OMA1 overexpression plasmid and 2.5 μg/mL cisplatin individually, or co-treated with both for 48 hours,ATP levels were detected by ATP assay kit. One-way analysis of variance was used to analyze the data and draw corresponding statistical charts. * * * P \u0026lt; 0.001; \u003csup\u003e# # \u003c/sup\u003eP \u0026lt; 0.01,\u003csup\u003e# # #\u003c/sup\u003eP \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8726309/v1/bed557ce2b7a1bb7c2c63cbb.jpeg"},{"id":104808405,"identity":"9224da39-7fe3-4897-9c3e-d9c97078de69","added_by":"auto","created_at":"2026-03-17 12:37:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3804284,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8726309/v1/32adda3c-f7bf-4ee3-98b7-0b55f0f6b72a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Baicalein improves cisplatin resistance in gastric cancer through OMA1/OPA1 axis mediated mitochondrial dynamics","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastric cancer is a fatal disease with low survival rate worldwide. It is reported that there are over one million new cases every year, and gastric cancer is the fifth largest diagnosed malignant tumor in the world\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Chemotherapy is an important means for the treatment of gastric cancer, but drug resistance often leads to treatment failure, which has become the main clinical problem of poor prognosis in patients with gastric cancer\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Therefore, re-sensitizing gastric cancer cells to chemotherapy and investigating the mechanism of drug resistance is of clinical significance.\u003c/p\u003e \u003cp\u003eMitochondria are highly dynamic organelles with constantly division, elongation and fusion to each other, forming networks or fragments\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.Mitochondrial fusion generates a tubular mitochondrial network that counteracts metabolic stress, maintains cellular integrity, and provides protection against cell death\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.In contrast, mitochondrial fission produces numerous small and fragmented mitochondria, a process linked to cellular stress or apoptotic signaling\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. In our previous studies, it was found that mitochondrial dynamics participate in cisplatin resistance in ovarian cancer, and the level of mitochondrial fusion was higher in cisplatin resistant ovarian cancer cells\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.Excessive mitochondrial fragmentation is linked to platinum sensitivity, whereas chemoresistant cells exhibit a more fused mitochondrial network.These suggest that mitochondrial fusion plays a key role in the acquisition of chemotherapy resistance.\u003c/p\u003e \u003cp\u003eMitochondrial dynamics regulate mitochondrial function through a balance of fusion and fission. The fusion process, mediated by Mitofusion 1/2 (Mfn1/2 )and Optic atrophy 1(OPA1), facilitates the exchange of mitochondrial contents and repairs damaged mtDNA, thereby maintaining oxidative phosphorylation efficiency and ATP synthesis capacity. Conversely, mitochondrial fission, driven by Dynamin-related protein 1 (Drp1)\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.Mitochondrial fusion enhances the efficiency of ATP synthesis and increases mitochondrial stability\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.However, excessive fission induced by apoptotic/stress signals produces too much reactive oxygen species (ROS), a prerequisite for apoptosis\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.Enhancing mitochondrial fission may be a promising strategy to induce apoptosis in gastric cancer cells.\u003c/p\u003e \u003cp\u003eStress-sensitive OMA1 mediates proteolytic cleavage of L-OPA1 into S-OPA1, which disrupts mitochondrial fusion and causes network fragmentation\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Mitochondrial protease OMA1 has been shown to be involved in tumor progression \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, but its role in promoting or inhibiting tumor growth has not been determined.Some studies have found that OMA1 activation increases the sensitivity of ovarian cancer to cisplatin both in vivo and in vitro. This effect may be mediated by the cleavage of OPA1, which disrupts the coordination between mitochondrial inner and outer membranes, ultimately leading to ovarian cancer cell death. Conversely, following OMA1 knockout, OPA1 levels increase and apoptosis is attenuated \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBaicalein is a natural product with antioxidant, antiviral, anti-tumor and other biological activities\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Studies have suggested that it plays an important role in the treatment of cancer \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. When combined with existing chemotherapy drugs, it can improve the therapeutic effect and reduce the toxicity of chemotherapy drugs \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The pathway and mechanism of baicalein's anti-tumor and improvement of chemotherapy resistance are still in the exploration stage. In lung cancer cells, baicalein can reduce mitochondrial membrane potential (MMP) and decrease the level of mitochondrial dynamic protein OPA1 \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhether baicalein plays a role in drug resistance of gastric cancer cells through OMA1-mediated mitochondrial dynamics remains unclear. The purpose of this study was to investigate the effect of baicalin on cisplatin resistance in AGS gastric cancer, and whether the dynamic mitochondrial function is mediated by affecting the OMA1/OPA1 axis, so as to provide new ideas and basis for the solution of chemotherapy resistance in gastric cancer.Here, we investigate the consequence of altered OMA1 expression on mitochondrial dynamics, ATP,ROS and therapeutic resistance in gastric cancer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\n\u003ch3\u003e1. Cell culture\u003c/h3\u003e\n\u003cp\u003eHuman gastric cancer AGS cells and cisplatin-resistant AGS/DDP cells were purchased from the Jinyuan Biotechnology(Shanghai, China). AGS and AGS/DDP cells were cultured in RPMI 1640 (BI, Israel) and supplemented with 1% penicillin/streptomycin (Solarbio, China) and 10% fetal bovine serum (FBS, BI, Israel) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified atmosphere.\u003c/p\u003e\n\u003ch3\u003e2. Cell viability assay\u003c/h3\u003e\n\u003cp\u003eCells were collected through pancreatic enzyme digestion and spread on 96-well cell culture plates with 5*103 cells/holes in complete medium (plates and cell numbers were selected according to specific experiments). The cells were incubated in a 37℃ temperature box overnight according to the cell adhesion conditions, and the treatment could be started after the cell adhesion was complete. Discard the old medium, add the drug-containing medium for treatment, and culture for 48h. Discard the supernatant, add 10%CCK-8(#CA1210,Solarbio, Beijing,China)detection reagent, culture for 1\u0026thinsp;~\u0026thinsp;2h, and then test with enzyme marker.\u003c/p\u003e\n\u003ch3\u003e3. Annexin V-FITC/PI apoptosis assay\u003c/h3\u003e\n\u003cp\u003eAfter the cells were treated according to the experimental requirements, the culture supernatant was discarded, washed with PBS((Solarbio, China) for 2 to 3 times, digested with pancreatic enzyme for 2 to 3 min, the reaction was terminated with cell culture medium, and cells were collected by centrifugation at 500 \u0026times;g for 5 min. Apoptosis was detected using Annexin V-FITC/PI kit(#E-CK-A211,Solarbio, Beijing,China). The cells were washed with pre-cooled PBS solution for 2\u0026ndash;3 times, centrifuged at 500 \u0026times;g for 5 min, and the supernatant was discarded and the cells were collected. Add 100\u0026micro;L pre-cooled Binding Buffer to the suspension cells, add 5\u0026micro;L An‑nexin V-FITC and 5\u0026micro;L PI solution, gently mix, avoid light at room temperature for 15 min, add 400\u0026micro;L pre-cooled binding buffer and mix well. The proportion of cells expressing Annexin V-FITC fluorescence was measured by flow cytometry, which was the apoptosis rate.Cells were analyzed using a flow cytometer equipped with a laser emitting at 488 nm and an optical filter FL1 (530/30 nm).\u003c/p\u003e\n\u003ch3\u003e4.Intracellular ROS Level Assay\u003c/h3\u003e\n\u003cp\u003eAfter treatment, the cells were digested and collected by pancreatic enzymes, centrifuged at 500\u0026times;g for 5 min, and the supernatant was discarded. DCFH-DA (#MA0219,Dalian Meilun Biotechnology Co., Ltd.,Dalian,China)was diluted in serum-free medium at 1:1000 to a final concentration of 10\u0026micro;M. After the cells were collected, they were suspended in the DCFH-DA working liquid with an appropriate volume of diluted DCFH-DA, and the cell density was 1.0\u0026times;10^6\u0026thinsp;~\u0026thinsp;2.0\u0026times;10^7/mL (500uL/ well). The cells were incubated in the cell incubator at 37℃ for 20\u0026thinsp;~\u0026thinsp;30 min in dark light, and mixed upside down every 3\u0026ndash;5 min. The probe was fully in contact with the cells, and then the cells were washed with serum-free culture solution 3 times. FITC channel was selected and samples were detected by flow cytometry.\u003c/p\u003e\n\u003ch3\u003e5. Intracellular ATP Level Assay\u003c/h3\u003e\n\u003cp\u003eThe cells of each group treated with experimental drugs were separated from the culture supernatant, and the culture supernatant was removed to obtain the bottom layer of precipitated cells. The collected cells were added to 300\u0026thinsp;~\u0026thinsp;500\u0026micro;L cold double steaming water and homogenized in an ice water bath for crushing (part of the cells were taken out for determination of protein concentration), and then the cell suspension was heated in a boiling water bath for 10 minutes, then the cells were mixed and extracted for 1 minute. The ATP levels were detected with commercial ATP assay kit (#A095-1-1, Nanjing Jiancheng Bioengineering Institute,Nanjing,China) following manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch3\u003e6.Transmission electron microscopy\u003c/h3\u003e\n\u003cp\u003eAGS/DDP and AGS cells were seeded at 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/culture dish (35mm) and treated with indicated reagents. Then cells were collected and fixed by 3% glutaraldehyde at 4\u0026deg;C followed by 1% osmium tetroxide. Next, cells were dehydrated in 30%~100% acetone, embedded with Epox 812 and cut to semithin sections. After stained with methylene bule, the ultrathin sections (60 nm\u0026thinsp;~\u0026thinsp;90 nm) were prepared by EM UC7 (Leica, Germany) and stained with lead citrate and uranyl acetate. The sections were detected by transmission electron microscope (FEI, Ltd, America).\u003c/p\u003e\n\u003ch3\u003e7.Intracellular MMP Level Assay\u003c/h3\u003e\n\u003cp\u003eAfter treating the cells according to the experimental requirements, pancreatic enzyme digestion and collection of cells, according to the JC-1(#E-CK-A301,Wuhan,China) staining kit operation is as follows: The cells to be tested in each group were added with JC-1 dye and incubated for 20 min at room temperature in the dark. After gradient washing with the buffer delivered with the kit, the cells were detected by upflow cytometry. The relative number of mitochondrial membrane potential polarization and depolarization of the cells was analyzed to reflect the changes of cell MMP.\u003c/p\u003e\n\u003ch3\u003e8.Western blot analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e8.Western blot analysis\u003c/div\u003e \u003cp\u003eAfter administration according to the above experimental groups, the proteins of the cells were extracted, the proteins were analyzed by BCA protein kit, SDS-PAGE glue, sample loading, electrophoresis, and membrane transfer were prepared, sealed with 5% skim milk powder, incubated overnight at 4℃ with primary antibody, incubated overnight at 37℃ with secondary antibody, TBST membrane washing, and then ECL light-producing liquid was added after TBST membrane washing. The protein expressions of MFN1(#13798-1-AP,proteintech), MFN2(#12186-1-AP,proteintech), Drp1( #DF7037,Affinity), OPA1 (#27733-1-AP,proteintech)and OMA1( #DF12435,Affinity) were detected in the gel imaging system, and the gray values of the bands were analyzed using Image J software for statistical analysis.\u003c/p\u003e\n\u003ch3\u003e9.OMA1 overexpression transfection\u003c/h3\u003e\n\u003cp\u003eAGS/DDP cells were transfected with OMA1 overexpression plasmid(GeneCreate,Wuhan, China).The cells were collected through pancreatic enzyme digestion, the cells were suspended in the medium, the cells were counted, the cell density was diluted to 3*10\u003csup\u003e^\u003c/sup\u003e5 cells /ml, and the 2ml holes were inoculated on the 6-well cell culture plate. The cells were incubated in a 37℃ temperature box overnight according to the cell adhesion conditions. After the cell adhesion was complete, the treatment could be started. The supernatant was discarded and replaced with 1mL basic medium. According to the transfection group, two sterile EP tubes were prepared for each group, 125ul Opti-MEM was added to each tube, 5uL lipofectamine 3000 was added to one tube, 2.5ug plasmid and 5ul lipofectamine 3000 was added to the other tube, and the tubes were gently mixed and left for 5min at room temperature. The above two EP tubes were mixed well, left at room temperature for 15min, the mixed liquid drops were placed into the corresponding holes in the six-well plate, and the cells were put back into the incubator for culture. 4-6h after transfection, 1ml complete medium containing 20% serum was added into the six-well plate. The culture plates were cultured in a cell incubator at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e for 24h, cell precipitate was collected, cleaned once with PBS, and 1mL Tranzol was added.\u003c/p\u003e\n\u003ch3\u003e10.Statistical analysis\u003c/h3\u003e\n\u003cp\u003eUsing Prism 6 statistical software, measurement data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (\u0026plusmn;\u0026thinsp;s), T-test was used for inter-group comparison, and one-way analysis of variance was used for inter-group comparison. All experiments were independently repeated 3 or more times. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\n\u003ch3\u003e1. Baicalein increased cisplatin-induced cell proliferation and apoptosis.\u003c/h3\u003e\n\u003cp\u003eCisplatin is one of the most commonly used chemotherapy drugs for gastric cancer. However, its toxic side effects greatly limit the clinical application of cisplatin. The dose of cisplatin needs to be reduced by combination with some synergist drugs. In order to investigate whether baicalein can enhance the sensitivity of cisplatin chemotherapy. First,AGS cells were treated with baicalein 20\u0026micro;mol/L, cisplatin 2.5\u0026micro;g/mL, baicalein 20\u0026micro;mol/L combined with cisplatin 2.5\u0026micro;g/mL for 48h, and the activity of the cells was detected by CCK8.As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,DDP treatment inhibited cell viability. While Baicalein had no effect on cell activity, DDP treatment in combination with Baicalein showed greater efficiency in inhibiting cell activity than treatment with DDP alone.Furthermore, cell apoptosis was examined in AGS cells treated with DDP and/or baicalein. Baicalein treatment combination with DDP efficiently promote cisplatin-induced apoptosis in AGS cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB,C).All these results indicate that baicalein may increase the sensitivity to cisplatin chemotherapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.Baicalein increased cisplatin-induced mitochondrial fission and ROS level ,while decreased the intracellular ATP level in AGC cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDisturbance of homeostasis results in ROS accumulation and ATP reduction, leading to mitochondrial damage, which is crucial in cancer progression\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. The intracellular ROS levels in AGS cells were measured by detection of the DCFH-DA staining.As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B,While Baicalein had no effect on the level of ROS, 2.5\u0026micro;g/l of cisplatin significantly increased the intracellular ROS level in AGS cells. DDP treatment in combination with Baicalein significantly increase cisplatin-induced intracellular ROS production in AGS cells.Mitochondria are the major source for ATP production. Therefore, intracellular ATP level was detected in AGS cells. Our results showed that Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Baicalein in combination with DDP significantly decreased the intracellular ATP levels in AGC cell.Mitochondrial fusion enhances the efficiency of ATP synthesis and increases mitochondrial stability\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. The results of TEM indicated that cisplatin treatment shifted mitochondrial morphology toward smaller, more rounded/oval shapes compared to the control, and this effect was further enhanced when cisplatin was combined with baicalein(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).All these results indicated that baicalein in combination with DDP impaire mitochondrial function, which might stimulated ROS accumulation and mitochondrial fission ,while prevent ATP generation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e3.Baicalein regulates the expression of mitochondrial dynamics proteins and OMA1 in cisplatin-treated AGC cells.\u003c/h3\u003e\n\u003cp\u003eOur previous research results showed that compared with AGS cells, OMA1 mRNA was low expressed in AGS/DDP cells, while OPA1 mRNA was high expressed\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD ,decreased mitochondrial fusion was detected in AGS cells upon cisplatin treatment.These suggest that OMA1and mitochondrial dynamics may be related to cisplatin resistance in GC.Thus, the mechanism of mitochondrial dynamics which baicalin reduces cisplatin resistance in AGS cells was further explored.It was found that cisplatin down-regulated the expression of MFN1,MFN2 and OPA1, which was attenuated by cisplatin in combination with baicalein( Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). In contrast, the expression of Drp1and OMA1 was significantly upregulated following cisplatin in combination with baicalein treatment, suggesting that mitochondria have undergone division( Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE,F). These results suggest that OMA1 and mitochondrial dynamics may be involved in the mechanism baicalein reverses cisplatin resistance in GC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e4.Overexpression of OMA1 stimulated cisplatin-induced mitochondrial fission in AGS/DDP cells by regulating OPA1.\u003c/h3\u003e\n\u003cp\u003eIt is reported that the activation of OMA1 can mediate the hydrolysis of OPA1 and the division of mitochondria\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.To further investigate the underlying mechanism of OMA1 mediated mitochondrial dynamics, the lentiviral plasmid was used to establish OMA1 overexpressed AGS/DDP cells.Western blot results showed that OPA1 expression was significantly decreased following OMA1overexpressed in AGS/DDP cells.After combined with cisplatin, the decrease in OPA1 level was greater than that when cisplatin was used alone,while OMA1 expression were increased(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C).Moreover, the results of TEM also indicated that compared with the control(NC), both OMA1 overexpression and cisplatin treatment alone induced a shift in mitochondrial morphology towards smaller, more rounded and oval shapes, and this effect was further enhanced when cisplatin was combined with OMA1 overexpression than cisplatin alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e5. Overexpression of OMA1 re-sensitized AGS/DDP cells to cisplatin-induced cytotoxicity.\u003c/h3\u003e\n \u003cp\u003eTo examine the role of OMA1 in cisplatin resistance, AGS/DDP cells were transfected with OMA1 overexpression plasmid or treated with cisplatin, cell viability and apoptosis assay were detected using CCK-8 assay and flow cytometry. As expected, overexpression of OMA1mediated suppression on cisplatin-induced growth inhibition and apoptosis was also significantly increased in AGS/DDP cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). In conclusion, all these results highlight the important role of OMA1/OPA1 axis-mediated mitochondrial fission in the development of cisplatin resistance in GC.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e6.OMA1/OPA1 axis enhanced cisplatin-induced cytotoxicity by mitochondrial dysfunction.\u003c/h3\u003e\n\u003cp\u003eAlthough the mitochondria are in a constant state of transition between fission and fusion, excessive mitochondrial fission leads to a decrease in metabolic function and an increase in oxidative stress\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,B,E,OMA1 overexpression reduces ATP levels and increases ROS levels in AGS/DDP cells. DDP in combination with OMA1 overexpression showed higher ROS levels and lower ATP levels than DDP treatment alone. ΔψM serves as an indirect indicator of mitochondrial ATP synthesis capability. The shape of mitochondria may directly affect biological energy production. For instance, longer mitochondria are sometimes linked to more efficient ATP production\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.We then evaluated the changes in ΔψM in mitochondria that were measured by flow cytometry using JC-1 staining. OMA1 overexpression reduces ΔψM levels in AGS/DDP cells. DDP in combination with OMA1 overexpression showed lower ψM levels than DDP treatment alone.It was also indirectly implied that OMA1overexpression caused damage to mitochondrial function.These results provide additional evidence that OMA1/OPA1 axis causes mitochondrial dysfunction via mitochondrial fragmentation. In conclusion, all these data suggest that OMA1 signaling pathway may be involved in cisplatin-induced mitochondrial dynamics, and that baicalein reduces cisplatin resistance in gastric cancer cells via the OMA1/OPA1 axis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGastric cancer represents one of the leading causes of cancer deaths worldwide. Cisplatin (DDP) is a basic chemotherapy drug for gastric cancer (GC). With the increase of DDP drug concentration in clinical treatment, cancer cells gradually became resistant. Therefore,identifying novel molecular targets and drugs capable of overcoming drug resistance is crucial for improving patient prognosis.\u003c/p\u003e \u003cp\u003eBaicalein showed anti-tumor effect on gynecological tumor, lung cancer, liver cancer and other tumor cells, and combined with chemotherapy drugs can enhance the effect of chemotherapy. The combination of baicalein and cisplatin has a synergistic effect on cervical cancer cells, which can promote cell apoptosis and inhibit cell vitality, and has the potential to improve the sensitivity of cervical cancer cells to cisplatin\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Similarly, baicalein treatment can increase the sensitivity of ovarian cancer cells to cisplatin and inhibit cell proliferation, metastasis and tumor growth\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. A number of studies have found that baicalin is an important candidate drug for the treatment of lung cancer, and its mechanism of action includes regulating cell proliferation, metastasis, apoptosis, autophagy, etc\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In addition, baicalein can induce apoptosis and autophagy of multi-drug resistant liver cancer cells and reverse multi-drug resistance of liver cancer, which may contribute to the treatment of drug-resistant liver cancer\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. In our previous study, we found that baicalin inhibited the proliferation and promoted apoptosis of gastric cancer resistant cells AGS/DDP through the mitochondrial pathway\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Here, baicalein combined with cisplatin can significantly increase the proliferation inhibition and induce apoptosis of AGS gastric cancer cells, which is consistent with the results of previous studies showing that baicalein enhances the chemotherapy sensitivity of tumor cells, providing further supporting evidence for the anti-tumor treatment of baicalein, especially in the treatment of gastric cancer.\u003c/p\u003e \u003cp\u003eAt present, the mechanism of baicalein enhancing tumor chemotherapy sensitivity has not been determined. Studies have shown that baicalein enhances the sensitivity of cervical cancer cells to cisplatin through the Akt pathway \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, improves the drug resistance of ovarian cancer by regulating CirSLC7A6 \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, and enhances the sensitivity of triple-negative breast cancer MDA-MB-231 cells to adriamycin through autophagy mediated down-regulation of CDK1\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Studies have also confirmed that baicalein inhibits the glutamine-mtor metabolic pathway, induces apoptosis of lung cancer cells\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, and triggers siderptosis of colorectal cancer cells by blocking the JAK2/STAT3/GPX4 axis\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. In gastric cancer cells, baicalin can induce apoptosis and autophagy by regulating miR-7/FAK/AKT\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, Akt/mTOR and Nrf2/ Keap1 pathways \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, thereby enhancing cisplatin sensitivity of gastric cancer cells. Studies have found that microRNA is a key factor in the cisplatin resistance mechanism of gastric cancer\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e, but interestingly, microRNA may enhance the sensitivity of cisplatin chemotherapy by promoting threadgrain fission \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.In recent years, researchers have observed that tumor cells manipulate mitochondrial dynamics to acquire proliferation and survival advantages \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. It has been reported that baicalein can resensitize tamoxifen-resistant breast cancer cells by inhibiting hypoxia-inducing factor-1 α, reducing aerobic glycolysis and reversing mitochondrial dysfunction\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. When the morphology of mitochondria tends to be prolonged and networked, it is beneficial to maintain the normal function of mitochondria, such as enhancing energy metabolism and reducing the excessive production of reactive oxygen species (ROS). In order to confirm that baicalein can improve the sensitivity of tumor chemotherapy through mitochondrial pathway, cisplatin combined with baicalein was applied to gastric cancer cells, and it was found that mitochondrial division increased,ROS production increased, and ATP decreased, indicating that baicalein affects mitochondrial dynamics and damages mitochondrial function in the process of anti-tumor cells.\u003c/p\u003e \u003cp\u003eThe morphology and ultrastructure of mitochondria are regulated by mitochondrial shaping proteins,and the dynamic changes of mitochondrial shape can in turn control mitochondrial function\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. OPA1, Mfn1, and Mfn2 regulate mitochondrial fusion, and Drp1 is the main protein regulating division \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. In lung cancer cells, baicalein activates AMPK by activating phosphorylated adenylate, and then promotes the phosphorylation of mitochondrial fission protein Drp1, inducing mitochondrial division and apoptosis\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. We determined proteins related to mitochondrial dynamics and found that mitochondrial fusion protein decreased and fission protein increased in gastric cancer cells, which further confirmed that baicalin reshaps mitochondrial dynamics by affecting mitochondrial regulatory proteins.OPA1 plays a crucial role in the dysregulation of key processes such as apoptosis and cell proliferation in cancer by directly controlling mitochondrial biology\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. In unbiased screening of the Venetoclaxresistance gene, it was found that OPA1 was up-regulated, and its deletion restored the sensitivity of cells to Venetoclax\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, suggesting that OPA1 may be a key target for overcoming resistance. In this study, OPA1 was significantly down-regulated after baicalein combined with cisplatin treatment of gastric cancer cells, and the sensitivity of gastric cancer cells to cisplatin was enhanced.Thus, we sought to identify upstream regulators of OPA1 to further clarify the precise mechanism through which baicalein sensitizes gastric cancer cells to cisplatin.\u003c/p\u003e \u003cp\u003eIt is well-known that OMA1 is the protease responsible for cleaving and regulating OPA1.Some studies have found that OMA1 activation increases the sensitivity of ovarian cancer to cisplatin in vivo and in vitro, possibly through the cleavage of OPA1, coordinating the inner and outer membrane of mitochondria, and ultimately inducing ovarian cancer cell death, while the OPA1 increases and apoptosis is reversed after OMA1 knockout \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. However, OMA1 can also promote the development of colorectal cancer by enhancing glycolysis and suppressing OXPHOS in colorectal cancer cells\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e.Therefore, we overexpressed OMA1 in AGS/DDP cells and found that OPA1 expression decreased, and mitochondrial fusion was reduced.OMA1 overexpression treatment was performed on AGS/DPP cells of gastric cancer, and it was found that overexpression of OMA1 combined with cisplatin treatment inhibited proliferation, induced apoptosis, and impaired mitochondrial function of AGS/DPP cells. In conclusion, our results indicate that baicalin may overcome cisplatin resistance in GC cells by OMA1/OPA1 axis-mediated mitochondrial dynamics.\u003c/p\u003e \u003cp\u003eIn summary, this study found that baicalein plays an important role in the cisplatin resistance of AGS gastric cancer. Baicalein, as a natural product, has the advantages of relatively low toxicity and wide sources, which is expected to become a new choice for adjuvant cisplatin chemotherapy, providing a new theoretical basis and potential treatment strategy for solving drug resistance in gastric cancer. In addition, the combined application of baicalein and cisplatin may reduce the clinical dose of cisplatin, reduce the toxic side effects of cisplatin while ensuring the therapeutic effect, and improve the quality of life of patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWe declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere.We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.We understand that the Corresponding Author is the sole contact for the Editorial process. She is responsible for communicatine with the other authors about progress, submissions of revisions and final approval ot proofs.All authors as follows：Chunmei Yu (First Author):Conceptualization,Funding Acquisition, Resources,Supervision,Experimental verification;Chunxia Yu: Wrote the main manuscript text,Data Curation,Formal Analysis, Investigation, Software;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSmyth EC, Nilsson M, Grabsch HI et al (2020) Gastric cancer[J] Lancet 396(10251):635\u0026ndash;648\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMora-Lagos B, Cartas-Espinel I, Riquelme I et al (2020) Functional and transcriptomic characterization of cisplatin-resistant AGS and MKN-28 gastric cancer cell lines[J]. 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EMBO Rep 22(1):e50827\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Baicalein, Gastric cancer, Cisplatin resistance, Mitochondrial dynamics, OMA1, OPA1","lastPublishedDoi":"10.21203/rs.3.rs-8726309/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8726309/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eResistance to chemotherapy represents a major obstacle to the successful treatment of gastric cancer (GC). This study aimed to investigate the role and underlying mechanism of baicalein in cisplatin resistance in gastric cancer through the OMA1/OPA1 axis.Baicalein treatment significantly increased mitochondrial fission and ROS levels in AGS cells, leading to reduced ATP production and consequently enhancing cisplatin-induced cytotoxicity in gastric cancer cells.Intriguingly,OMA1 expression was most significantly increased upon combined treatment with baicalein and cisplatin in AGS cells.In contrast,OPA1 expression was significantly reduced.Mechanistically, we define a signaling axis linking OMA1 to OPA1 and mitochondrial fission in Gastric cancer. Moreover, overexpression of OMA1 in AGS/DDP cells also significantly increased mitochondrial fission and ROS levels, decreased OPA1 protein expression and mitochondrial membrane potential, and reduced ATP production.Consistently, the cell viability and cell apoptosis assay showed that OMA1 overexpression synergistically enhancing cisplatin sensitivity in AGS/DDP cells.Taken together, all data in this study indicate that baicalein improves cisplatin resistance in gastric cancer through OMA1/OPA1 axis mediated mitochondrial dynamics,and repurpose baicalein as a potential agent to inhibit cisplatin resistance in GC.Furthermore,improve our knowledge about how OMA1 regulates mitochondria and provide justification for combining OMA1 overexpression with chemotherapy in GC.\u003c/p\u003e","manuscriptTitle":"Baicalein improves cisplatin resistance in gastric cancer through OMA1/OPA1 axis mediated mitochondrial dynamics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-12 20:24:37","doi":"10.21203/rs.3.rs-8726309/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-01T20:43:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T06:56:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-16T07:56:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-12T00:53:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326904523177896665115813892012210133582","date":"2026-03-11T23:57:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"116237969094244149721151512191733885519","date":"2026-03-11T03:16:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174296287359859885887581298966468030928","date":"2026-03-09T23:46:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-09T22:41:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-09T16:22:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-07T05:53:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Biochemistry","date":"2026-01-29T02:39:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"56d53fa2-5fbe-4cd2-af7c-b6e7b6ec0b86","owner":[],"postedDate":"March 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T15:38:34+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-12 20:24:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8726309","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8726309","identity":"rs-8726309","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}