Indirubin inhibits cervical cancer by inhibiting apoptosis and autophagy through the PI3K/AKT and MAPK pathways

Indirubin inhibits cervical cancer by inhibiting apoptosis and autophagy through the PI3K/AKT and MAPK pathways | 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 Article Indirubin inhibits cervical cancer by inhibiting apoptosis and autophagy through the PI3K/AKT and MAPK pathways Xiuchao Xie, Chang Liu, Xiaoling Liu, Lin Hou, Ya Zhang, Lin Huang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7541331/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract This study investigated the effects of indirubin on autophagy and apoptosis in cervical cancer models, with a focus on elucidating the underlying molecular mechanisms. A xenograft tumor model in BALB/c-Nude mice and in vitro experiments using HeLa cell lines were employed. Indirubin treatment demonstrated concentration-dependent inhibition of cervical cancer cell growth, accompanied by induction of apoptosis and autophagy. Mechanistic analysis revealed that indirubin exerted its effects through dual modulation of the PI3K/AKT signaling axis. It enhanced autophagic flux, as evidenced by increased LC3-II/LC3-I ratio and decreased P62 expression. Concurrently, indirubin induced mitochondrial apoptosis through upregulation of pro-apoptotic Bax expression and downregulation of anti-apoptotic Bcl-2 levels. These coordinated molecular alterations ultimately led to programmed cell death execution. Additionally, indirubin inhibited the activation of the MEKK1/SEK1/JNK/AP-1 signaling pathway, further contributing to its anti-tumor effects. These findings suggest that indirubin inhibits cervical cancer cell growth by regulating the PI3K/AKT pathway, enhancing autophagy, and promoting apoptosis, providing a theoretical basis for its therapeutic potential in cervical cancer treatment. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Drug discovery Health sciences/Oncology Indirubin Cervical cancer Hela cells PI3K/AKT MAPK Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Cervical cancer, also known as uterine cervix carcinoma, is a severe tumor primarily affecting the cervical area, and it stands as the second most frequently diagnosed cancer in the female population across the world 1 . This condition represents a significant peril to the health of women worldwide, attributed to its substantial prevalence and fatality statistics. As reported by the World Health Organization (WHO) in 2022, approximately 660,000 fresh incidents and 350,000 fatalities due to cervical cancer were recorded worldwide. The main cause of cervical cancer is HPV, particularly HPV16 and HPV18. Women who fall into high-risk categories for cervical cancer are those who have engaged in sexual activity with numerous partners, early onset of sexual activity, and immunodeficiency 2 . In the early stages, cervical cancer may not present with noticeable symptoms. However, as the disease progresses, symptoms such as vaginal bleeding, abnormal vaginal discharge, and tumor invasion of surrounding tissues or organs may occur 3 . Current treatment modalities for cervical cancer encompass surgical resection, chemotherapy, and radiotherapy 4 . Nevertheless, these strategies exhibit limited efficacy in patients with advanced-stage disease, and the adverse effects associated with chemotherapy agents further constrain their clinical applicability 5 . In recent years, traditional Chinese medicine has demonstrated clear benefits in mitigating symptoms associated with cancer and lowering the likelihood of recurrence following surgical intervention, and mitigating the toxic side effects and drug resistance associated with radiotherapy 6 – 8 . Indirubin, the principal active constituent of the traditional Chinese medicine indigo naturalis, demonstrates promising potential for antitumor applications 9 . However, the underlying mechanisms of its action against cervical cancer remain poorly understood. To date, only a study conducted by Wu Qiwei has indicated that indirubin inhibits the proliferation of cervical cancer cells 10 , highlighting the necessity for further investigation into its mechanisms of action. The PI3K/AKT signaling pathway is crucial for cancer initiation and progression. It impacts various cellular functions, such as viability, growth, differentiation, motility, and metabolism 11 . Abnormal activation of PI3K/AKT signaling is commonly observed in cancer and promotes tumor occurrence and progression. In cervical cancer, abnormal activation of the PI3K/AKT signaling pathway links closely to tumor cell proliferation, migration, and invasion 12 . Moreover, research shows targeting the mTOR/PI3K/AKT signaling pathway can trigger apoptosis and autophagy in cervical cancer cells, suppressing tumor growth 13 . Multiple inhibitors for the PI3K/AKT pathway have been developed, such as pan - PI3K inhibitors, subtype - specific PI3K inhibitors, dual PI3K/mTOR inhibitors, and Akt inhibitors. These pharmacological agents act on critical nodes within the PI3K/AKT pathway, effectively impeding the growth and proliferation of tumor cells 14 . Additionally, the PI3K/AKT/mTOR pathway is significantly associated with the pathogenesis of gastric cancer, where its activation inhibits apoptosis, accelerates cell cycle progression, promotes angiogenesis, and contributes to the invasion and metastasis of gastric cancer 15 . In the treatment of gynecological malignant tumors, activation of the PI3K/AKT signaling pathway is implicated in enhanced proliferation and metastasis of tumor cells and induces treatment resistance 16 . Therefore, drug targets within this pathway presents a promising avenue for effective clinical interventions in cancer therapy. Autophagy, a critical mechanism for maintaining protein homeostasis and organelle integrity, exhibits a complex and multifaceted role in cancer. It can both prevent early tumor development and maintain and adapt to the metabolic adaptation in established and metastatic tumors 17 . The MEKK1/SEK1/JNK/AP-1 signaling axis, as a core stress response pathway, plays a dual role in cell fate determination. Under the background of stress and apoptosis, continuous JNK activation can induce the expression of pro-apoptotic genes by phosphorylating the transcription factor AP-1, thereby exerting its classic tumor suppressive function. However, this pathway also has cancer-promoting potential. Its abnormal activation can promote the survival, invasion and metastasis of tumor cells, showing a significant cancer-promoting phenotype. In addition, this pathway is also deeply involved in inflammatory regulation, and its activation can drive the expression of various pro-inflammatory factors. Given its core position in the regulation of cellular stress, inflammation and apoptosis, targeting the key nodes of the MEKK1/SEK1/JNK/AP-1 pathway has become an important direction for developing new therapeutic strategies for various pathological conditions such as cancer and inflammatory diseases 18 – 19 . This study was initiated to investigate the therapeutic potential of indirubin in a nude mouse model of cervical cancer. We specifically focused on elucidating the molecular mechanisms by which indirubin modulates autophagy and apoptosis in cervical cancer cells through the PI3K/AKT and MAPK signaling pathways. The aim of this research is to provide a deeper understanding of indirubin’s anti-cancer actions and to establish a novel mechanistic basis for its potential application in the treatment of cervical cancer. Materials and methods Cell lines and animals Hela cells (Cyagen, Shanghai) were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a 5% CO₂ incubator. U14 mouse cervical cancer cells (Cyagen, Shanghai) were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a 5% CO₂ incubator. A total of twenty-one female BALB/c-Nude mice, classified as Specific Pathogen-Free (SPF) grade, were acquired from Shanghai BiKaikeyi Biotechnology Co., Ltd. on July 7, 2023. The mice, which weighed between 18 and 20 grams, were accompanied by two additional spare specimens. The acquisition was conducted under license number SCXK(Shanghai)2018-0006 and certificate number 20180006050891. All animal procedures were approved by the Ethics Committee of Laboratory Animals of Hubei Bainite Biotechnology Co., LTD. (Ethics Number: IACUC-BNT-2023-007). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.At the endpoint of the experiment, mice were anesthetized with 3–4% isoflurane in oxygen until loss of consciousness was achieved, followed by maintenance at 1.5–2% isoflurane. Once deeply anesthetized, euthanasia was performed via cervical dislocation by trained personnel to ensure rapid and humane death, in accordance with the AVMA Guidelines for the Euthanasia of Animals. Immediately following euthanasia, blood samples were collected via cardiac puncture for subsequent analysis. Tumors were then carefully excised, photographed for morphological documentation, and weighed using an analytical balance. Finally, tumor tissues were snap-frozen in liquid nitrogen and stored at − 80°C for future molecular analysis. All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) and conducted in strict compliance with the national guidelines for the care and use of laboratory animals. Mice model The right forelimb of each mouse was disinfected and inoculated with 0.1 mL of U14 cell suspension (5 million cells) using an insulin syringe. Tumor growth in mice was monitored daily, with body weight and tumor dimensions recorded every 2–3 days to calculate tumor volume 20 (V = 0.5 × a × b²). Control mice were not inoculated. Reagents and instruments Indirubin (MCE, Shanghai, China), DMEM medium (SEVEN, Beijing, China), PBS (Life, Shanghai, China), TsingZol Total RNA Extraction Reagent (TSINGKE, Beijing, China), RIPA Lysis Buffer (Beyotime, Shanghai, China). Inverted phase contrast microscope (Olympus, Beijing, China), Desktop high-speed refrigerated microcentrifuge (DLAB, Beijing, China), Analytical flow cytometer (Beckman, Fullerton, United States), Real-time fluorescence quantitative PCR instrument (ABI, Foster City, United States), Gel imaging system (Bio-rad, California, United States). Method of experiment When the average tumor volume reached 50 mm³, mice were grouped (three per group) and treated daily with various drugs: IR (10, 20, 40 mg/kg), DDP (5 mg/kg), or IFO (40 mg/kg). Body weight and tumor dimensions were monitored every 2–3 days for growth curves 21 . Treatment ceased when the positive control showed efficacy. When the experiment was terminated, anesthetized mice were subjected to tumor-bearing blood collection, tumor-stripping tissue photo collection, tumor tissue weighing, and tumor tissue preservation at -80 degrees. Tumor tissues of mice were taken for Q-PCR and Western Blot to detect the expression of genes and proteins. Cell Counting Kit-8 (CCK-8) assay Hela cells, after PBS wash and 1–3 min trypsinization, were centrifuged, resuspended, and counted with trypan blue. They were then seeded at 7,000 per well in a 96-well plate with blanks. Post CCK-8 addition, the plates were incubated for 3 hours and read at 450 nm using a microplate reader 22 . The cells were subjected to treatment using the suitable drug concentrations according to the experimental groups. Group 1: Hela cells Hela cells + IR (0, 1.25, 2.5, 5, 10, 20, 40, 80 µmol/L) Measured cell viability at 24, 48, 72 h post drug treatment. Group 2: Hela cells Hela cells + IR (low, medium, high dose) Hela cells + DDP Hela cells + IFO Cell viability was assessed according to the optimal time and concentration established in the preceding experiment. Flow Cytometry Analysis Cells were harvested, washed, digested, and centrifuged, then resuspended and counted 23 . Following seeding of 3.5 × 10⁵ cells per well in 6-well plates. Cells were then treated according to experimental groups and incubated as specified. Groups: Hela cells Hela cells + IR (5 µmol/L) Hela cells + IR (20 µmol/L) Hela cells + IR (80 µmol/L) Hela cells + DDP (25 µmol/L) Hela cells + IFO (4000 µmol/L) Cells were collected for further analysis after 48 h of drug treatment. Apoptosis Detection Cells were harvested via trypsinization, centrifuged at 200 g for 5 minutes at 4°C, and rinsed twice with pre-cooled PBS. After resuspending in 100 µL Binding Buffer with Annexin FITC and PI, the sample was incubated in the dark for 15 minutes, then mixed with an additional 300 µL Binding Buffer. Analyzed via flow cytometry within one hour while kept on ice 24 . Quantitative real-time PCR Total RNA of hela cells was extracted using Trizol and reverse transcribed into cDNA using 5×SynScriptTM RT SuperMix. The expressions of MEKK1, AP-1, JNK1, SEK1 genes in mouse tumor tissues and LC3, P62, PI3K, AKT, BCL2, Bax and GAPDH in cells were detected by multiplex real-time RT-PCR on the LightCycler®480 system 23 . Western blotting Rinsed hela cells in cold PBS were lysed in PMSF-buffer, centrifuged, and supernatant protein quantified by BCA. Samples were denatured, cooled, and stored at -20°C. Proteins were separated by SDS-PAGE based on molecular weight and analyzed by Western Blot using specific antibodies for MEKK1, AP-1, JNK1, SEK1, LC3, p62, PI3K, p-PI3K, AKT, p-AKT, BCL2, BAX, and GAPDH 24 . Statistical Analysis Graphs were generated using GraphPad Prism 8.0.2, with data shown as mean ± SD. Data handling and analysis were conducted with SPSS 19.0, and statistical significance was determined by one-way ANOVA ( P < 0.05). Results Indirubin inhibits the growth of U14 cervical cancer in the body and regulates the tumor microenvironment Indirubin significantly inhibited cervical cancer growth without affecting mouse weight (Fig. 1 A). Tumor weight (Fig. 1 B) and volume (Fig. 1 C) decreased dose-dependently with indirubin (10, 20, 40 mg/kg), with the most significant effect at 40 mg/kg. The results demonstrate indirubin’s efficacy in suppressing tumor growth in vivo. The specific data show that the model group had the highest tumor weight (0.8 g), which decreased to 0.68 g, 0.52 g, and 0.12 g for indirubin doses of 10, 20, and 40 mg/kg, respectively. The tumor volume in the model group (852.7 mm³) reduced to 452.4 mm³, 262.0 mm³, and 73.1 mm³ for the same doses. The most pronounced effects were observed at 40 mg/kg indirubin. Figure 1 D shows the flow cytometry analysis of CD4 + CD25 + Foxp3 + . It can be found that the proportion of the indirubin treatment group and the positive control group increased, indicating that indirubin treatment increased the proportion of regulatory T cells (Treg). Treg cells can reduce inflammatory responses by inhibiting the activation and proliferation of effector T cells 25 – 26 . These indicate that indirubin may inhibit the occurrence of cervical cancer by improving the tumor microenvironment. Indirubin inhibits the proliferation of Hela cells and induces apoptosis in vitro The CCK-8 assay (Fig. 2 A) showed that indirubin reduced hela cell viability in a time- and concentration-dependent manner. Higher concentrations (25 and 50 µmol/L) significantly decreased viability, with stronger effects at 48 and 72 hours compared to 24 hours. In Fig. 2 B, after 48-hour treatment, indirubin reduced viability dose-dependently (83.08% at 5 µmol/L, P < 0.05; 52.06% at 20 µmol/L, P < 0.01; 25.48% at 80 µmol/L, P < 0.001). Controls DDP (44.60%, P < 0.001) and IFO (49.28%, P < 0.001) also significantly decreased viability. Figure 2 C shows that indirubin increases apoptosis in hela cells in a dose-dependent manner, from 13.85% in controls to 65.49% at 80 µmol/L, indicating its potential to inhibit cervical cancer cell growth. Studies by Shi et al. and Wu et al. support indirubin’s role in promoting apoptosis and inhibiting proliferation in various cancer cells, including hela 27 – 28 . Indirubin exerts anti-tumor effects in vitro by regulating autophagy and apoptosis-related proteins Q-PCR results (Fig. 3 C) indicate that 20 µmol/L and 80 µmol/L indirubin significantly upregulated LC3 mRNA and downregulated P62 mRNA in hela cells, suggesting autophagy activation. This may facilitate the removal of damaged components and inhibit proliferation. Indirubin reduced anti-apoptotic BCL2 expression in all groups, most significantly at 20 µmol/L and 80 µmol/L, potentially enhancing apoptosis 29 – 30 . Concurrently, pro-apoptotic Bax was upregulated at these same concentrations 31 . Furthermore, indirubin decreased AKT and PI3K mRNA levels at 20 µmol/L and 80 µmol/L, suggesting PI3K/AKT pathway inhibition and apoptosis promotion 32 . These findings confirm indirubin induces autophagy and apoptosis in hela cells, likely via PI3K/AKT modulation. Western Blot (Fig. 3 B) showed 80 µmol/L indirubin increased the LC3-II/LC3-I ratio and decreased P62, indicating enhanced autophagy 33 . It also reduced p-PI3K/PI3K and p-AKT/AKT ratios, especially at 80 µmol/L, suggesting PI3K/AKT pathway suppression. Higher IR doses decreased BCL2 and increased BAX, pointing to apoptosis induction 34 . These results confirm protein changes in IR-treated hela cells, demonstrating indirubin modulates autophagy, inhibits the PI3K/AKT pathway, and induces apoptosis, particularly at higher concentrations. Indirubin inhibits the activation of the MEKK1/SEK1/JNK/AP-1 signaling pathway To elucidate the molecular mechanism by which indirubin exerts anti-tumor effects in cervical cancer, we examined the expression and activation status of key components in the MEKK1/SEK1/JNK/AP-1 signaling pathway in U14 tumor tissues from nude mice. Western blotting (Fig. 4 A) revealed that indirubin treatment dose-dependently reduced protein levels of phosphorylated AP-1, JNK1, SEK1, and MEKK1 compared to the model group. Quantitative analysis (Fig. 4 B) further confirmed significant downregulation of these proteins at both low (10 mg/kg) and high (40 mg/kg) doses of indirubin. Notably, the positive control drugs cisplatin (DDP, 5 mg/kg) and ifosfamide (IFO, 40 mg/kg) also suppressed pathway activation, consistent with indirubin’s effect. At the mRNA level, qRT-PCR results (Fig. 4 C) demonstrated that indirubin significantly decreased the transcriptional expression of MEKK1, SEK1, JNK1, and AP-1 in a dose-dependent manner. These data collectively indicate that indirubin potently inhibits the MEKK1/SEK1/JNK/AP-1 signaling cascade, suggesting this pathway as a key target mediating its anti-tumor activity in cervical cancer. Discussion The present study provides compelling evidence that indirubin, a natural compound extracted from indigo naturalis, exhibits significant anti-tumor activity against cervical cancer both in vivo and in vitro. The observed growth inhibition, apoptosis induction, and autophagy activation suggest that indirubin holds promise as a novel therapeutic agent for cervical cancer treatment. Our findings demonstrate that indirubin exerts its anti-tumor effects primarily through modulation of the PI3K/AKT signaling pathway. By downregulating key components of this pathway, indirubin effectively inhibits cancer cell proliferation and survival, leading to increased apoptosis and autophagy. This is consistent with previous studies demonstrating the critical role of the PI3K/AKT pathway in cancer development and progression 35 – 37 . Furthermore, our study reveals that indirubin also targets the MEKK1/SEK1/JNK/AP-1 signaling pathway, further contributing to its anti-tumor activity. Inhibition of this pathway has been shown to suppress tumor growth, promote apoptosis, and reduce inflammation in various cancer types. It is important to note that indirubin treatment did not significantly affect the body weight of mice, indicating its potential safety profile. This is a crucial advantage over conventional chemotherapy drugs, which often cause severe side effects and limit their clinical applicability. Conclusion In conclusion, this study demonstrates that indirubin inhibits cervical cancer cell growth by regulating the PI3K/AKT and MAPK signaling pathways, enhancing autophagy, and promoting apoptosis. These findings provide a theoretical basis for the therapeutic potential of indirubin in cervical cancer treatment and offer new insights for the development of novel anti-cancer strategies. Further investigation, including clinical trials, is warranted to explore the clinical applicability of indirubin in the treatment of cervical cancer. Declarations Competing interests The authors declare no competing interests. Institutional review board statement This study was approved by the Ethics Committee of Laboratory Animals of Hubei Bainite Biotechnology Co., LTD. (Ethics Number: IACUC-BNT-2023-007). All participants were provided informed consent and signed a document. Funding This work was supported by Project of Sichuan Provincial Administration of Traditional Chinese Medicine (No. 2021MS479) & Scientific Research Project of Sichuan Provincial Maternal and Child Health Association (No. 2024FX24) of China. Author Contribution X.X., C.L., designed the experiments and drafted the manuscript. X.L., L.H., investigated the literature. Y.Z., L.H., W.P., analyzed the data. Y.L., reviewed and edited. All authors approved the final version of the manuscript for publication. Data Availability All data generated or analysed during this study are included in this published article [and its supplementary information files]. References Zhou, J. et al. Chrysotoxine regulates Ferroptosis and the PI3K/AKT/mTOR pathway to prevent cervical cancer. J. Ethnopharmacol. 338 (P3), 119126 (2024). Peng, C. et al. A literature review on signaling pathways of cervical cancer cell death-apoptosis induced by Traditional Chinese Medicine. J. Ethnopharmacol. 334 , 118491 (2024). Florence, G. et al. Global and regional estimates of orphans attributed to maternal cancer mortality in 2020. Nat. Med. 28 (12), 2563–2572 (2022). Bray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 74 (3), 229–263 (2024). Fernandes, A. et al. Human papillomavirus-independent cervical cancer. Int. J. Gynecol. Cancer . 32 (1), 1–7 (2021). Burmeister, C. et al. Cervical cancer therapies: current challenges and future perspectives. Tumour Virus Res. 13 , 200238–200238 (2022). Yadav, N., Deshmukh, R. & Mazumder, R. A comprehensive review on the use of traditional Chinese medicine for cancer treatmen. Pharmacol. Research-Modern Chin. Med. 11100423 (2024). Mayadev, J. S. et al. Global challenges of radiotherapy for the treatment of locally advanced cervical cancer. Int. J. Gynecol. Cancer . 32 (3), 436–445 (2022). Zhang, X. et al. The positive role of traditional Chinese medicine as an adjunctive therapy for cancer. Biosci. Trends . 15 (5), 283–298 (2021). Yang, L. et al. Pharmacological properties of indirubin and its derivatives. Biomed. Pharmacother. 151 , 113112–113112 (2022). Wu, Q. Study on the Inhibitory Effect of Indirubin, an Active Component of Traditional Chinese Medicine, on Tumor Cells (Master dissertation). Beijing University of Chinese Medicine, Beijing (2007). Khezri, M. R. et al. The PI3K/AKT signaling pathway in cancer: Molecular mechanisms and possible therapeutic interventions. Exp. Mol. Pathol. 127 , 104787–104787 (2022). Glaviano, A. et al. PI3K/AKT/mTOR signaling transduction pathway and targeted therapies in cancer. Mol Cancer. 22(1), 138–138(2023). (2023). Rascio, F. et al. The Pathogenic Role of PI3K/AKT Pathway in Cancer Onset and Drug Resistance. Updated Rev. Cancers . 13 (16), 3949–3949 (2021). Saad, K. A. et al. PI3K/Akt/mTOR Pathways Inhibitors with Potential Prospects in Non-Small-Cell Lung Cancer. J. Environ. Pathol. Toxicol. Oncol. 41 (4), 185 – 102(2022). Yu, W. et al. ACTL8 Promotes the Progression of Gastric Cancer Through PI3K/AKT/mTOR Signaling Pathway. Dig. Dis. Sci. 1–13 (2024). Wang, L. et al. TYMS Knockdown Suppresses Cells Proliferation, Promotes Ferroptosis via Inhibits PI3K/Akt/mTOR Signaling Pathway Activation in Triple Negative Breast Cancer. Cell. Biochem. Biophys. 82 (3), 2717–2726 (2024). Su, L. et al. Xihuang pill promotes apoptosis of Treg cells in the tumor microenvironment in 4T1 mouse breast cancer by upregulating MEKK1/SEK1/JNK1/AP-1 pathway. Biomed Pharmacother, 1021111-1119(2018). (2018). Song, L. et al. Resveratrol inhibits autophagy against myocardial ischemia-reperfusion injury through the DJ-1/MEKK1/JNK pathway. Eur. J. Pharmacol. , 951175748–951175748 (2023). Li, Q. et al. TRPV2 calcium channel promotes breast cancer progression potential by activating autophagy. Cancer Cell. Int. 24 (1), 324–324 (2024). Qin, X. et al. Anti-tumour and radiosensitising effects of PARP inhibitor on cervical cancer xenografts. J. Obstet. Gynaecol. 43 (1), 2171783–2171783 (2023). Tao, H. et al. Galaxamide alleviates cisplatin-induced premature ovarian insufficiency via the PI3K signaling pathway in HeLa tumor-bearing mice. BMC Cancer . 24 (1), 1060–1060 (2024). Ying, H. et al. Development of an optimal protocol for isolation and purification of human granulosa cells in patients with different ovarian reserves. Exp. Ther. Med. 22 (3), 938–938 (2021). Lordon, B. et al. Impact of trypsin on cell cytoplasm during detachment of cells studied by terahertz sensing. Biophys. J. 123 (16), 2476–2483 (2024). Li, Z. H. et al. Indirubin exerts anticancer effects on human glioma cells by inducing apoptosis and autophagy. AMB Express . 10 (1), 171–171 (2020). Youngmu, K. et al. RNF2 regulates Wnt/ß-catenin signaling via TCF7L1 destabilization. Sci. Rep. 13 (1), 19750–19750 (2023). Shi, H., Ding, K. & Zhang, X. Research Progress on the Apoptosis Mechanism of Traditional Chinese Medicine Active Components Affecting Cervical Cancer HeLa Cells. Global Traditional Chin. Med. 8 (01), 124–127 (2015). Wu, Q. et al. Study on the Inhibitory Effect of Indirubin on Tumor Cells and the Related Mechanisms. Tianjin J. Traditional Chin. Med. (01), 55–58 (2008). Wong, T. et al. Expression of LC3A, LC3B and p62/SQSTM1 autophagy proteins in hepatocellular carcinoma (HCC) tissues and the predicted microRNAs involved in the autophagy-related pathway. J. Mol. Histol. 55 (3), 317–328 (2024). Danish, F. et al. Investigating the Association between the Autophagy Markers LC3B, SQSTM1/p62, and DRAM and Autophagy-Related Genes in Glioma. Int. J. Mol. Sci. 25 (1) (2024). Ljubiša, J. et al. Prognostic relevance of autophagy-related markers p62, LC3, and Beclin1 in ovarian cancer. Croat Med. J. 63 (5), 453–460 (2022). Motie, M. F., Howyzeh, S. M. & Ghanbariasad, A. Synergic effects of DL-limonene, R-limonene, and cisplatin on AKT, PI3K, and mTOR gene expression in MDA-MB-231 and 5637 cell lines. Int. J. Biol. Macromol. 280 (P4), 136216–136216 (2024). Ma, Y. M. & Xu, W. Tanshinone reverses androgen deprivation-induced invasion of prostate cancer cells by suppressing PI3K and AKT signaling pathways. Zhonghua nan ke xue = National. J. Androl. 30 (2), 104–110 (2024). Song, C. et al. H2S alleviated sepsis-induced acute kidney injury by inhibiting PERK/Bax-Bcl2 pathway. Nitric Oxide . 15 , 211–218 (2024). Lin, L. et al. NLRC5 promotes tumorigenesis by regulating the PI3K/AKT signaling pathway in cervical cancer. Sci. Rep. 14 (1), 15353 (2024). Wang, S. et al. NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling. Cell Death Dis. 15 (8), 565–565 (2024). Chen, J. et al. MYBL2 promotes cell proliferation and inhibits cell apoptosis via PI3K/AKT and BCL2/BAX/Cleaved-caspase-3 signaling pathway in gastric cancer cells. Sci. Rep. 15 (1), 9148 (2025). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 13 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 12 Nov, 2025 Reviews received at journal 11 Nov, 2025 Reviews received at journal 10 Nov, 2025 Reviewers agreed at journal 05 Nov, 2025 Reviewers agreed at journal 03 Nov, 2025 Reviewers invited by journal 02 Nov, 2025 Editor assigned by journal 23 Oct, 2025 Editor invited by journal 01 Oct, 2025 Submission checks completed at journal 15 Sep, 2025 First submitted to journal 15 Sep, 2025 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-7541331","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":543108585,"identity":"391b2811-e56f-402b-a94a-eff22efb3a12","order_by":0,"name":"Xiuchao Xie","email":"","orcid":"","institution":"The First People's Hospital of Suining","correspondingAuthor":false,"prefix":"","firstName":"Xiuchao","middleName":"","lastName":"Xie","suffix":""},{"id":543108589,"identity":"15225916-dc53-4cb2-9e83-0e467605b7fb","order_by":1,"name":"Chang Liu","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Chang","middleName":"","lastName":"Liu","suffix":""},{"id":543108590,"identity":"35afa60d-6e69-4c55-9afc-4606b3b07e98","order_by":2,"name":"Xiaoling Liu","email":"","orcid":"","institution":"The First People's Hospital of Suining","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Liu","suffix":""},{"id":543108592,"identity":"e24950f5-7e26-4a88-9eda-f365c15daca5","order_by":3,"name":"Lin Hou","email":"","orcid":"","institution":"The First People's Hospital of Suining","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Hou","suffix":""},{"id":543108594,"identity":"239fdbc2-0a0f-4a1e-8b9a-1746f81fb687","order_by":4,"name":"Ya Zhang","email":"","orcid":"","institution":"The First People's Hospital of Suining","correspondingAuthor":false,"prefix":"","firstName":"Ya","middleName":"","lastName":"Zhang","suffix":""},{"id":543108595,"identity":"916ca8ce-497d-4047-ad78-e411c49ca45c","order_by":5,"name":"Lin Huang","email":"","orcid":"","institution":"The First People's Hospital of Suining","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Huang","suffix":""},{"id":543108597,"identity":"52794fd9-fa57-49b9-976a-65ae53f965b5","order_by":6,"name":"Weidong Peng","email":"","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Weidong","middleName":"","lastName":"Peng","suffix":""},{"id":543108603,"identity":"67495af1-cebf-43e2-98c9-1e976f381278","order_by":7,"name":"Yu Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIie3PsYoCMRCA4VkC2SbbjxzoK2RJ44Hgq2Q52EpEsLGQu8DCXmm7hfoMVtpOCFyVB9jWsxb2CUQfQMxed0U+pkgxP0wAougfkgw4wgqH04oRdY/HqF/iJ0qmdWEbP1a5CSUATCZ1WRyEVy6rVwVQKElFfhHcJScstcv2qBPDzr/ty8OEdEI49t58kN0dcZ4CV2oWTNBxaEtN1yMuEyP4Wzh5DLQzSdkWC0O9El2i9F7azPRK+MJuaSIH37W2zQ+qvAr8ZbpxB7re8GvDmOu69edwlFbny6vkCfa39SiKouiJO41kTc6DtJbRAAAAAElFTkSuQmCC","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":true,"prefix":"","firstName":"Yu","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-09-05 06:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7541331/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7541331/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-35382-z","type":"published","date":"2026-01-13T16:31:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":95800866,"identity":"5ffacf9d-da05-4e9c-b11a-099d604b2ea9","added_by":"auto","created_at":"2025-11-13 08:23:44","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1014253,"visible":true,"origin":"","legend":"","description":"","filename":"IndirubininhibitscervicalcancerbyinhibitingapoptosisandautophagythroughthePI3KAKTandMAPKpathways.docx","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/d68ee572e792db9013925464.docx"},{"id":95764047,"identity":"902ada01-f0bc-499b-b4b7-dba42018f824","added_by":"auto","created_at":"2025-11-12 18:56:04","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8500,"visible":true,"origin":"","legend":"","description":"","filename":"e9900a54c4fc477ab4f357ba63196f9e.json","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/773a81695fc22ea24fb5a92a.json"},{"id":95801369,"identity":"69f96fa6-3d27-460d-88f6-03c238b03db8","added_by":"auto","created_at":"2025-11-13 08:25:13","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":77245,"visible":true,"origin":"","legend":"","description":"","filename":"e9900a54c4fc477ab4f357ba63196f9e1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/ba31f8092617a5f10f4409f3.xml"},{"id":95764051,"identity":"ae2b38d1-d4d8-4e9b-8f97-b4fee94155f3","added_by":"auto","created_at":"2025-11-12 18:56:04","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":35920,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/1f4f2098baedbc2246d130cf.png"},{"id":95802326,"identity":"86731df3-609c-4e60-a058-34a10fb1ea07","added_by":"auto","created_at":"2025-11-13 08:27:28","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":58283,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/663343f2e8b7094ede492da8.png"},{"id":95764058,"identity":"106d961c-bf40-4f51-b4eb-50f3946fbf03","added_by":"auto","created_at":"2025-11-12 18:56:04","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":47111,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/30a8082f1e7bba8991581249.png"},{"id":95764055,"identity":"c7b9dd85-110d-4c8f-b149-e15c2ce85289","added_by":"auto","created_at":"2025-11-12 18:56:04","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52754,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/d0a676a1df9a490df77da16a.png"},{"id":95764060,"identity":"8ccb5adb-9b5c-4911-b684-4f0d83298f87","added_by":"auto","created_at":"2025-11-12 18:56:04","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":75175,"visible":true,"origin":"","legend":"","description":"","filename":"e9900a54c4fc477ab4f357ba63196f9e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/3ad8c16d9cc8478dfa10052f.xml"},{"id":95764056,"identity":"cdf59f1e-0c43-4f29-a41b-d4515f1136e2","added_by":"auto","created_at":"2025-11-12 18:56:04","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86125,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/bfcfad29482eb703cf18db4c.html"},{"id":95764048,"identity":"ff11f239-0090-430b-b3a2-0b3182b10039","added_by":"auto","created_at":"2025-11-12 18:56:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":168539,"visible":true,"origin":"","legend":"\u003cp\u003eIndirubin safely reduces tumor weight and volume in mice without affecting normal body weight.\u003c/p\u003e\n\u003cp\u003eA: Mouse weight; B: Tumor weight; C: Tumor volume. D: Ratio of Treg. (***\u003cem\u003eP\u003c/em\u003e≤0.001, **\u003cem\u003eP\u003c/em\u003e≤0.01, *\u003cem\u003eP\u003c/em\u003e≤0.05 vs Model, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.0001 vs Model)\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/3aee1e31a070392ec7ceb288.png"},{"id":95764050,"identity":"065adf5e-2466-464c-9025-2691d512a1fb","added_by":"auto","created_at":"2025-11-12 18:56:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":308350,"visible":true,"origin":"","legend":"\u003cp\u003eIndirubin reduced hela cell viability dose-dependently.\u003c/p\u003e\n\u003cp\u003eA: Group 1 cells viability; B: Group 2 cells viability. C: Apoptosis in hela Cells. (\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.001\u003cem\u003e, \u003c/em\u003e\u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.05 vs Blank)\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/763be6f2f5a4b10c790f3689.png"},{"id":95802270,"identity":"4c4dc773-38e0-4068-aad9-522410e0c886","added_by":"auto","created_at":"2025-11-13 08:27:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":312854,"visible":true,"origin":"","legend":"\u003cp\u003eIndirubin inhibits PI3K/AKT signaling pathway in hela cells.\u003c/p\u003e\n\u003cp\u003eA: Western blotting bands; B: Relative protein expression; C: Relative mRNA expression (\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.001\u003cem\u003e, \u003c/em\u003e\u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.05 vs Control)\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/678762f3144b33f566cb60ea.png"},{"id":95800773,"identity":"1133f6f0-9965-4814-940f-599f4b6ad3d4","added_by":"auto","created_at":"2025-11-13 08:23:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":301695,"visible":true,"origin":"","legend":"\u003cp\u003eIndirubin inhibits the activation of the MEKK1/SEK1/JNK/AP-1 signaling pathway.\u003c/p\u003e\n\u003cp\u003eA: Western blotting bands; B: Relative protein expression; C: Relative mRNA expression. (\u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.001\u003cem\u003e, \u003c/em\u003e\u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.05 vs Model)\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/ee17d859b8ee99d711324949.png"},{"id":100614870,"identity":"4797aa78-33b1-4a43-9431-ae9a7f73f14a","added_by":"auto","created_at":"2026-01-19 17:27:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1634151,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7541331/v1/869e852a-3a77-4a64-8704-012b3f21eae3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Indirubin inhibits cervical cancer by inhibiting apoptosis and autophagy through the PI3K/AKT and MAPK pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCervical cancer, also known as uterine cervix carcinoma, is a severe tumor primarily affecting the cervical area, and it stands as the second most frequently diagnosed cancer in the female population across the world\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This condition represents a significant peril to the health of women worldwide, attributed to its substantial prevalence and fatality statistics. As reported by the World Health Organization (WHO) in 2022, approximately 660,000 fresh incidents and 350,000 fatalities due to cervical cancer were recorded worldwide. The main cause of cervical cancer is HPV, particularly HPV16 and HPV18. Women who fall into high-risk categories for cervical cancer are those who have engaged in sexual activity with numerous partners, early onset of sexual activity, and immunodeficiency\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In the early stages, cervical cancer may not present with noticeable symptoms. However, as the disease progresses, symptoms such as vaginal bleeding, abnormal vaginal discharge, and tumor invasion of surrounding tissues or organs may occur\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCurrent treatment modalities for cervical cancer encompass surgical resection, chemotherapy, and radiotherapy\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Nevertheless, these strategies exhibit limited efficacy in patients with advanced-stage disease, and the adverse effects associated with chemotherapy agents further constrain their clinical applicability\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In recent years, traditional Chinese medicine has demonstrated clear benefits in mitigating symptoms associated with cancer and lowering the likelihood of recurrence following surgical intervention, and mitigating the toxic side effects and drug resistance associated with radiotherapy\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Indirubin, the principal active constituent of the traditional Chinese medicine indigo naturalis, demonstrates promising potential for antitumor applications\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, the underlying mechanisms of its action against cervical cancer remain poorly understood. To date, only a study conducted by Wu Qiwei has indicated that indirubin inhibits the proliferation of cervical cancer cells\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, highlighting the necessity for further investigation into its mechanisms of action.\u003c/p\u003e\u003cp\u003eThe PI3K/AKT signaling pathway is crucial for cancer initiation and progression. It impacts various cellular functions, such as viability, growth, differentiation, motility, and metabolism\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Abnormal activation of PI3K/AKT signaling is commonly observed in cancer and promotes tumor occurrence and progression. In cervical cancer, abnormal activation of the PI3K/AKT signaling pathway links closely to tumor cell proliferation, migration, and invasion\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Moreover, research shows targeting the mTOR/PI3K/AKT signaling pathway can trigger apoptosis and autophagy in cervical cancer cells, suppressing tumor growth\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Multiple inhibitors for the PI3K/AKT pathway have been developed, such as pan - PI3K inhibitors, subtype - specific PI3K inhibitors, dual PI3K/mTOR inhibitors, and Akt inhibitors. These pharmacological agents act on critical nodes within the PI3K/AKT pathway, effectively impeding the growth and proliferation of tumor cells\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Additionally, the PI3K/AKT/mTOR pathway is significantly associated with the pathogenesis of gastric cancer, where its activation inhibits apoptosis, accelerates cell cycle progression, promotes angiogenesis, and contributes to the invasion and metastasis of gastric cancer\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In the treatment of gynecological malignant tumors, activation of the PI3K/AKT signaling pathway is implicated in enhanced proliferation and metastasis of tumor cells and induces treatment resistance\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Therefore, drug targets within this pathway presents a promising avenue for effective clinical interventions in cancer therapy. Autophagy, a critical mechanism for maintaining protein homeostasis and organelle integrity, exhibits a complex and multifaceted role in cancer. It can both prevent early tumor development and maintain and adapt to the metabolic adaptation in established and metastatic tumors\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The MEKK1/SEK1/JNK/AP-1 signaling axis, as a core stress response pathway, plays a dual role in cell fate determination. Under the background of stress and apoptosis, continuous JNK activation can induce the expression of pro-apoptotic genes by phosphorylating the transcription factor AP-1, thereby exerting its classic tumor suppressive function. However, this pathway also has cancer-promoting potential. Its abnormal activation can promote the survival, invasion and metastasis of tumor cells, showing a significant cancer-promoting phenotype. In addition, this pathway is also deeply involved in inflammatory regulation, and its activation can drive the expression of various pro-inflammatory factors. Given its core position in the regulation of cellular stress, inflammation and apoptosis, targeting the key nodes of the MEKK1/SEK1/JNK/AP-1 pathway has become an important direction for developing new therapeutic strategies for various pathological conditions such as cancer and inflammatory diseases\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This study was initiated to investigate the therapeutic potential of indirubin in a nude mouse model of cervical cancer. We specifically focused on elucidating the molecular mechanisms by which indirubin modulates autophagy and apoptosis in cervical cancer cells through the PI3K/AKT and MAPK signaling pathways. The aim of this research is to provide a deeper understanding of indirubin\u0026rsquo;s anti-cancer actions and to establish a novel mechanistic basis for its potential application in the treatment of cervical cancer.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell lines and animals\u003c/h2\u003e\u003cp\u003eHela cells (Cyagen, Shanghai) were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin at 37\u0026deg;C in a 5% CO₂ incubator.\u003c/p\u003e\u003cp\u003eU14 mouse cervical cancer cells (Cyagen, Shanghai) were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin at 37\u0026deg;C in a 5% CO₂ incubator.\u003c/p\u003e\u003cp\u003eA total of twenty-one female BALB/c-Nude mice, classified as Specific Pathogen-Free (SPF) grade, were acquired from Shanghai BiKaikeyi Biotechnology Co., Ltd. on July 7, 2023. The mice, which weighed between 18 and 20 grams, were accompanied by two additional spare specimens. The acquisition was conducted under license number SCXK(Shanghai)2018-0006 and certificate number 20180006050891. All animal procedures were approved by the Ethics Committee of Laboratory Animals of Hubei Bainite Biotechnology Co., LTD. (Ethics Number: IACUC-BNT-2023-007). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.At the endpoint of the experiment, mice were anesthetized with 3\u0026ndash;4% isoflurane in oxygen until loss of consciousness was achieved, followed by maintenance at 1.5\u0026ndash;2% isoflurane. Once deeply anesthetized, euthanasia was performed via cervical dislocation by trained personnel to ensure rapid and humane death, in accordance with the AVMA Guidelines for the Euthanasia of Animals. Immediately following euthanasia, blood samples were collected via cardiac puncture for subsequent analysis. Tumors were then carefully excised, photographed for morphological documentation, and weighed using an analytical balance. Finally, tumor tissues were snap-frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for future molecular analysis. All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) and conducted in strict compliance with the national guidelines for the care and use of laboratory animals.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMice model\u003c/h3\u003e\n\u003cp\u003eThe right forelimb of each mouse was disinfected and inoculated with 0.1 mL of U14 cell suspension (5\u0026nbsp;million cells) using an insulin syringe. Tumor growth in mice was monitored daily, with body weight and tumor dimensions recorded every 2\u0026ndash;3 days to calculate tumor volume\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e (V\u0026thinsp;=\u0026thinsp;0.5 \u0026times; a \u0026times; b\u0026sup2;). Control mice were not inoculated.\u003c/p\u003e\n\u003ch3\u003eReagents and instruments\u003c/h3\u003e\n\u003cp\u003eIndirubin (MCE, Shanghai, China), DMEM medium (SEVEN, Beijing, China), PBS (Life, Shanghai, China), TsingZol Total RNA Extraction Reagent (TSINGKE, Beijing, China), RIPA Lysis Buffer (Beyotime, Shanghai, China).\u003c/p\u003e\u003cp\u003eInverted phase contrast microscope (Olympus, Beijing, China), Desktop high-speed refrigerated microcentrifuge (DLAB, Beijing, China), Analytical flow cytometer (Beckman, Fullerton, United States), Real-time fluorescence quantitative PCR instrument (ABI, Foster City, United States), Gel imaging system (Bio-rad, California, United States).\u003c/p\u003e\n\u003ch3\u003eMethod of experiment\u003c/h3\u003e\n\u003cp\u003eWhen the average tumor volume reached 50 mm\u0026sup3;, mice were grouped (three per group) and treated daily with various drugs: IR (10, 20, 40 mg/kg), DDP (5 mg/kg), or IFO (40 mg/kg). Body weight and tumor dimensions were monitored every 2\u0026ndash;3 days for growth curves\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Treatment ceased when the positive control showed efficacy. When the experiment was terminated, anesthetized mice were subjected to tumor-bearing blood collection, tumor-stripping tissue photo collection, tumor tissue weighing, and tumor tissue preservation at -80 degrees. Tumor tissues of mice were taken for Q-PCR and Western Blot to detect the expression of genes and proteins.\u003c/p\u003e\n\u003ch3\u003eCell Counting Kit-8 (CCK-8) assay\u003c/h3\u003e\n\u003cp\u003eHela cells, after PBS wash and 1\u0026ndash;3 min trypsinization, were centrifuged, resuspended, and counted with trypan blue. They were then seeded at 7,000 per well in a 96-well plate with blanks. Post CCK-8 addition, the plates were incubated for 3 hours and read at 450 nm using a microplate reader\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe cells were subjected to treatment using the suitable drug concentrations according to the experimental groups.\u003c/p\u003e\u003cp\u003eGroup 1:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHela cells\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHela cells\u0026thinsp;+\u0026thinsp;IR (0, 1.25, 2.5, 5, 10, 20, 40, 80 \u0026micro;mol/L)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eMeasured cell viability at 24, 48, 72 h post drug treatment.\u003c/p\u003e\u003cp\u003eGroup 2:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHela cells\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHela cells\u0026thinsp;+\u0026thinsp;IR (low, medium, high dose)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHela cells\u0026thinsp;+\u0026thinsp;DDP\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHela cells\u0026thinsp;+\u0026thinsp;IFO\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eCell viability was assessed according to the optimal time and concentration established in the preceding experiment.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eFlow Cytometry Analysis\u003c/h2\u003e\u003cp\u003eCells were harvested, washed, digested, and centrifuged, then resuspended and counted\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Following seeding of 3.5 \u0026times; 10⁵ cells per well in 6-well plates. Cells were then treated according to experimental groups and incubated as specified.\u003c/p\u003e\u003cp\u003eGroups:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHela cells\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHela cells\u0026thinsp;+\u0026thinsp;IR (5 \u0026micro;mol/L)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHela cells\u0026thinsp;+\u0026thinsp;IR (20 \u0026micro;mol/L)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHela cells\u0026thinsp;+\u0026thinsp;IR (80 \u0026micro;mol/L)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHela cells\u0026thinsp;+\u0026thinsp;DDP (25 \u0026micro;mol/L)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHela cells\u0026thinsp;+\u0026thinsp;IFO (4000 \u0026micro;mol/L)\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eCells were collected for further analysis after 48 h of drug treatment.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eApoptosis Detection\u003c/h3\u003e\n\u003cp\u003eCells were harvested via trypsinization, centrifuged at 200 g for 5 minutes at 4\u0026deg;C, and rinsed twice with pre-cooled PBS. After resuspending in 100 \u0026micro;L Binding Buffer with Annexin FITC and PI, the sample was incubated in the dark for 15 minutes, then mixed with an additional 300 \u0026micro;L Binding Buffer. Analyzed via flow cytometry within one hour while kept on ice\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA of hela cells was extracted using Trizol and reverse transcribed into cDNA using 5\u0026times;SynScriptTM RT SuperMix. The expressions of MEKK1, AP-1, JNK1, SEK1 genes in mouse tumor tissues and LC3, P62, PI3K, AKT, BCL2, Bax and GAPDH in cells were detected by multiplex real-time RT-PCR on the LightCycler\u0026reg;480 system\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eWestern blotting\u003c/h2\u003e\u003cp\u003eRinsed hela cells in cold PBS were lysed in PMSF-buffer, centrifuged, and supernatant protein quantified by BCA. Samples were denatured, cooled, and stored at -20\u0026deg;C. Proteins were separated by SDS-PAGE based on molecular weight and analyzed by Western Blot using specific antibodies for MEKK1, AP-1, JNK1, SEK1, LC3, p62, PI3K, p-PI3K, AKT, p-AKT, BCL2, BAX, and GAPDH\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eGraphs were generated using GraphPad Prism 8.0.2, with data shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Data handling and analysis were conducted with SPSS 19.0, and statistical significance was determined by one-way ANOVA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIndirubin inhibits the growth of U14 cervical cancer in the body and regulates the tumor microenvironment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIndirubin significantly inhibited cervical cancer growth without affecting mouse weight (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Tumor weight (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB) and volume (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC) decreased dose-dependently with indirubin (10, 20, 40 mg/kg), with the most significant effect at 40 mg/kg. The results demonstrate indirubin\u0026rsquo;s efficacy in suppressing tumor growth in vivo. The specific data show that the model group had the highest tumor weight (0.8 g), which decreased to 0.68 g, 0.52 g, and 0.12 g for indirubin doses of 10, 20, and 40 mg/kg, respectively. The tumor volume in the model group (852.7 mm\u0026sup3;) reduced to 452.4 mm\u0026sup3;, 262.0 mm\u0026sup3;, and 73.1 mm\u0026sup3; for the same doses. The most pronounced effects were observed at 40 mg/kg indirubin. Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD shows the flow cytometry analysis of CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e. It can be found that the proportion of the indirubin treatment group and the positive control group increased, indicating that indirubin treatment increased the proportion of regulatory T cells (Treg). Treg cells can reduce inflammatory responses by inhibiting the activation and proliferation of effector T cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. These indicate that indirubin may inhibit the occurrence of cervical cancer by improving the tumor microenvironment.\u003c/p\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eIndirubin inhibits the proliferation of Hela cells and induces apoptosis in vitro\u003c/h2\u003e\n \u003cp\u003eThe CCK-8 assay (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) showed that indirubin reduced hela cell viability in a time- and concentration-dependent manner. Higher concentrations (25 and 50 \u0026micro;mol/L) significantly decreased viability, with stronger effects at 48 and 72 hours compared to 24 hours. In Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, after 48-hour treatment, indirubin reduced viability dose-dependently (83.08% at 5 \u0026micro;mol/L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; 52.06% at 20 \u0026micro;mol/L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; 25.48% at 80 \u0026micro;mol/L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Controls DDP (44.60%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and IFO (49.28%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) also significantly decreased viability.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC shows that indirubin increases apoptosis in hela cells in a dose-dependent manner, from 13.85% in controls to 65.49% at 80 \u0026micro;mol/L, indicating its potential to inhibit cervical cancer cell growth. Studies by Shi et al. and Wu et al. support indirubin\u0026rsquo;s role in promoting apoptosis and inhibiting proliferation in various cancer cells, including hela\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eIndirubin exerts anti-tumor effects in vitro by regulating autophagy and apoptosis-related proteins\u003c/h2\u003e\n \u003cp\u003eQ-PCR results (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC) indicate that 20 \u0026micro;mol/L and 80 \u0026micro;mol/L indirubin significantly upregulated LC3 mRNA and downregulated P62 mRNA in hela cells, suggesting autophagy activation. This may facilitate the removal of damaged components and inhibit proliferation. Indirubin reduced anti-apoptotic BCL2 expression in all groups, most significantly at 20 \u0026micro;mol/L and 80 \u0026micro;mol/L, potentially enhancing apoptosis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Concurrently, pro-apoptotic Bax was upregulated at these same concentrations\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Furthermore, indirubin decreased AKT and PI3K mRNA levels at 20 \u0026micro;mol/L and 80 \u0026micro;mol/L, suggesting PI3K/AKT pathway inhibition and apoptosis promotion\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. These findings confirm indirubin induces autophagy and apoptosis in hela cells, likely via PI3K/AKT modulation.\u003c/p\u003e\n \u003cp\u003eWestern Blot (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB) showed 80 \u0026micro;mol/L indirubin increased the LC3-II/LC3-I ratio and decreased P62, indicating enhanced autophagy\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. It also reduced p-PI3K/PI3K and p-AKT/AKT ratios, especially at 80 \u0026micro;mol/L, suggesting PI3K/AKT pathway suppression. Higher IR doses decreased BCL2 and increased BAX, pointing to apoptosis induction\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. These results confirm protein changes in IR-treated hela cells, demonstrating indirubin modulates autophagy, inhibits the PI3K/AKT pathway, and induces apoptosis, particularly at higher concentrations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eIndirubin inhibits the activation of the MEKK1/SEK1/JNK/AP-1 signaling pathway\u003c/h2\u003e\n \u003cp\u003eTo elucidate the molecular mechanism by which indirubin exerts anti-tumor effects in cervical cancer, we examined the expression and activation status of key components in the MEKK1/SEK1/JNK/AP-1 signaling pathway in U14 tumor tissues from nude mice. Western blotting (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA) revealed that indirubin treatment dose-dependently reduced protein levels of phosphorylated AP-1, JNK1, SEK1, and MEKK1 compared to the model group. Quantitative analysis (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB) further confirmed significant downregulation of these proteins at both low (10 mg/kg) and high (40 mg/kg) doses of indirubin. Notably, the positive control drugs cisplatin (DDP, 5 mg/kg) and ifosfamide (IFO, 40 mg/kg) also suppressed pathway activation, consistent with indirubin\u0026rsquo;s effect.\u003c/p\u003e\n \u003cp\u003eAt the mRNA level, qRT-PCR results (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC) demonstrated that indirubin significantly decreased the transcriptional expression of MEKK1, SEK1, JNK1, and AP-1 in a dose-dependent manner. These data collectively indicate that indirubin potently inhibits the MEKK1/SEK1/JNK/AP-1 signaling cascade, suggesting this pathway as a key target mediating its anti-tumor activity in cervical cancer.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study provides compelling evidence that indirubin, a natural compound extracted from indigo naturalis, exhibits significant anti-tumor activity against cervical cancer both in vivo and in vitro. The observed growth inhibition, apoptosis induction, and autophagy activation suggest that indirubin holds promise as a novel therapeutic agent for cervical cancer treatment.\u003c/p\u003e\u003cp\u003eOur findings demonstrate that indirubin exerts its anti-tumor effects primarily through modulation of the PI3K/AKT signaling pathway. By downregulating key components of this pathway, indirubin effectively inhibits cancer cell proliferation and survival, leading to increased apoptosis and autophagy. This is consistent with previous studies demonstrating the critical role of the PI3K/AKT pathway in cancer development and progression\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFurthermore, our study reveals that indirubin also targets the MEKK1/SEK1/JNK/AP-1 signaling pathway, further contributing to its anti-tumor activity. Inhibition of this pathway has been shown to suppress tumor growth, promote apoptosis, and reduce inflammation in various cancer types.\u003c/p\u003e\u003cp\u003eIt is important to note that indirubin treatment did not significantly affect the body weight of mice, indicating its potential safety profile. This is a crucial advantage over conventional chemotherapy drugs, which often cause severe side effects and limit their clinical applicability.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study demonstrates that indirubin inhibits cervical cancer cell growth by regulating the PI3K/AKT and MAPK signaling pathways, enhancing autophagy, and promoting apoptosis. These findings provide a theoretical basis for the therapeutic potential of indirubin in cervical cancer treatment and offer new insights for the development of novel anti-cancer strategies. Further investigation, including clinical trials, is warranted to explore the clinical applicability of indirubin in the treatment of cervical cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eInstitutional review board statement\u003c/h2\u003e\u003cp\u003e This study was approved by the Ethics Committee of Laboratory Animals of Hubei Bainite Biotechnology Co., LTD. (Ethics Number: IACUC-BNT-2023-007). All participants were provided informed consent and signed a document.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by Project of Sichuan Provincial Administration of Traditional Chinese Medicine (No. 2021MS479) \u0026amp; Scientific Research Project of Sichuan Provincial Maternal and Child Health Association (No. 2024FX24) of China.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX.X., C.L., designed the experiments and drafted the manuscript. X.L., L.H., investigated the literature. Y.Z., L.H., W.P., analyzed the data. Y.L., reviewed and edited. All authors approved the final version of the manuscript for publication.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhou, J. et al. Chrysotoxine regulates Ferroptosis and the PI3K/AKT/mTOR pathway to prevent cervical cancer. \u003cem\u003eJ. Ethnopharmacol.\u003c/em\u003e \u003cb\u003e338\u003c/b\u003e (P3), 119126 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeng, C. et al. A literature review on signaling pathways of cervical cancer cell death-apoptosis induced by Traditional Chinese Medicine. \u003cem\u003eJ. Ethnopharmacol.\u003c/em\u003e \u003cb\u003e334\u003c/b\u003e, 118491 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFlorence, G. et al. Global and regional estimates of orphans attributed to maternal cancer mortality in 2020. \u003cem\u003eNat. Med.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e (12), 2563\u0026ndash;2572 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA Cancer J. Clin.\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e (3), 229\u0026ndash;263 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFernandes, A. et al. Human papillomavirus-independent cervical cancer. \u003cem\u003eInt. J. Gynecol. Cancer\u003c/em\u003e. \u003cb\u003e32\u003c/b\u003e (1), 1\u0026ndash;7 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBurmeister, C. et al. Cervical cancer therapies: current challenges and future perspectives. \u003cem\u003eTumour Virus Res.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 200238\u0026ndash;200238 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYadav, N., Deshmukh, R. \u0026amp; Mazumder, R. A comprehensive review on the use of traditional Chinese medicine for cancer treatmen. \u003cem\u003ePharmacol. Research-Modern Chin. Med.\u003c/em\u003e \u003cb\u003e11100423\u003c/b\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMayadev, J. S. et al. Global challenges of radiotherapy for the treatment of locally advanced cervical cancer. \u003cem\u003eInt. J. Gynecol. Cancer\u003c/em\u003e. \u003cb\u003e32\u003c/b\u003e (3), 436\u0026ndash;445 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang, X. et al. The positive role of traditional Chinese medicine as an adjunctive therapy for cancer. \u003cem\u003eBiosci. Trends\u003c/em\u003e. \u003cb\u003e15\u003c/b\u003e (5), 283\u0026ndash;298 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang, L. et al. Pharmacological properties of indirubin and its derivatives. \u003cem\u003eBiomed. Pharmacother.\u003c/em\u003e \u003cb\u003e151\u003c/b\u003e, 113112\u0026ndash;113112 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu, Q. Study on the Inhibitory Effect of Indirubin, an Active Component of Traditional Chinese Medicine, on Tumor Cells (Master dissertation). Beijing University of Chinese Medicine, Beijing (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhezri, M. R. et al. The PI3K/AKT signaling pathway in cancer: Molecular mechanisms and possible therapeutic interventions. \u003cem\u003eExp. Mol. Pathol.\u003c/em\u003e \u003cb\u003e127\u003c/b\u003e, 104787\u0026ndash;104787 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGlaviano, A. et al. PI3K/AKT/mTOR signaling transduction pathway and targeted therapies in cancer. Mol Cancer. 22(1), 138\u0026ndash;138(2023). (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRascio, F. et al. The Pathogenic Role of PI3K/AKT Pathway in Cancer Onset and Drug Resistance. \u003cem\u003eUpdated Rev. Cancers\u003c/em\u003e. \u003cb\u003e13\u003c/b\u003e (16), 3949\u0026ndash;3949 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaad, K. A. et al. PI3K/Akt/mTOR Pathways Inhibitors with Potential Prospects in Non-Small-Cell Lung Cancer. \u003cem\u003eJ. Environ. Pathol. Toxicol. Oncol.\u003c/em\u003e \u003cb\u003e41\u003c/b\u003e(4), 185\u0026thinsp;\u0026ndash;\u0026thinsp;102(2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu, W. et al. ACTL8 Promotes the Progression of Gastric Cancer Through PI3K/AKT/mTOR Signaling Pathway. \u003cem\u003eDig. Dis. Sci.\u003c/em\u003e 1\u0026ndash;13 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, L. et al. TYMS Knockdown Suppresses Cells Proliferation, Promotes Ferroptosis via Inhibits PI3K/Akt/mTOR Signaling Pathway Activation in Triple Negative Breast Cancer. \u003cem\u003eCell. Biochem. Biophys.\u003c/em\u003e \u003cb\u003e82\u003c/b\u003e (3), 2717\u0026ndash;2726 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSu, L. et al. Xihuang pill promotes apoptosis of Treg cells in the tumor microenvironment in 4T1 mouse breast cancer by upregulating MEKK1/SEK1/JNK1/AP-1 pathway. Biomed Pharmacother, 1021111-1119(2018). (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong, L. et al. Resveratrol inhibits autophagy against myocardial ischemia-reperfusion injury through the DJ-1/MEKK1/JNK pathway. \u003cem\u003eEur. J. Pharmacol.\u003c/em\u003e, 951175748\u0026ndash;951175748 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, Q. et al. TRPV2 calcium channel promotes breast cancer progression potential by activating autophagy. \u003cem\u003eCancer Cell. Int.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e (1), 324\u0026ndash;324 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQin, X. et al. Anti-tumour and radiosensitising effects of PARP inhibitor on cervical cancer xenografts. \u003cem\u003eJ. Obstet. Gynaecol.\u003c/em\u003e \u003cb\u003e43\u003c/b\u003e (1), 2171783\u0026ndash;2171783 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTao, H. et al. Galaxamide alleviates cisplatin-induced premature ovarian insufficiency via the PI3K signaling pathway in HeLa tumor-bearing mice. \u003cem\u003eBMC Cancer\u003c/em\u003e. \u003cb\u003e24\u003c/b\u003e (1), 1060\u0026ndash;1060 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYing, H. et al. Development of an optimal protocol for isolation and purification of human granulosa cells in patients with different ovarian reserves. \u003cem\u003eExp. Ther. Med.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e (3), 938\u0026ndash;938 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLordon, B. et al. Impact of trypsin on cell cytoplasm during detachment of cells studied by terahertz sensing. \u003cem\u003eBiophys. J.\u003c/em\u003e \u003cb\u003e123\u003c/b\u003e (16), 2476\u0026ndash;2483 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, Z. H. et al. Indirubin exerts anticancer effects on human glioma cells by inducing apoptosis and autophagy. \u003cem\u003eAMB Express\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e (1), 171\u0026ndash;171 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoungmu, K. et al. RNF2 regulates Wnt/\u0026szlig;-catenin signaling via TCF7L1 destabilization. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (1), 19750\u0026ndash;19750 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi, H., Ding, K. \u0026amp; Zhang, X. Research Progress on the Apoptosis Mechanism of Traditional Chinese Medicine Active Components Affecting Cervical Cancer HeLa Cells. \u003cem\u003eGlobal Traditional Chin. Med.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (01), 124\u0026ndash;127 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu, Q. et al. Study on the Inhibitory Effect of Indirubin on Tumor Cells and the Related Mechanisms. \u003cem\u003eTianjin J. Traditional Chin. Med.\u003c/em\u003e (01), 55\u0026ndash;58 (2008).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWong, T. et al. Expression of LC3A, LC3B and p62/SQSTM1 autophagy proteins in hepatocellular carcinoma (HCC) tissues and the predicted microRNAs involved in the autophagy-related pathway. \u003cem\u003eJ. Mol. Histol.\u003c/em\u003e \u003cb\u003e55\u003c/b\u003e (3), 317\u0026ndash;328 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDanish, F. et al. Investigating the Association between the Autophagy Markers LC3B, SQSTM1/p62, and DRAM and Autophagy-Related Genes in Glioma. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e(1) (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLjubiša, J. et al. Prognostic relevance of autophagy-related markers p62, LC3, and Beclin1 in ovarian cancer. \u003cem\u003eCroat Med. J.\u003c/em\u003e \u003cb\u003e63\u003c/b\u003e (5), 453\u0026ndash;460 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMotie, M. F., Howyzeh, S. M. \u0026amp; Ghanbariasad, A. Synergic effects of DL-limonene, R-limonene, and cisplatin on AKT, PI3K, and mTOR gene expression in MDA-MB-231 and 5637 cell lines. \u003cem\u003eInt. J. Biol. Macromol.\u003c/em\u003e \u003cb\u003e280\u003c/b\u003e (P4), 136216\u0026ndash;136216 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa, Y. M. \u0026amp; Xu, W. Tanshinone reverses androgen deprivation-induced invasion of prostate cancer cells by suppressing PI3K and AKT signaling pathways. Zhonghua nan ke xue\u0026thinsp;=\u0026thinsp;National. \u003cem\u003eJ. Androl.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e (2), 104\u0026ndash;110 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong, C. et al. H2S alleviated sepsis-induced acute kidney injury by inhibiting PERK/Bax-Bcl2 pathway. \u003cem\u003eNitric Oxide\u003c/em\u003e. \u003cb\u003e15\u003c/b\u003e, 211\u0026ndash;218 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin, L. et al. NLRC5 promotes tumorigenesis by regulating the PI3K/AKT signaling pathway in cervical cancer. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (1), 15353 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, S. et al. NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling. \u003cem\u003eCell Death Dis.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (8), 565\u0026ndash;565 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen, J. et al. MYBL2 promotes cell proliferation and inhibits cell apoptosis via PI3K/AKT and BCL2/BAX/Cleaved-caspase-3 signaling pathway in gastric cancer cells. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (1), 9148 (2025).\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Indirubin, Cervical cancer, Hela cells, PI3K/AKT, MAPK","lastPublishedDoi":"10.21203/rs.3.rs-7541331/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7541331/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the effects of indirubin on autophagy and apoptosis in cervical cancer models, with a focus on elucidating the underlying molecular mechanisms. A xenograft tumor model in BALB/c-Nude mice and in vitro experiments using HeLa cell lines were employed. Indirubin treatment demonstrated concentration-dependent inhibition of cervical cancer cell growth, accompanied by induction of apoptosis and autophagy. Mechanistic analysis revealed that indirubin exerted its effects through dual modulation of the PI3K/AKT signaling axis. It enhanced autophagic flux, as evidenced by increased LC3-II/LC3-I ratio and decreased P62 expression. Concurrently, indirubin induced mitochondrial apoptosis through upregulation of pro-apoptotic Bax expression and downregulation of anti-apoptotic Bcl-2 levels. These coordinated molecular alterations ultimately led to programmed cell death execution. Additionally, indirubin inhibited the activation of the MEKK1/SEK1/JNK/AP-1 signaling pathway, further contributing to its anti-tumor effects. These findings suggest that indirubin inhibits cervical cancer cell growth by regulating the PI3K/AKT pathway, enhancing autophagy, and promoting apoptosis, providing a theoretical basis for its therapeutic potential in cervical cancer treatment.\u003c/p\u003e","manuscriptTitle":"Indirubin inhibits cervical cancer by inhibiting apoptosis and autophagy through the PI3K/AKT and MAPK pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 18:55:59","doi":"10.21203/rs.3.rs-7541331/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-12T17:40:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-11T06:43:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-10T07:01:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"261528664675846616612195176446022690382","date":"2025-11-05T05:26:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322248333263424253576786284966419721618","date":"2025-11-03T07:26:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-03T01:30:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-23T12:48:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-01T10:51:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-16T03:44:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-16T03:41:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f8c4254-f6f5-4a1c-95ec-fe788ec663a9","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":57777271,"name":"Biological sciences/Cancer"},{"id":57777272,"name":"Biological sciences/Cell biology"},{"id":57777273,"name":"Biological sciences/Drug discovery"},{"id":57777274,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-01-19T16:49:59+00:00","versionOfRecord":{"articleIdentity":"rs-7541331","link":"https://doi.org/10.1038/s41598-026-35382-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-13 16:31:02","publishedOnDateReadable":"January 13th, 2026"},"versionCreatedAt":"2025-11-12 18:55:59","video":"","vorDoi":"10.1038/s41598-026-35382-z","vorDoiUrl":"https://doi.org/10.1038/s41598-026-35382-z","workflowStages":[]},"version":"v1","identity":"rs-7541331","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7541331","identity":"rs-7541331","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}