Bufalin targets the SRC-3/c-Myc pathway in chemoresistant cells to regulate metastasis induced by chemoresistance in colorectal cancer

Bufalin targets the SRC-3/c-Myc pathway in chemoresistant cells to regulate metastasis induced by chemoresistance in colorectal cancer | 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 Bufalin targets the SRC-3/c-Myc pathway in chemoresistant cells to regulate metastasis induced by chemoresistance in colorectal cancer Jinbao Chen, Chenqi Wu, Kun Yu, Xiaoxia Tang, Ke Xu, Yi Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2536022/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Metastasis and chemoresistance are often major challenges in advanced-stage colorectal cancer. Recent studies have found extensive crosstalk between them. Previous studies have shown that bufalin has a therapeutic effect on both metastasis and drug resistance, but how bufalin affects chemoresistance-mediated metastasis remains unclear. In our study, we found that bufalin inhibited resistance-induced epithelial-mesenchymal transition (EMT) and angiogenesis, which in turn inhibited the resulting metastasis. In addition, we demonstrated that targeting of the SRC-3 protein by bufalin reduced the expression level of c-Myc and inhibited the prometastatic effect mediated by chemoresistance. Overexpression of SRC-3 or c-Myc reversed the inhibitory effect of bufalin on chemotherapeutic resistance, promoting metastasis. More interestingly, we also found that the clinical drug cinobufacini and its main active monomer bufalin reduced liver metastasis of colorectal cancer caused by chemoresistance in vivo. In conclusion, bufalin can target the SRC-3/c-Myc signaling pathway to affect the prometastatic effect of chemoresistant cells, suggesting that bufalin may be used as a new adjuvant antimetastatic therapy for colorectal cancer. bufalin SRC-3/c-Myc metastasis chemoresistance colorectal cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Colorectal cancer (CRC) is a common clinical cancer that is a serious threat to human health [ 1 ] . Chemotherapy is an important and effective method for the treatment of CRC. However, patients often exhibit chemoresistance in the advanced stages of CRC. Chemoresistance severely limits the clinical use of drugs and is a major cause of cancer-related deaths [ 2 , 3 ] . In the advanced stages of CRC, chemoresistance induced by different causes occurs in almost all patients [ 4 ] . Both chemoresistance and metastasis are common adverse events in advanced CRC, and there a strong association between them has previously been discovered. Metastatic tumors are a major contributor to human mortality from cancers, including CRC [ 5 ] . Many studies have shown that chemoresistance is closely related to tumor EMT and angiogenesis [ 6 – 7 ] . Therefore, it is very important to find a scheme that can inhibit chemoresistance that promotes metastasis. Bufalin (BU) is the main active monomer of cinobufacini. Cinobufacini has clear efficacy in clinical practice. The addition of cinobufacini to combination chemotherapy can significantly improve the efficacy of chemotherapeutic drugs, improve drug sensitivity and inhibit recurrence and metastasis [ 8 ] . Previous studies have shown that BU plays an antitumor role by affecting the metastasis, proliferation and chemoresistance of tumor cells [ 9 – 12 ] . In addition, the team previously found that BU can inhibit chemoresistance [ 10 ] , and BU can inhibit tumor microenvironment-mediated angiogenesis [ 13 ] . Therefore, we hypothesized that BU may affect metastasis caused by chemoresistance. Here, we explored the related mechanism of BU in reversing CRC metastasis induced by chemoresistance in vitro and in vivo, providing a theoretical basis for the treatment of CRC with BU as the main active monomer. Materials And Methods Cell lines and culture Cells were cultured in an incubator at 37°C and 5% CO 2 . Human umbilical vein endothelial cells were grown in endothelial cell medium, and only cells at early passages (< p6) were used. HCT8 cells were obtained from the Cell Bank of the Chinese Academy of Sciences and were cultured in RPMI-1640 medium containing 10% Fetal Bovine Serum(FBS) and 1% penicillin/streptomycin. Tube formation assay Matrigel (BD, #356234, USA) was thawed overnight at 4°C. The wells of a 96-well plate were coated with 50 µl of Matrigel, which was allowed to polymerize at 37°C for 30 min. HUVECs were pretreated with conditioned medium for 24 h, and then 3×10 4 HUVECs in 50 µl of ECM were inoculated into each well and incubated in an incubator (37℃, 5% CO 2 ) for 4 h. HUVECs were photographed under a microscope. Cell migration assay In the cell migration assay, 1×10 5 cells were inoculated in 12-well plates and cultured for 1 day under suitable environmental conditions. Then, the monolayer of confluent cells was scraped to form a linear wound of a specific size. After thoroughly washing with PBS, culture was continued in the medium. Images were acquired at the time of wound formation (time 0) and after 6 hours (HUVECs) or 24 hours (HCT8 cells). The images were analyzed to determine wound closure and indirectly infer cell migration. Cell invasion assay Cells were placed in the upper compartment of a Transwell insert containing a 6.5 mm polycarbonate film membrane with an 8.0 mm pore size (Corning, New York, USA), and the membrane was coated with 200 mg/ml MatrigelTM (BD, Sparks, MD, USA). The cells were plated in basal medium in the upper compartment, and the attractant was medium containing serum. After incubation at 37°C for a period of time, the number of cells that migrated to the lower compartment was determined. The upper surface of the membrane was wiped with cotton wool to remove nonmetastatic cells. Cells on the bottom of the membrane were fixed with 100% methanol for 10 min and stained with 0.1% crystal violet. The number of cells was determined by microscopy (Ti-E; Nikon). Each experiment was repeated 3 times, and the average value was calculated. Conditioned medium (CM) preparation HCT8/5FU cells were cultured to 80% confluence, and the medium was replaced with FBS-free medium. After 48 h of treatment, cell suspensions were collected as the CM. The CM was collected after high-speed centrifugation, filtered through a 0.22 µm microporous membrane and stored at -20°C. In the same way, cells were treated with 20 nM BU for 24 h and then treated as described above to obtain the relevant conditioned medium after drug treatment. Immunofluorescence staining HUVECs (2 × 10 4 ) were seeded and cultured overnight on microscope coverslips. After 24 h of treatment under different medium conditions, the cells were washed twice with PBS and fixed with methanol for 15 min, permeabilized with 0.2% Triton X-100 (Beyotime, Shanghai, China)/PBS for 5 min and blocked with 5% BSA for 1 h at room temperature. The coverslips were incubated with primary antibodies at 4°C overnight (VCAM, E-Selectin and P-Selectin) and then with secondary antibodies for 2 h at 37°C in the dark. Nuclear localization was assessed with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China). Tissue sections were permeabilized with cold methanol for 5 min and incubated with 5% BSA in PBS for 1 h. Primary antibodies were applied in blocking buffer and incubated overnight at 4°C. Dye-conjugated secondary antibodies were added to the blocking buffer and incubated for 2 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China). Images were acquired using a Zeiss LSM880 confocal microscope at the same voltage setting and analyzed using ZEN software. Western blot (WB) analysis Cultured cells were lysed with RIPA buffer, and the protein content was quantitatively analyzed by a BCA kit (Beyotime) prior to SDS‒PAGE. In the immunoblotting process, an enhanced fluorescence substrate (Merck Millipore) was used for detection based on the manufacturer’s instructions. A chemiluminescence detection system (Bio Rad) was applied for spectral band analysis. The antibodies required for the experiment—α-primary antibodies specific for E-cadherin, N-cadherin, Vimentin, MMP2, SNAIL, VCAM, E-selectin, P-selectin and β-actin—were purchased from Cell Signaling (Beverly, MA, USA). Quantitative RT‒PCR (RT-qPCR) Total RNA was extracted with TRIzol (Invitrogen), and an RT‒PCR kit (TaKaRa Biotechnology) was used for reverse transcription based on the relevant instructions to determine the corresponding mRNA expression levels. The corresponding primer sequences were as follows: CDH1, 5′-GAATGACAACAAGCCCGAA-3′ and 5′-GACCTCCATCACAGAGGTTCC-3′; E-Sele, 5′-ATGTTCAAGCCTGGCAGT TCCG-3′ and 5′-GCAGAGCCATTGAGCGTCCATC-3′; P-Sele, 5′-CGCTCT GGACCAACCCTGTTTC-3′ and 5′-CTCCTGGCTTCTGTGGCTTGTG-3′; MMP2, 5′-GCTATGGACTTGGGAGAA-3′ and 5′-TGGAACGGAATGGAAACC-3′; Vimentin, 5′-GAGAACTTTGCCGTTGAAGC-3′ and 5′-TCCAGCAGCTTCCTGT AGGT-3′; SNAIL, 5′-TCGGAAGCCTAACTACAGCCA-3′ and 5′-AGATGAGCA TTGGCAGCGAG-3′; VCAM, 5′-CGAAAGGCCCAGTTGAAGGA-3′ and 5′-GA GCACGAGAAGCTCAGGAGAAA-3′; CDH2, 5′-GTGCCATTAGCCAAGGGA ATTCAGC-3′ and 5′-GCGTTCCTGTTCCACTCATAGGAGG-3′; GAPDH, 5′-CCGGGAAACTGT GGCGTGATGG-3′ and 5′-AGGTGGAGGAGTGGGTGTCGHCTGTT- 3′. Splenic injection liver metastasis model HCT8/5-Fu/Luc cells (8×10 6 ) were injected into the spleens of male nude mice aged 5 to 6 weeks. The mice were divided into the control group, bufalin group (1 mg/kg) and cinobufacini group (3 mL/kg). These drugs were given by i.p. injection 5 days per week for 3 weeks. Body weight was measured at the initial injection and every 7 days thereafter, and the results were accurately recorded. Intravital imaging was performed 1 week after injection. After the course of treatment, the experimental animals were killed, and the collected liver tissues and tumors were fixed with formalin to provide material for subsequent experiments. All animal experiments were conducted in accordance with guidelines and protocols approved by the Institutional Animal Care and Use Committee of Putuo Hospital, Shanghai University of Traditional Chinese Medicine, China. In vivo optical imaging Sodium phenobarbital was injected into the abdominal cavities of mice as an anesthetic. D-fluorescein solution was injected into the abdominal cavity 5 minutes before imaging. The exposure time set during was 20 seconds and could be adjusted appropriately according to the exposure intensity. The corresponding wavelengths were 490 nm and 535 nm. Immunohistochemistry (IHC) Collected tissue samples were fixed with 10% formalin, embedded in paraffin and sliced to a thickness of 5 mm. During the experimental study, the expression levels of Ki67, CD31, Vimentin, SRC-3 and c-Myc were analyzed by IHC. Thirty random images (400×) were collected, and the cells with positive expression were identified by software analysis. Enzyme-linked immunosorbent assays (ELISAs) CXCL16, PDGFAA, VEGFA, IL-6 and ANG levels were measured with a Human ELISA Kit (BOSTER, China) following the manufacturer’s instructions. Statistical analysis GraphPad Prism 6.02 software was applied to conduct statistical analysis on relevant experimental data. The experimental data are described as the means ± standard deviations. The significance of differences between the two groups were determined by an unpaired t test. ANOVA with Tukey’s test was used to determine the significance of differences among multiple groups, and the levels of significance were set as 0.05, 0.01, and 0.001. Results 1. BU reverses EMT induced by chemoresistance in CRC The development of chemoresistance in tumor cells is accompanied by changes in many phenomena, such as EMT and angiogenesis. Studies have found that with the onset of EMT, chemoresistant cells undergo cytomorphological changes, and the migration ability of cells is enhanced, which promotes the metastasis of tumor cells [ 14 ] . BU, as an effective antitumor drug, also has clear effects on chemoresistance, tumor invasion and metastasis. In previous work, we established a chemoresistant subcutaneous tumor model and found that BU reversed the effects of chemoresistance [ 15 ] . In addition, we previously showed that BU inhibits angiogenesis [ 13 ] . Epithelial-mesenchymal transition (EMT) is a key step in local invasion and distant metastasis [ 16 ] . Studies have determined that EMT in cancer cells can promote metastasis and chemoresistance [ 17 ] , while BU can influence EMT. We observed changes in metastasis induced by chemoresistance after stimulation of chemoresistant cells by BU. To confirm the effect of BU, we chose a low concentration (20 nM) to conduct the experiment (Fig. 1 A, B). Studies have shown that chemoresistant cells tend to undergo metastasis and EMT [ 18 ] . We explored whether BU can reverse this process. We found that BU reversed HCT8/5-FU-mediated EMT (Fig. 1 C, D). The wound healing assays and invasion assays showed that BU reduced the ability of chemoresistant cells to promote migration and invasion (Fig. 1 E-F). Therefore, we determined that BU can reverse EMT induced by chemoresistance in CRC. 2. BU reverses angiogenesis induced by chemoresistance in CRC On the other hand, many studies have shown that chemoresistant tumor cells can promote angiogenesis in vascular endothelial cells by secreting proangiogenic factors [ 19 ] . Angiogenesis is an important factor in the development of metastasis and a common target for the treatment of metastatic tumors [ 20 ] . In this work, we explored the role of BU in the inhibition of angiogenesis induced by chemoresistant cells. The HUVEC tube formation and adhesion assays showed that BU reversed HCT8/5-FU-mediated angiogenesis (Fig. 2 A, B). The wound healing assays and invasion assays showed that BU reversed HCT8/5-FU-mediated HUVEC migration and invasion (Fig. 2 C, D). In addition, we further found that BU altered the protein and mRNA expression levels of angiogenesis-related factors (Fig. 2 E, F). The same results were obtained by immunofluorescence analysis (Fig. 2 G). Taken together, these results show that BU reverses angiogenesis induced by chemoresistance in CRC. 3. BU inhibits the SRC-3/c-Myc signaling pathway to reduce the levels of a series of tumor metastasis-related factors Chemoresistant cells can promote metastasis through a range of factors. Through previous studies, we identified 5 factors that play important roles in metastasis and are also associated with the development of chemoresistance [ 21 – 25 ] . To determine whether BU can influence these factors, we focused on expression changes by quantitative PCR and ELISA. The results showed that BU significantly downregulated the expression of these factors in HCT8/5-Fu cells (Fig. 3 A, B). In addition, we found that the clinical drug cinobufacini reduced the levels of tumor metastasis-related factors in the serum of CRC patients (Fig. 3 C). As an oncogene, SRC-3 is involved in the occurrence and development of many cancers. SRC-3 has been shown to promote drug resistance in cancer cells, leading to reduced disease-free survival in breast and lung cancer patients [ 26 ] . c-Myc is a key factor in prometastatic behaviors. It has been noted that regulation of SRC-3 in breast cancer can affect the expression of c-Myc [ 27 ] . Studies have shown that SRC-3 could function as a target of BU to regulate the development of cancer [ 15 , 28 ] . Therefore, we investigated whether BU can ameliorate metastasis induced by chemoresistance via SRC-3. In this study, we found that BU reduced the expression levels of SRC-3 and c-Myc (Fig. 3 D, E). Next, we found that c-Myc expression was significantly restored after overexpression of SRC-3 (Fig. 3 F, G). We further found that after rescuing the expression of SRC-3 or c-Myc, the expression of the metastasis-related factors PDGFAA and IL-6 was also restored (Fig. 3 H, I). These results suggest that BU may mediate the effects on metastasis induced by chemoresistance through the SRC-3/c-Myc pathway. 4. BU reverses metastasis induced by chemoresistance through the SRC-3/c-Myc signaling pathway To discover whether SRC-3 and c-Myc are key factors in the influence of BU on metastasis induced by chemoresistance, we performed rescue experiments by overexpressing SRC-3 or c-Myc in HCT8/5-Fu cells treated with BU. First, we collected conditioned medium after overexpression of SRC-3 or c-Myc. After stimulating HCT8 cells with this conditioned medium, we observed that the migration and invasion abilities of HCT8 cells were enhanced (Fig. 4 A, B). In addition, WB analysis also showed that the protein levels of EMT-related factors were restored (Fig. 4 C). These data suggest that BU could reverse EMT induced by chemoresistance through the SRC-3/c-Myc signaling pathway. On the other hand, we performed rescue experiments to determine whether the SRC-3/c-Myc pathway is related to chemoresistance-induced angiogenesis. We collected conditioned medium to stimulate HUVECs and tested the changes in the angiogenic capacity. As expected, the migration and invasion abilities of HUVECs were enhanced after overexpression of SRC-3 or c-Myc (Fig. 4 D, E). Through adhesion experiments, it was found that with overexpression of SRC-3 or c-Myc, HUVECs adhered to more tumor cells (Fig. 4 G, SFig 1A). Similarly, overexpression of SRC-3 or c-Myc restored the tube-forming ability of HUVECs (Fig. 4 H, SFig 1B). This suggests that BU could reverse angiogenesis induced by chemoresistance through the SRC-3/c-Myc signaling pathway. 5. BU inhibits CRC metastasis induced by chemoresistance in vivo To study the role of BU, the main active monomer of cinobufacini, in overcoming chemoresistance-induced metastasis, we established a liver metastasis model using chemoresistant cells. We explored the effects of BU as well as its clinical agent, cinobufacini, and selected drug concentrations that were not toxic (Fig. 5 A). The progression of liver metastasis from Day 7 to Day 21 was observed with an in vivo imaging system. The in vivo imaging results showed that after chemotherapy administration, the drug inhibited liver metastasis compared with that in the vehicle group (Fig. 5 B, C). HE-stained spleen sections were observed under a microscope, and the sizes of the splenic tumors were slightly reduced after BU or cinobufacini treatment (Fig. 5 D). On the other hand, observation of hematoxylin-eosin-stained liver sections revealed an approximately 50% reduction in metastasis formation following drug treatment (Fig. 5 E). Large areas of metastasis were seen in the livers of vehicle-treated mice. The results of IHC showed that the expression of the cell proliferation marker Ki67 and the angiogenesis marker CD31 was decreased in the tumors of the treated group (Fig. 6 A, C, D). The expression of c-Myc and SRC-3 in tumors was also significantly reduced after drug administration (Fig. 6 A, B, E-H). The results further suggest that cinobufacini and its active monomer BU inhibit liver metastasis by targeting the SRC-3/c-Myc pathway in vivo. Discussion CRC is the third most common cancer worldwide and poses a great threat to human life. Many studies have shown that in the advanced stage of cancer, metastasis often occurs in patients who develop chemoresistance. 5-Fluorouracil is a common chemotherapeutic agent in CRC treatment, and chemoresistance is an important obstacle to its therapeutic effect. Studies have shown that CRC cells resistant to 5-fluorouracil have a greater ability to metastasize [ 29 ] . Liver metastasis is an important marker of poor prognosis in CRC patients and is one of the most important factors affecting the survival of CRC patients. Therefore, there is an urgent need to find a treatment modality that is effective against both metastasis and chemoresistance. BU is the main active monomer of the clinical drug cinobufacini [ 15 , 30 ] . Previously, we investigated a series of molecular mechanisms of BU in the treatment of CRC. It was found that BU had effects on chemoresistance, stemness, and angiogenesis in CRC. Previous studies have shown that BU can reduce EMT in hepatocellular carcinoma cells and thus affect metastasis [ 31 ] . Additionally, BU can produce antiangiogenic effects by targeting HUVECs. In our current study, we found that BU not only reduced EMT and angiogenesis but also suppressed the causes of metastasis. We found experimentally that administration of BU reversed metastasis and angiogenesis induced by chemoresistant cells. BU also regulated the expression of the corresponding EMT and angiogenic factors. These findings suggest that BU can affect metastasis and angiogenesis induced by drug resistance. c-Myc is a common proto-oncogene in cancer. High expression of c-Myc has been detected in a variety of cancers, and c-Myc plays an important role in tumor angiogenesis and metastasis [ 32 ] . c-Myc is also one of the key clinical drug targets. We found that BU can reduce metastasis by influencing c-Myc expression. More interestingly, BU could directly regulate the expression of c-Myc through its target SRC-3. After treatment with BU, the expression of SRC-3 and c-Myc in chemoresistant cells was reduced. Through rescue experiments, we found that the expression of metastasis-related factors was altered by reversing the decrease in SRC-3 or c-Myc expression. Furthermore, this reversal also altered EMT and angiogenesis. By inhibiting SRC-3 and thus targeting c-Myc, BU further exerts a therapeutic effect on tumor metastasis. Numerous studies have reported the clinical application of cinobufacini in the treatment of malignant tumors, especially in combination with chemotherapy, and it can significantly enhance the therapeutic effect and reduce the severity of adverse reactions after chemotherapy [ 33 , 34 ] . More importantly, in vivo experiments have further revealed that both BU and cinobufacini can ameliorate drug resistance-promoted EMT and angiogenic effects. These results suggest that BU and its clinical drug cinobufacini can ameliorate chemoresistance-induced metastasis by reversing EMT and angiogenesis. In summary, our findings suggest that BU can inhibit EMT and angiogenesis induced by chemoresistance. Moreover, BU can precisely regulate c-Myc expression by targeting SRC-3 in chemoresistant cells, which in turn affects the occurrence of metastasis (Fig. 6 I). This study elucidates the molecular mechanism by which BU alleviates metastasis by targeting chemoresistance. Conclusion Overall, we found that BU targets the SRC-3/c-Myc pathway in chemoresistant cells to regulate metastasis induced by chemoresistance in colorectal cancer. BU may be an adjuvant drug for the treatment of metastasis induced by chemoresistance in advanced-stage clinical settings in the future. Abbreviations CRC:colorectal cancer; BU:Bufalin; CM:Conditioned medium; ELISA:Enzyme-linked immunosorbent assays; 5-FU: 5-Fluorouracil; CCK-8: Cell Counting Kit-8 assay; IHC:Immunohistochemistry; OE:overexpressing; qPCR: quantitative PCR; WB: Western blot; HUVECs: Human umbilical vein endothelial cells. Declarations ACKNOWLEDGMENTS Not applicable. AUTHOR CONTRIBUTIONS WL, YC and KX: Conceived or designed the study. JC, CW and KY: Collected the data, analysed and interpreted the data. JC and CW: drafted the article. JC, CW KY, and XT: Did part of experiments and aided in the construction of tumour model. WL, YC and KX: Critically revised the article. All authors approved the final version to be published. FUNDING This project was sponsored by the Natural Science Foundation of Shanghai (20ZR1450500), Clinical Specialized Disease Construction Project of Shanghai Putuo District Municipal Health Commission (NO.2020tszb03), Shanghai Rising-Star Program (Sailing special Project, 22YF1441400), the One Hundred Talents Project of Putuo Hospital, Shanghai University of Traditional Chinese Medicine (2022-RCLH-03) and Science and Technology Innovation Project of Putuo District Health System(Nos.ptkwws202315). DATA AVAILABILITY STATEMENT The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. ETHICS STATEMENT All patient samples were obtained after informed consent and approval of the ethics committee of Putuo District Center Hospital,Shanghai(Putuo Hospital, Shanghai University of Traditional Chinese Medicine). All animal experiments were conducted in accordance with guidelines and protocol approved by the institutional animal care and use committee of Putuo Hospital, Shanghai University of Traditional Chinese Medicine, China. COMPETING INTERESTS The authors declare that they have no conficts of interest. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al (2021) Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 71:209-249. Lage H (2008) An overview of cancer multidrug resistance: a still unsolved problem. Cellular and Molecular Life Sciences 65:3145-3167. Wang Y, Liu YF, Cheng Y, Yi DH, Li P, Song WQ, et al (2010) Prognosis of colorectal cancer with liver metastasis: value of a prognostic index. Brazilian Journal of Medical and Biological Research 43:1116-1122. Hu T, Li Z, Gao CY, Cho CH (2016) Mechanisms of drug resistance in colon cancer and its therapeutic strategies. World Journal of Gastroenterology 22:6876-6889. Tournigand C, André T, Achille E, Lledo G, Flesh M, Mery-Mignard D, et al (2004) FOLFIRI followed by FOLFOX6 or the reverse sequence in advanced colorectal cancer: A randomized GERCOR study. Journal of Clinical Oncology 22:229-237. Liu Y, DU F, Zhao Q, Jin J, Ma X, Li H (2015) Acquisition of 5-fluorouracil resistance induces epithelial-mesenchymal transitions through the Hedgehog signaling pathway in HCT-8 colon cancer cells.Oncology Letters 9:2675-2679. Zhang H, Deng T, Liu R, Bai M, Zhou L, Wang X, et al (2017) Exosome-delivered EGFR regulates liver microenvironment to promote gastric cancer liver metastasis. Nature Communications 8:15016. Kai S, Lu JH, Hui PP, Zhao H (2014) Pre-clinical evaluation of cinobufotalin as a potential anti-lung cancer agent. Biochemical and Biophysical Research Communications 452:768-774. Sun J, Xu K, Qiu Y, Gao H, Xu J, Tang Q, et al (2017) Bufalin reverses acquired drug resistance by inhibiting stemness in colorectal cancer cells. Oncology Reports 38:1420-1430. Yuan ZT, Shi XJ, Yuan YX, Qiu YY, Zou Y, Liu C, et al (2017) Bufalin reverses ABCB1-mediated drug resistance in colorectal cancer. Oncotarget 8:48012-48026. Qiu YY, Hu Q, Tang QF, Feng W, Hu SJ, Liang B, et al (2014) MicroRNA-497 and bufalin act synergistically to inhibit colorectal cancer metastasis. Tumor Biology 35:2599-2606. Zhan Y, Qiu Y, Wang H, Wang Z, Xu J, Fan G, et al (2020) Bufalin reverses multidrug resistance by regulating stemness through the CD133/nuclear factor-kappa B/MDR1 pathway in colorectal cancer. Cancer Science 111:1619-1630. Fang K, Zhan Y, Zhu R, Wang Y, Wu C, Sun M, et al (2021) Bufalin suppresses tumour microenvironment-mediated angiogenesis by inhibiting the STAT3 signalling pathway. Journal of Translational Medicine 19:383. Liu Y, DU F, Zhao Q, Jin J, Ma X, Li H (2015) Acquisition of 5-fluorouracil resistance induces epithelial-mesenchymal transitions through the Hedgehog signaling pathway in HCT-8 colon cancer cells. Oncology Letters 9:2675-2679. Chen J, Wang H, Jia L, He J, Li Y, Liu H, et al (2021) Bufalin targets the SRC-3/MIF pathway in chemoresistant cells to regulate M2 macrophage polarization in colorectal cancer. Cancer letters 513:63-74. De Wever O, Pauwels P, De Craene B, Sabbah M, Emami S, Redeuilh G, et al (2008) Molecular and pathological signatures of epithelial-mesenchymal transitions at the cancer invasion front. Histochemistry and Cell Biology 130:481-494. Rosanò L, Cianfrocca R, Spinella F, Di Castro V, Nicotra MR, Lucidi A, et al (2011) Acquisition of Chemoresistance and EMT Phenotype Is Linked with Activation of the Endothelin A Receptor Pathway in Ovarian Carcinoma Cells. Clinical Cancer Research 17:2350-2360. De Las Rivas J, Brozovic A, Izraely S, Casas-Pais A, Witz IP, Figueroa A (2021) Cancer drug resistance induced by EMT: novel therapeutic strategies. Archives of Toxicology 95:2279-2297. Zheng X, Liu J, Li X, Tian R, Shang K, Dong X, et al (2021) Angiogenesis is promoted by exosomal DPP4 derived from 5-fluorouracil-resistant colon cancer cells. Cancer letters 497:190-201. de Gramont A, Van Cutsem E, Schmoll HJ, Tabernero J, Clarke S, Moore MJ, et al (2012) Bevacizumab plus oxaliplatin-based chemotherapy as adjuvant treatment for colon cancer (AVANT): a phase 3 randomised controlled trial. Lancet Oncology 13:1225-1233. Li S, Shi X, Chen M, Xu N, Sun D, Bai R, et al (2019) Angiogenin promotes colorectal cancer metastasis via tiRNA production. International Journal of Cancer 145:1395-1407. Rokavec M, Öner MG, Li H, Jackstadt R, Jiang L, Lodygin D, et al (2014) IL-6R/STAT3/miR-34a feedback loop promotes EMT-mediated colorectal cancer invasion and metastasis. Journal of Clinical Investigation 124:1853-1867. Ikeda T, Nishita M, Hoshi K, Honda T, Kakeji Y, Minami Y (2020) Mesenchymal stem cell-derived CXCL16 promotes progression of gastric cancer cells by STAT3-mediated expression of Ror1. Cancer Science 111:1254-1265. Zhang Q, Lu S, Li T, Yu L, Zhang Y, Zeng H, et al (2019) ACE2 inhibits breast cancer angiogenesis via suppressing the VEGFa/VEGFR2/ERK pathway. Journal of Experimental & Clinical Cancer Research 38:173. Sahraei M, Roy LD, Curry JM, Teresa TL, Nath S, Besmer D, et al (2012) MUC1 regulates PDGFA expression during pancreatic cancer progression. Oncogene 31:4935-4945. Pollack JR, Girard L, Sullivan JP, Gao B, Peyton M, Nanjundan M, et al (2010) Steroid Receptor Coactivator-3 Expression in Lung Cancer and Its Role in the Regulation of Cancer Cell Survival and Proliferation. Cancer research 70:6477-6485. Al-azawi D, Ilroy MM, Kelly G, Redmond AM, Bane FT, Cocchiglia S, et al (2008) Ets-2 and p160 proteins collaborate to regulate c-Myc in endocrine resistant breast cancer. Oncogene 27:3021-3031. Wang Y, Lonard DM, Yu Y, Chow DC, Palzkill TG, Wang J, et al (2014) Bufalin Is a Potent Small-Molecule Inhibitor of the Steroid Receptor Coactivators SRC-3 and SRC-1. Cancer research 5:1506-1517. Lai Z, Yan Z, Chen W, Peng J, Feng J, Li Q, et al (2017) Hedyotis diffusa Willd suppresses metastasis in 5-fluorouracil-resistant colorectal cancer cells by regulating the TGF-beta signaling pathway. Molecular Medicine Reports 16:7752-7758. Cheng CS, Wang J, Chen J, Kuo KT, Tang J, Gao H, et al (2019) New therapeutic aspects of steroidal cardiac glycosides: the anticancer properties of Huachansu and its main active constituent Bufalin. Cancer Cell Int 19:92. Gai JQ, Sheng X, Qin JM, Sun K, Zhao W, Ni L (2016). The effect and mechanism of bufalin on regulating hepatocellular carcinoma cell invasion and metastasis via Wnt/beta-catenin signaling pathway. International Journal of Oncology 48: 338-348. Duffy MJ, O'Grady S, Tang M, Crown J (2021) MYC as a target for cancer treatment. Cancer Treatment Reviews 94:102154. Xie M, Chen X, Qin S, Bao Y, Bu K, Lu Y (2018) Clinical study on thalidomide combined with cinobufagin to treat lung cancer cachexia. J Cancer Res Ther 14:226-232. Wang T, Zhang L, Han L, Liu X, Zhang H, Zhang J, et al (2018) Clinical effect of intravenous infusion of zoledronic acid combined with oral medication of cinobufagin in the treatment of metastatic bone tumors, Pak. J. Pharm. Sci 31: 1609-1612. Additional Declarations No competing interests reported. Supplementary Files SFigure1.tif Fig. S1. (A) Statistical graph of the ability of (HCT8/5-FU+BU) CM to stimulate HUVEC adhesion to tumor cells after overexpression of SRC-3 or c-Myc, as evaluated by an adhesion assay. (B) Statistical graph of the tube formation ability of HUVECs stimulated with (HCT8/5-FU+BU) CM after overexpression of SRC-3 or c-Myc. The results are presented as the means ± SDs. **P<0.01 , ***P<0.001 . Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-2536022","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":172546113,"identity":"40a0c056-7e14-4a51-8b8a-d046b683b488","order_by":0,"name":"Jinbao Chen","email":"","orcid":"","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinbao","middleName":"","lastName":"Chen","suffix":""},{"id":172546118,"identity":"59078328-20cc-4e03-8bad-f8bffa1a647a","order_by":1,"name":"Chenqi Wu","email":"","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chenqi","middleName":"","lastName":"Wu","suffix":""},{"id":172546121,"identity":"c24ce50a-6a0a-4942-8e84-f5bba452be65","order_by":2,"name":"Kun Yu","email":"","orcid":"","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Yu","suffix":""},{"id":172546124,"identity":"7817fb6b-399d-419e-8145-a64f939bf0b2","order_by":3,"name":"Xiaoxia Tang","email":"","orcid":"","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoxia","middleName":"","lastName":"Tang","suffix":""},{"id":172546126,"identity":"85cd073e-be34-4d2a-9daa-fd88b1f0eb5b","order_by":4,"name":"Ke Xu","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Xu","suffix":""},{"id":172546129,"identity":"94ddf949-abba-44e6-aac6-1c94a97276b7","order_by":5,"name":"Yi Chen","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Chen","suffix":""},{"id":172546130,"identity":"95ed0ac5-4f6b-4ed3-90a8-ad38f3a9b369","order_by":6,"name":"Wei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYBACPmYgkWBgI2ff3sAGEzTAq4UNrKUgzdiA5wBQSwIxWsDkh8OJGyQSiNXCzmP44IFBGuN2yefPHvP+sItmYG/eJsFQcwePw3iMDYB+YbacnWNuzJOQnNvAc6xMguHYMzxaeLdJJBiksTHczmGT5kk4kNsgkWMmwdhwGJ+W7T8SDA7zMNw8/gyiRf4NQS3bgIF8WMLgBoMZ1BYeQlr4P4McZiDZk2MmOSctObeNJ63YIuEYbi38/McSP/74Y1Pfz378mcQbG7vcfvbDG298qMGtBYu9ICKBBA2jYBSMglEwCjABACdWTF6mP9y0AAAAAElFTkSuQmCC","orcid":"","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2023-02-01 01:14:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2536022/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2536022/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":32442470,"identity":"d16b0ccc-1c94-4c49-81f4-431ab8986e05","added_by":"auto","created_at":"2023-02-03 15:51:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":587568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBU reverses EMT induced by chemoresistance in CRC.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Chemical structure of BU. \u003cstrong\u003e(B) \u003c/strong\u003eCell viability after 48 h of treatment of chemoresistant cells with BU.\u003cstrong\u003e (C, D) \u003c/strong\u003eThe effects of BU on the protein and mRNA expression of EMT-related factors induced by chemoresistant cells was tested by RT-qPCR and WB. \u003cstrong\u003e(E)\u003c/strong\u003e The effect of BU on cell migration induced by chemoresistant cells was examined by a scratch assay. \u003cstrong\u003e(F) \u003c/strong\u003eThe effect of BU on cell invasion induced by chemoresistant cells was determined by a Transwell assay. The results are presented as the means ± SDs.\u003cem\u003e *P\u0026lt;0.05, **P\u0026lt;0.01\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2536022/v1/4d2c0e29ec80056bc01b9e69.png"},{"id":32444124,"identity":"68d0b5a2-3236-497a-baa5-97c267751af4","added_by":"auto","created_at":"2023-02-03 15:59:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1001778,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBU reverses angiogenesis induced by chemoresistance in CRC.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eThe effect of BU on tube formation.\u003cstrong\u003e (B)\u003c/strong\u003e An adhesion assay was used to detect the changes in the ability of HUVECs to adhere to tumor cells after stimulation with different conditioned media. \u003cstrong\u003e(C) \u003c/strong\u003eThe migration ability of HUVECs stimulated with different conditioned media was determined by a scratch assay.\u003cstrong\u003e (D)\u003c/strong\u003e An invasion assay was used to detect the change in the invasion ability of HUVECs stimulated with different conditioned media. \u003cstrong\u003e(E) \u003c/strong\u003eWB analysis was used to detect changes in the expression of angiogenesis -related proteins after HUVECs were stimulated with different conditioned media. \u003cstrong\u003e(F)\u003c/strong\u003e RT-qPCR was used to measure the mRNA expression of angiogenesis-related factors after HUVECs were stimulated with different conditioned media. \u003cstrong\u003e(G) \u003c/strong\u003eImmunofluorescence was used to evaluate the expression of VCAM, E-selectin and P-selectin factors after HUVECs were stimulated with different conditioned media. The results are presented as the means ± SDs.\u003cem\u003e *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2536022/v1/f34f13d4b0e62744f7cefc9c.png"},{"id":32442473,"identity":"de7f72e7-ab48-43d0-af26-6dd5e36d7a3c","added_by":"auto","created_at":"2023-02-03 15:51:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":483646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBU inhibits the SRC-3/c-Myc signaling pathway to reduce the levels a series of tumor metastasis-related factors. (A, B)\u003c/strong\u003e Effect of BU on the expression of metastasis-related factors in chemoresistant cells. \u003cstrong\u003e(C) \u003c/strong\u003eEffect of cinobufacini on the expression of metastasis-related factors in the serum of patients with CRC. \u003cstrong\u003e(D)\u003c/strong\u003e WB analysis was used to evaluate the protein expression of SRC-3 and c-Myc after BU treatment.\u003cstrong\u003e (E)\u003c/strong\u003e RT-qPCR was used to measure the mRNA expression of c-Myc after BU treatment. \u003cstrong\u003e(F, G) \u003c/strong\u003eAfter changing the expression of SRC-3 or c-Myc, the expression level of c-Myc in chemoresistant cells treated with BU was evaluated. \u003cstrong\u003e(H, I)\u003c/strong\u003e ELISA was used to detect the expression of PDGFAA and IL-6 in different conditioned media. The results are presented as the means ± SDs. *\u003cem\u003eP\u0026lt;0.05, **P\u0026lt;0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2536022/v1/b2be29f1f022f39f73f462f8.png"},{"id":32442469,"identity":"71f0eda3-d91b-4031-adc5-d130e92f86c7","added_by":"auto","created_at":"2023-02-03 15:51:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1265413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBU reverses metastasis induced by chemoresistance through the SRC-3/c-Myc signaling pathway. (A, B) \u003c/strong\u003eThe migration and invasion abilities of HCT8 cells stimulated with (HCT8/5-FU+BU)\u003csub\u003eCM\u003c/sub\u003e after overexpression of SRC-3 or c-Myc were evaluated by a scratch assay and an invasion assay.\u003cstrong\u003e (C) \u003c/strong\u003eWB analysis was used to evaluate the protein expression of EMT-related factors after HCT8 cells were stimulated with (HCT8/5-FU+BU)\u003csub\u003eCM\u003c/sub\u003e after overexpression of SRC-3 or c-Myc. \u003cstrong\u003e(D, E) \u003c/strong\u003eThe changes in the migration and invasion abilities of HUVECs stimulated with (HCT8/5-FU+BU)\u003csub\u003eCM\u003c/sub\u003e after overexpression of SRC-3 or c-Myc were detected by a scratch assay and an invasion assay. \u003cstrong\u003e(F) \u003c/strong\u003eWB analysis was used to evaluate the protein expression of angiogenesis-related factors after HUVECs were stimulated with (HCT8/5-FU+BU)\u003csub\u003eCM\u003c/sub\u003e after overexpression of SRC-3 or c-Myc. \u003cstrong\u003e(G)\u003c/strong\u003e The ability of (HCT8/5-FU+BU)\u003csub\u003eCM\u003c/sub\u003e to stimulate HUVECs to adhere to tumor cells after overexpression of SRC-3 or c-Myc was evaluated by an adhesion assay.\u003cstrong\u003e (H)\u003c/strong\u003e A tube formation assay was used to detect the changes in the tube formation ability of HUVECs stimulated with (HCT8/5-FU+BU)\u003csub\u003eCM\u003c/sub\u003e after overexpression of SRC-3 or c-Myc. The results are presented as the means ± SDs.\u003cem\u003e *P\u0026lt;0.05, **P\u0026lt;0.01\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2536022/v1/9f9ff6b97321a247d8395acf.png"},{"id":32442471,"identity":"4c4505a7-5817-4d58-8292-358e7a89f19b","added_by":"auto","created_at":"2023-02-03 15:51:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1001910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBU inhibits CRC metastasis induced by chemoresistance in vivo. (A) \u003c/strong\u003eCurve showing changes in the body weight of mice in each group.\u003cstrong\u003e (B, C) \u003c/strong\u003eTumor metastasis was visualized by an in vivo imaging system from Day 7 to Day 21.\u003cstrong\u003e (D) \u003c/strong\u003eEvaluation of liver metastasis in mice in each group by HE staining. \u003cstrong\u003e(E) \u003c/strong\u003eHE staining was used to evaluate splenic tumors in each group. The results are presented as the means ± SDs.\u003cem\u003e *P\u0026lt;0.05, **P\u0026lt;0.01\u003c/em\u003e,\u003cem\u003e ***P\u0026lt;0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2536022/v1/87b4be47a695cee7da444519.png"},{"id":32444129,"identity":"d16ee0cf-f53b-4083-a0cb-eee3083a73ef","added_by":"auto","created_at":"2023-02-03 15:59:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":904177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBU inhibits chemoresistance induced CRC metastasis in vivo through the SRC-3/c-Myc signaling pathway. (A)\u003c/strong\u003e Representative images of immunohistochemical staining of Ki67, CD31, SRC-3 and c-Myc in tissues. Scale bars in all images: 10 μm. \u003cstrong\u003e(B)\u003c/strong\u003e The expression of SRC-3 and c-Myc in different groups was observed by an immunofluorescence assay.\u003cstrong\u003e (C-F)\u003c/strong\u003e The positive rates of Ki67, CD31, SRC-3 and c-Myc expression were based on immunohistochemical staining and compared with those in the control group.\u003cstrong\u003e (G, H) \u003c/strong\u003eStatistical analysis of SRC-3 and c-Myc fluorescence in the different groups.\u003cstrong\u003e (I) \u003c/strong\u003eSchematic diagram of the mechanism by which BU inhibits chemoresistance-mediated metastasis. The results are presented as the means ± SDs.\u003cem\u003e **P\u0026lt;0.01\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2536022/v1/4ff16d99262995b3baeb43a3.png"},{"id":34876297,"identity":"96314fed-a3fb-41e1-9cc8-fab8c70796f8","added_by":"auto","created_at":"2023-03-27 16:59:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4278060,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2536022/v1/960472c1-44b5-43ae-8b79-030c3abb0bb4.pdf"},{"id":32442474,"identity":"2a596e8b-dd5a-403e-9c14-0df4e817d25e","added_by":"auto","created_at":"2023-02-03 15:51:23","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2418272,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eStatistical graph of the ability of (HCT8/5-FU+BU)\u003csub\u003eCM\u003c/sub\u003e to stimulate HUVEC adhesion to tumor cells after overexpression of SRC-3 or c-Myc, as evaluated by an adhesion assay. \u003cstrong\u003e(B) \u003c/strong\u003eStatistical graph of the tube formation ability of HUVECs stimulated with (HCT8/5-FU+BU)\u003csub\u003eCM\u003c/sub\u003e after overexpression of SRC-3 or c-Myc. The results are presented as the means ± SDs.\u003cem\u003e **P\u0026lt;0.01\u003c/em\u003e,\u003cem\u003e ***P\u0026lt;0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"SFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-2536022/v1/fc0c024006e2578d2c40dccc.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bufalin targets the SRC-3/c-Myc pathway in chemoresistant cells to regulate metastasis induced by chemoresistance in colorectal cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is a common clinical cancer that is a serious threat to human health\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Chemotherapy is an important and effective method for the treatment of CRC. However, patients often exhibit chemoresistance in the advanced stages of CRC. Chemoresistance severely limits the clinical use of drugs and is a major cause of cancer-related deaths\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. In the advanced stages of CRC, chemoresistance induced by different causes occurs in almost all patients\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Both chemoresistance and metastasis are common adverse events in advanced CRC, and there a strong association between them has previously been discovered. Metastatic tumors are a major contributor to human mortality from cancers, including CRC\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Many studies have shown that chemoresistance is closely related to tumor EMT and angiogenesis\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is very important to find a scheme that can inhibit chemoresistance that promotes metastasis.\u003c/p\u003e \u003cp\u003eBufalin (BU) is the main active monomer of cinobufacini. Cinobufacini has clear efficacy in clinical practice. The addition of cinobufacini to combination chemotherapy can significantly improve the efficacy of chemotherapeutic drugs, improve drug sensitivity and inhibit recurrence and metastasis\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown that BU plays an antitumor role by affecting the metastasis, proliferation and chemoresistance of tumor cells\u003csup\u003e[\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In addition, the team previously found that BU can inhibit chemoresistance\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, and BU can inhibit tumor microenvironment-mediated angiogenesis\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Therefore, we hypothesized that BU may affect metastasis caused by chemoresistance.\u003c/p\u003e \u003cp\u003eHere, we explored the related mechanism of BU in reversing CRC metastasis induced by chemoresistance in vitro and in vivo, providing a theoretical basis for the treatment of CRC with BU as the main active monomer.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003eCell lines and culture\u003c/h2\u003e\n \u003cp\u003eCells were cultured in an incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Human umbilical vein endothelial cells were grown in endothelial cell medium, and only cells at early passages (\u0026lt;\u0026thinsp;p6) were used. HCT8 cells were obtained from the Cell Bank of the Chinese Academy of Sciences and were cultured in RPMI-1640 medium containing 10% Fetal Bovine Serum(FBS) and 1% penicillin/streptomycin.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003eTube formation assay\u003c/h2\u003e\n \u003cp\u003eMatrigel (BD, #356234, USA) was thawed overnight at 4\u0026deg;C. The wells of a 96-well plate were coated with 50 \u0026micro;l of Matrigel, which was allowed to polymerize at 37\u0026deg;C for 30 min. HUVECs were pretreated with conditioned medium for 24 h, and then 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e HUVECs in 50 \u0026micro;l of ECM were inoculated into each well and incubated in an incubator (37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e) for 4 h. HUVECs were photographed under a microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003eCell migration assay\u003c/h2\u003e\n \u003cp\u003eIn the cell migration assay, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were inoculated in 12-well plates and cultured for 1 day under suitable environmental conditions. Then, the monolayer of confluent cells was scraped to form a linear wound of a specific size. After thoroughly washing with PBS, culture was continued in the medium. Images were acquired at the time of wound formation (time 0) and after 6 hours (HUVECs) or 24 hours (HCT8 cells). The images were analyzed to determine wound closure and indirectly infer cell migration.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003eCell invasion assay\u003c/h2\u003e\n \u003cp\u003eCells were placed in the upper compartment of a Transwell insert containing a 6.5 mm polycarbonate film membrane with an 8.0 mm pore size (Corning, New York, USA), and the membrane was coated with 200 mg/ml MatrigelTM (BD, Sparks, MD, USA). The cells were plated in basal medium in the upper compartment, and the attractant was medium containing serum. After incubation at 37\u0026deg;C for a period of time, the number of cells that migrated to the lower compartment was determined. The upper surface of the membrane was wiped with cotton wool to remove nonmetastatic cells. Cells on the bottom of the membrane were fixed with 100% methanol for 10 min and stained with 0.1% crystal violet. The number of cells was determined by microscopy (Ti-E; Nikon). Each experiment was repeated 3 times, and the average value was calculated.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003eConditioned medium (CM) preparation\u003c/h2\u003e\n \u003cp\u003eHCT8/5FU cells were cultured to 80% confluence, and the medium was replaced with FBS-free medium. After 48 h of treatment, cell suspensions were collected as the CM. The CM was collected after high-speed centrifugation, filtered through a 0.22 \u0026micro;m microporous membrane and stored at -20\u0026deg;C. In the same way, cells were treated with 20 nM BU for 24 h and then treated as described above to obtain the relevant conditioned medium after drug treatment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\n \u003cp\u003eHUVECs (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) were seeded and cultured overnight on microscope coverslips. After 24 h of treatment under different medium conditions, the cells were washed twice with PBS and fixed with methanol for 15 min, permeabilized with 0.2% Triton X-100 (Beyotime, Shanghai, China)/PBS for 5 min and blocked with 5% BSA for 1 h at room temperature. The coverslips were incubated with primary antibodies at 4\u0026deg;C overnight (VCAM, E-Selectin and P-Selectin) and then with secondary antibodies for 2 h at 37\u0026deg;C in the dark. Nuclear localization was assessed with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China).\u003c/p\u003e\n \u003cp\u003eTissue sections were permeabilized with cold methanol for 5 min and incubated with 5% BSA in PBS for 1 h. Primary antibodies were applied in blocking buffer and incubated overnight at 4\u0026deg;C. Dye-conjugated secondary antibodies were added to the blocking buffer and incubated for 2 h. Nuclei were stained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China). Images were acquired using a Zeiss LSM880 confocal microscope at the same voltage setting and analyzed using ZEN software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003eWestern blot (WB) analysis\u003c/h2\u003e\n \u003cp\u003eCultured cells were lysed with RIPA buffer, and the protein content was quantitatively analyzed by a BCA kit (Beyotime) prior to SDS‒PAGE. In the immunoblotting process, an enhanced fluorescence substrate (Merck Millipore) was used for detection based on the manufacturer\u0026rsquo;s instructions. A chemiluminescence detection system (Bio Rad) was applied for spectral band analysis. The antibodies required for the experiment\u0026mdash;\u0026alpha;-primary antibodies specific for E-cadherin, N-cadherin, Vimentin, MMP2, SNAIL, VCAM, E-selectin, P-selectin and \u0026beta;-actin\u0026mdash;were purchased from Cell Signaling (Beverly, MA, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003eQuantitative RT‒PCR (RT-qPCR)\u003c/h2\u003e\n \u003cp\u003eTotal RNA was extracted with TRIzol (Invitrogen), and an RT‒PCR kit (TaKaRa Biotechnology) was used for reverse transcription based on the relevant instructions to determine the corresponding mRNA expression levels. The corresponding primer sequences were as follows: CDH1, 5\u0026prime;-GAATGACAACAAGCCCGAA-3\u0026prime; and 5\u0026prime;-GACCTCCATCACAGAGGTTCC-3\u0026prime;; E-Sele, 5\u0026prime;-ATGTTCAAGCCTGGCAGT TCCG-3\u0026prime; and 5\u0026prime;-GCAGAGCCATTGAGCGTCCATC-3\u0026prime;; P-Sele, 5\u0026prime;-CGCTCT GGACCAACCCTGTTTC-3\u0026prime; and 5\u0026prime;-CTCCTGGCTTCTGTGGCTTGTG-3\u0026prime;; MMP2, 5\u0026prime;-GCTATGGACTTGGGAGAA-3\u0026prime; and 5\u0026prime;-TGGAACGGAATGGAAACC-3\u0026prime;;\u003c/p\u003e\n \u003cp\u003eVimentin, 5\u0026prime;-GAGAACTTTGCCGTTGAAGC-3\u0026prime; and 5\u0026prime;-TCCAGCAGCTTCCTGT AGGT-3\u0026prime;; SNAIL, 5\u0026prime;-TCGGAAGCCTAACTACAGCCA-3\u0026prime; and 5\u0026prime;-AGATGAGCA TTGGCAGCGAG-3\u0026prime;; VCAM, 5\u0026prime;-CGAAAGGCCCAGTTGAAGGA-3\u0026prime; and 5\u0026prime;-GA\u003c/p\u003e\n \u003cdiv class=\"Section3\" id=\"Sec11\"\u003e\n \u003cp\u003eGCACGAGAAGCTCAGGAGAAA-3\u0026prime;; CDH2, 5\u0026prime;-GTGCCATTAGCCAAGGGA\u003c/p\u003e\n \u003cp\u003eATTCAGC-3\u0026prime; and 5\u0026prime;-GCGTTCCTGTTCCACTCATAGGAGG-3\u0026prime;; GAPDH, 5\u0026prime;-CCGGGAAACTGT GGCGTGATGG-3\u0026prime; and 5\u0026prime;-AGGTGGAGGAGTGGGTGTCGHCTGTT- 3\u0026prime;.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003eSplenic injection liver metastasis model\u003c/h2\u003e\n \u003cp\u003eHCT8/5-Fu/Luc cells (8\u0026times;10\u003csup\u003e6\u003c/sup\u003e) were injected into the spleens of male nude mice aged 5 to 6 weeks. The mice were divided into the control group, bufalin group (1 mg/kg) and cinobufacini group (3 mL/kg). These drugs were given by i.p. injection 5 days per week for 3 weeks. Body weight was measured at the initial injection and every 7 days thereafter, and the results were accurately recorded. Intravital imaging was performed 1 week after injection. After the course of treatment, the experimental animals were killed, and the collected liver tissues and tumors were fixed with formalin to provide material for subsequent experiments.\u003c/p\u003e\n \u003cp\u003eAll animal experiments were conducted in accordance with guidelines and protocols approved by the Institutional Animal Care and Use Committee of Putuo Hospital, Shanghai University of Traditional Chinese Medicine, China.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003eIn vivo optical imaging\u003c/h2\u003e\n \u003cp\u003eSodium phenobarbital was injected into the abdominal cavities of mice as an anesthetic. D-fluorescein solution was injected into the abdominal cavity 5 minutes before imaging. The exposure time set during was 20 seconds and could be adjusted appropriately according to the exposure intensity. The corresponding wavelengths were 490 nm and 535 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e\n \u003cp\u003eCollected tissue samples were fixed with 10% formalin, embedded in paraffin and sliced to a thickness of 5 mm. During the experimental study, the expression levels of Ki67, CD31, Vimentin, SRC-3 and c-Myc were analyzed by IHC. Thirty random images (400\u0026times;) were collected, and the cells with positive expression were identified by software analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003eEnzyme-linked immunosorbent assays (ELISAs)\u003c/h2\u003e\n \u003cp\u003eCXCL16, PDGFAA, VEGFA, IL-6 and ANG levels were measured with a Human ELISA Kit (BOSTER, China) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eGraphPad Prism 6.02 software was applied to conduct statistical analysis on relevant experimental data. The experimental data are described as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations. The significance of differences between the two groups were determined by an unpaired t test. ANOVA with Tukey\u0026rsquo;s test was used to determine the significance of differences among multiple groups, and the levels of significance were set as 0.05, 0.01, and 0.001.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e1. BU reverses EMT induced by chemoresistance in CRC\u003c/h2\u003e \u003cp\u003eThe development of chemoresistance in tumor cells is accompanied by changes in many phenomena, such as EMT and angiogenesis. Studies have found that with the onset of EMT, chemoresistant cells undergo cytomorphological changes, and the migration ability of cells is enhanced, which promotes the metastasis of tumor cells\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. BU, as an effective antitumor drug, also has clear effects on chemoresistance, tumor invasion and metastasis. In previous work, we established a chemoresistant subcutaneous tumor model and found that BU reversed the effects of chemoresistance\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In addition, we previously showed that BU inhibits angiogenesis\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEpithelial-mesenchymal transition (EMT) is a key step in local invasion and distant metastasis\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Studies have determined that EMT in cancer cells can promote metastasis and chemoresistance\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, while BU can influence EMT. We observed changes in metastasis induced by chemoresistance after stimulation of chemoresistant cells by BU. To confirm the effect of BU, we chose a low concentration (20 nM) to conduct the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Studies have shown that chemoresistant cells tend to undergo metastasis and EMT\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. We explored whether BU can reverse this process. We found that BU reversed HCT8/5-FU-mediated EMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). The wound healing assays and invasion assays showed that BU reduced the ability of chemoresistant cells to promote migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). Therefore, we determined that BU can reverse EMT induced by chemoresistance in CRC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2. BU reverses angiogenesis induced by chemoresistance in CRC\u003c/h2\u003e \u003cp\u003eOn the other hand, many studies have shown that chemoresistant tumor cells can promote angiogenesis in vascular endothelial cells by secreting proangiogenic factors\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Angiogenesis is an important factor in the development of metastasis and a common target for the treatment of metastatic tumors\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. In this work, we explored the role of BU in the inhibition of angiogenesis induced by chemoresistant cells. The HUVEC tube formation and adhesion assays showed that BU reversed HCT8/5-FU-mediated angiogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). The wound healing assays and invasion assays showed that BU reversed HCT8/5-FU-mediated HUVEC migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). In addition, we further found that BU altered the protein and mRNA expression levels of angiogenesis-related factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). The same results were obtained by immunofluorescence analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Taken together, these results show that BU reverses angiogenesis induced by chemoresistance in CRC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3. BU inhibits the SRC-3/c-Myc signaling pathway to reduce the levels of a series of tumor metastasis-related factors\u003c/b\u003e \u003c/p\u003e \u003cp\u003eChemoresistant cells can promote metastasis through a range of factors. Through previous studies, we identified 5 factors that play important roles in metastasis and are also associated with the development of chemoresistance\u003csup\u003e[\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. To determine whether BU can influence these factors, we focused on expression changes by quantitative PCR and ELISA. The results showed that BU significantly downregulated the expression of these factors in HCT8/5-Fu cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). In addition, we found that the clinical drug cinobufacini reduced the levels of tumor metastasis-related factors in the serum of CRC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs an oncogene, SRC-3 is involved in the occurrence and development of many cancers. SRC-3 has been shown to promote drug resistance in cancer cells, leading to reduced disease-free survival in breast and lung cancer patients\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. c-Myc is a key factor in prometastatic behaviors. It has been noted that regulation of SRC-3 in breast cancer can affect the expression of c-Myc\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that SRC-3 could function as a target of BU to regulate the development of cancer\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Therefore, we investigated whether BU can ameliorate metastasis induced by chemoresistance via SRC-3. In this study, we found that BU reduced the expression levels of SRC-3 and c-Myc (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). Next, we found that c-Myc expression was significantly restored after overexpression of SRC-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G). We further found that after rescuing the expression of SRC-3 or c-Myc, the expression of the metastasis-related factors PDGFAA and IL-6 was also restored (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, I). These results suggest that BU may mediate the effects on metastasis induced by chemoresistance through the SRC-3/c-Myc pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4. BU reverses metastasis induced by chemoresistance through the SRC-3/c-Myc signaling pathway\u003c/h2\u003e \u003cp\u003eTo discover whether SRC-3 and c-Myc are key factors in the influence of BU on metastasis induced by chemoresistance, we performed rescue experiments by overexpressing SRC-3 or c-Myc in HCT8/5-Fu cells treated with BU. First, we collected conditioned medium after overexpression of SRC-3 or c-Myc. After stimulating HCT8 cells with this conditioned medium, we observed that the migration and invasion abilities of HCT8 cells were enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). In addition, WB analysis also showed that the protein levels of EMT-related factors were restored (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These data suggest that BU could reverse EMT induced by chemoresistance through the SRC-3/c-Myc signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the other hand, we performed rescue experiments to determine whether the SRC-3/c-Myc pathway is related to chemoresistance-induced angiogenesis. We collected conditioned medium to stimulate HUVECs and tested the changes in the angiogenic capacity. As expected, the migration and invasion abilities of HUVECs were enhanced after overexpression of SRC-3 or c-Myc (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). Through adhesion experiments, it was found that with overexpression of SRC-3 or c-Myc, HUVECs adhered to more tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, SFig 1A). Similarly, overexpression of SRC-3 or c-Myc restored the tube-forming ability of HUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, SFig 1B). This suggests that BU could reverse angiogenesis induced by chemoresistance through the SRC-3/c-Myc signaling pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e5. BU inhibits CRC metastasis induced by chemoresistance in vivo\u003c/h2\u003e \u003cp\u003eTo study the role of BU, the main active monomer of cinobufacini, in overcoming chemoresistance-induced metastasis, we established a liver metastasis model using chemoresistant cells. We explored the effects of BU as well as its clinical agent, cinobufacini, and selected drug concentrations that were not toxic (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The progression of liver metastasis from Day 7 to Day 21 was observed with an in vivo imaging system. The in vivo imaging results showed that after chemotherapy administration, the drug inhibited liver metastasis compared with that in the vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). HE-stained spleen sections were observed under a microscope, and the sizes of the splenic tumors were slightly reduced after BU or cinobufacini treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). On the other hand, observation of hematoxylin-eosin-stained liver sections revealed an approximately 50% reduction in metastasis formation following drug treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Large areas of metastasis were seen in the livers of vehicle-treated mice. The results of IHC showed that the expression of the cell proliferation marker Ki67 and the angiogenesis marker CD31 was decreased in the tumors of the treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, C, D). The expression of c-Myc and SRC-3 in tumors was also significantly reduced after drug administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B, E-H). The results further suggest that cinobufacini and its active monomer BU inhibit liver metastasis by targeting the SRC-3/c-Myc pathway in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCRC is the third most common cancer worldwide and poses a great threat to human life. Many studies have shown that in the advanced stage of cancer, metastasis often occurs in patients who develop chemoresistance. 5-Fluorouracil is a common chemotherapeutic agent in CRC treatment, and chemoresistance is an important obstacle to its therapeutic effect. Studies have shown that CRC cells resistant to 5-fluorouracil have a greater ability to metastasize\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Liver metastasis is an important marker of poor prognosis in CRC patients and is one of the most important factors affecting the survival of CRC patients. Therefore, there is an urgent need to find a treatment modality that is effective against both metastasis and chemoresistance.\u003c/p\u003e \u003cp\u003eBU is the main active monomer of the clinical drug cinobufacini\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Previously, we investigated a series of molecular mechanisms of BU in the treatment of CRC. It was found that BU had effects on chemoresistance, stemness, and angiogenesis in CRC. Previous studies have shown that BU can reduce EMT in hepatocellular carcinoma cells and thus affect metastasis\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Additionally, BU can produce antiangiogenic effects by targeting HUVECs. In our current study, we found that BU not only reduced EMT and angiogenesis but also suppressed the causes of metastasis. We found experimentally that administration of BU reversed metastasis and angiogenesis induced by chemoresistant cells. BU also regulated the expression of the corresponding EMT and angiogenic factors. These findings suggest that BU can affect metastasis and angiogenesis induced by drug resistance.\u003c/p\u003e \u003cp\u003ec-Myc is a common proto-oncogene in cancer. High expression of c-Myc has been detected in a variety of cancers, and c-Myc plays an important role in tumor angiogenesis and metastasis\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. c-Myc is also one of the key clinical drug targets. We found that BU can reduce metastasis by influencing c-Myc expression. More interestingly, BU could directly regulate the expression of c-Myc through its target SRC-3. After treatment with BU, the expression of SRC-3 and c-Myc in chemoresistant cells was reduced. Through rescue experiments, we found that the expression of metastasis-related factors was altered by reversing the decrease in SRC-3 or c-Myc expression. Furthermore, this reversal also altered EMT and angiogenesis. By inhibiting SRC-3 and thus targeting c-Myc, BU further exerts a therapeutic effect on tumor metastasis.\u003c/p\u003e \u003cp\u003eNumerous studies have reported the clinical application of cinobufacini in the treatment of malignant tumors, especially in combination with chemotherapy, and it can significantly enhance the therapeutic effect and reduce the severity of adverse reactions after chemotherapy\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. More importantly, in vivo experiments have further revealed that both BU and cinobufacini can ameliorate drug resistance-promoted EMT and angiogenic effects. These results suggest that BU and its clinical drug cinobufacini can ameliorate chemoresistance-induced metastasis by reversing EMT and angiogenesis.\u003c/p\u003e \u003cp\u003eIn summary, our findings suggest that BU can inhibit EMT and angiogenesis induced by chemoresistance. Moreover, BU can precisely regulate c-Myc expression by targeting SRC-3 in chemoresistant cells, which in turn affects the occurrence of metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). This study elucidates the molecular mechanism by which BU alleviates metastasis by targeting chemoresistance.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, we found that BU targets the SRC-3/c-Myc pathway in chemoresistant cells to regulate metastasis induced by chemoresistance in colorectal cancer. BU may be an adjuvant drug for the treatment of metastasis induced by chemoresistance in advanced-stage clinical settings in the future.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCRC:colorectal cancer; BU:Bufalin; CM:Conditioned medium; ELISA:Enzyme-linked immunosorbent assays; 5-FU: 5-Fluorouracil; CCK-8: Cell Counting Kit-8 assay; IHC:Immunohistochemistry; OE:overexpressing; qPCR: quantitative PCR; WB: Western blot; HUVECs: Human umbilical vein endothelial cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWL, YC and\u0026nbsp;KX: Conceived or designed the study. JC, CW and\u0026nbsp;KY: Collected the data, analysed and interpreted the data. JC and CW: drafted the article.\u0026nbsp;JC, CW\u0026nbsp;KY,\u0026nbsp;and XT: Did part of experiments and aided in the construction of tumour model. WL, YC and\u0026nbsp;KX: Critically revised the article. All authors approved the final version to be published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was sponsored by the Natural Science Foundation of Shanghai (20ZR1450500), Clinical Specialized Disease Construction Project of Shanghai Putuo District Municipal Health Commission (NO.2020tszb03), Shanghai Rising-Star Program (Sailing special Project, 22YF1441400), the One Hundred Talents Project of Putuo Hospital, Shanghai University of Traditional Chinese Medicine (2022-RCLH-03) and Science and Technology Innovation Project of Putuo District Health System(Nos.ptkwws202315).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patient samples were obtained after informed consent and approval of the ethics committee of Putuo District Center Hospital,Shanghai(Putuo Hospital, Shanghai University of Traditional Chinese Medicine). All animal experiments were conducted in accordance with guidelines and protocol approved by the institutional animal care and use committee of Putuo Hospital, Shanghai University of Traditional Chinese Medicine, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conficts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al (2021) Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 71:209-249. \u003c/li\u003e\n\u003cli\u003eLage H (2008) An overview of cancer multidrug resistance: a still unsolved problem. Cellular and Molecular Life Sciences 65:3145-3167. \u003c/li\u003e\n\u003cli\u003eWang Y, Liu YF, Cheng Y, Yi DH, Li P, Song WQ, et al (2010) Prognosis of colorectal cancer with liver metastasis: value of a prognostic index. Brazilian Journal of Medical and Biological Research 43:1116-1122. \u003c/li\u003e\n\u003cli\u003eHu T, Li Z, Gao CY, Cho CH (2016) Mechanisms of drug resistance in colon cancer and its therapeutic strategies. World Journal of Gastroenterology 22:6876-6889. \u003c/li\u003e\n\u003cli\u003eTournigand C, Andr\u0026eacute; T, Achille E, Lledo G, Flesh M, Mery-Mignard D, et al (2004) FOLFIRI followed by FOLFOX6 or the reverse sequence in advanced colorectal cancer: A randomized GERCOR study. Journal of Clinical Oncology 22:229-237. \u003c/li\u003e\n\u003cli\u003eLiu Y, DU F, Zhao Q, Jin J, Ma X, Li H (2015) Acquisition of 5-fluorouracil resistance induces epithelial-mesenchymal transitions through the Hedgehog signaling pathway in HCT-8 colon cancer cells.Oncology Letters 9:2675-2679. \u003c/li\u003e\n\u003cli\u003eZhang H, Deng T, Liu R, Bai M, Zhou L, Wang X, et al (2017) Exosome-delivered EGFR regulates liver microenvironment to promote gastric cancer liver metastasis. Nature Communications 8:15016. \u003c/li\u003e\n\u003cli\u003eKai S, Lu JH, Hui PP, Zhao H (2014) Pre-clinical evaluation of cinobufotalin as a potential anti-lung cancer agent. Biochemical and Biophysical Research Communications 452:768-774. \u003c/li\u003e\n\u003cli\u003eSun J, Xu K, Qiu Y, Gao H, Xu J, Tang Q, et al (2017) Bufalin reverses acquired drug resistance by inhibiting stemness in colorectal cancer cells. Oncology Reports 38:1420-1430.\u003c/li\u003e\n\u003cli\u003eYuan ZT, Shi XJ, Yuan YX, Qiu YY, Zou Y, Liu C, et al (2017) Bufalin reverses ABCB1-mediated drug resistance in colorectal cancer. Oncotarget 8:48012-48026. \u003c/li\u003e\n\u003cli\u003eQiu YY, Hu Q, Tang QF, Feng W, Hu SJ, Liang B, et al (2014) MicroRNA-497 and bufalin act synergistically to inhibit colorectal cancer metastasis. Tumor Biology 35:2599-2606. \u003c/li\u003e\n\u003cli\u003eZhan Y, Qiu Y, Wang H, Wang Z, Xu J, Fan G, et al (2020) Bufalin reverses multidrug resistance by regulating stemness through the CD133/nuclear factor-kappa B/MDR1 pathway in colorectal cancer. Cancer Science 111:1619-1630. \u003c/li\u003e\n\u003cli\u003eFang K, Zhan Y, Zhu R, Wang Y, Wu C, Sun M, et al (2021) Bufalin suppresses tumour microenvironment-mediated angiogenesis by inhibiting the STAT3 signalling pathway. Journal of Translational Medicine 19:383.\u003c/li\u003e\n\u003cli\u003eLiu Y, DU F, Zhao Q, Jin J, Ma X, Li H (2015) Acquisition of 5-fluorouracil resistance induces epithelial-mesenchymal transitions through the Hedgehog signaling pathway in HCT-8 colon cancer cells. Oncology Letters 9:2675-2679. \u003c/li\u003e\n\u003cli\u003eChen J, Wang H, Jia L, He J, Li Y, Liu H, et al (2021) Bufalin targets the SRC-3/MIF pathway in chemoresistant cells to regulate M2 macrophage polarization in colorectal cancer. Cancer letters 513:63-74. \u003c/li\u003e\n\u003cli\u003eDe Wever O, Pauwels P, De Craene B, Sabbah M, Emami S, Redeuilh G, et al (2008) Molecular and pathological signatures of epithelial-mesenchymal transitions at the cancer invasion front. Histochemistry and Cell Biology 130:481-494. \u003c/li\u003e\n\u003cli\u003eRosan\u0026ograve; L, Cianfrocca R, Spinella F, Di Castro V, Nicotra MR, Lucidi A, et al (2011) Acquisition of Chemoresistance and EMT Phenotype Is Linked with Activation of the Endothelin A Receptor Pathway in Ovarian Carcinoma Cells. Clinical Cancer Research 17:2350-2360. \u003c/li\u003e\n\u003cli\u003eDe Las Rivas J, Brozovic A, Izraely S, Casas-Pais A, Witz IP, Figueroa A (2021) Cancer drug resistance induced by EMT: novel therapeutic strategies. Archives of Toxicology 95:2279-2297. \u003c/li\u003e\n\u003cli\u003eZheng X, Liu J, Li X, Tian R, Shang K, Dong X, et al (2021) Angiogenesis is promoted by exosomal DPP4 derived from 5-fluorouracil-resistant colon cancer cells. Cancer letters 497:190-201. \u003c/li\u003e\n\u003cli\u003ede Gramont A, Van Cutsem E, Schmoll HJ, Tabernero J, Clarke S, Moore MJ, et al (2012) Bevacizumab plus oxaliplatin-based chemotherapy as adjuvant treatment for colon cancer (AVANT): a phase 3 randomised controlled trial. Lancet Oncology 13:1225-1233. \u003c/li\u003e\n\u003cli\u003eLi S, Shi X, Chen M, Xu N, Sun D, Bai R, et al (2019) Angiogenin promotes colorectal cancer metastasis via tiRNA production. International Journal of Cancer 145:1395-1407. \u003c/li\u003e\n\u003cli\u003eRokavec M, \u0026Ouml;ner MG, Li H, Jackstadt R, Jiang L, Lodygin D, et al (2014) IL-6R/STAT3/miR-34a feedback loop promotes EMT-mediated colorectal cancer invasion and metastasis. Journal of Clinical Investigation 124:1853-1867. \u003c/li\u003e\n\u003cli\u003eIkeda T, Nishita M, Hoshi K, Honda T, Kakeji Y, Minami Y (2020) Mesenchymal stem cell-derived CXCL16 promotes progression of gastric cancer cells by STAT3-mediated expression of Ror1. Cancer Science 111:1254-1265.\u003c/li\u003e\n\u003cli\u003eZhang Q, Lu S, Li T, Yu L, Zhang Y, Zeng H, et al (2019) ACE2 inhibits breast cancer angiogenesis via suppressing the VEGFa/VEGFR2/ERK pathway. Journal of Experimental \u0026amp; Clinical Cancer Research 38:173. \u003c/li\u003e\n\u003cli\u003eSahraei M, Roy LD, Curry JM, Teresa TL, Nath S, Besmer D, et al (2012) MUC1 regulates PDGFA expression during pancreatic cancer progression. Oncogene 31:4935-4945. \u003c/li\u003e\n\u003cli\u003ePollack JR, Girard L, Sullivan JP, Gao B, Peyton M, Nanjundan M, et al (2010) Steroid Receptor Coactivator-3 Expression in Lung Cancer and Its Role in the Regulation of Cancer Cell Survival and Proliferation. Cancer research 70:6477-6485. \u003c/li\u003e\n\u003cli\u003eAl-azawi D, Ilroy MM, Kelly G, Redmond AM, Bane FT, Cocchiglia S, et al (2008) Ets-2 and p160 proteins collaborate to regulate c-Myc in endocrine resistant breast cancer. Oncogene 27:3021-3031.\u003c/li\u003e\n\u003cli\u003eWang Y, Lonard DM, Yu Y, Chow DC, Palzkill TG, Wang J, et al (2014) Bufalin Is a Potent Small-Molecule Inhibitor of the Steroid Receptor Coactivators SRC-3 and SRC-1. Cancer research 5:1506-1517. \u003c/li\u003e\n\u003cli\u003eLai Z, Yan Z, Chen W, Peng J, Feng J, Li Q, et al (2017) Hedyotis diffusa Willd suppresses metastasis in 5-fluorouracil-resistant colorectal cancer cells by regulating the TGF-beta signaling pathway. Molecular Medicine Reports 16:7752-7758. \u003c/li\u003e\n\u003cli\u003eCheng CS, Wang J, Chen J, Kuo KT, Tang J, Gao H, et al (2019) New therapeutic aspects of steroidal cardiac glycosides: the anticancer properties of Huachansu and its main active constituent Bufalin. Cancer Cell Int 19:92. \u003c/li\u003e\n\u003cli\u003eGai JQ, Sheng X, Qin JM, Sun K, Zhao W, Ni L (2016). The effect and mechanism of bufalin on regulating hepatocellular carcinoma cell invasion and metastasis via Wnt/beta-catenin signaling pathway. International Journal of Oncology 48: 338-348.\u003c/li\u003e\n\u003cli\u003eDuffy MJ, O\u0026apos;Grady S, Tang M, Crown J (2021) MYC as a target for cancer treatment. Cancer Treatment Reviews 94:102154. \u003c/li\u003e\n\u003cli\u003eXie M, Chen X, Qin S, Bao Y, Bu K, Lu Y (2018) Clinical study on thalidomide combined with cinobufagin to treat lung cancer cachexia. J Cancer Res Ther 14:226-232. \u003c/li\u003e\n\u003cli\u003eWang T, Zhang L, Han L, Liu X, Zhang H, Zhang J, et al (2018) Clinical effect of intravenous infusion of zoledronic acid combined with oral medication of cinobufagin in the treatment of metastatic bone tumors, Pak. J. Pharm. Sci 31: 1609-1612.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"bufalin, SRC-3/c-Myc, metastasis, chemoresistance, colorectal cancer","lastPublishedDoi":"10.21203/rs.3.rs-2536022/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2536022/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMetastasis and chemoresistance are often major challenges in advanced-stage colorectal cancer. Recent studies have found extensive crosstalk between them. Previous studies have shown that bufalin has a therapeutic effect on both metastasis and drug resistance, but how bufalin affects chemoresistance-mediated metastasis remains unclear. In our study, we found that bufalin inhibited resistance-induced epithelial-mesenchymal transition (EMT) and angiogenesis, which in turn inhibited the resulting metastasis. In addition, we demonstrated that targeting of the SRC-3 protein by bufalin reduced the expression level of c-Myc and inhibited the prometastatic effect mediated by chemoresistance. Overexpression of SRC-3 or c-Myc reversed the inhibitory effect of bufalin on chemotherapeutic resistance, promoting metastasis. More interestingly, we also found that the clinical drug cinobufacini and its main active monomer bufalin reduced liver metastasis of colorectal cancer caused by chemoresistance in vivo. In conclusion, bufalin can target the SRC-3/c-Myc signaling pathway to affect the prometastatic effect of chemoresistant cells, suggesting that bufalin may be used as a new adjuvant antimetastatic therapy for colorectal cancer.\u003c/p\u003e","manuscriptTitle":"Bufalin targets the SRC-3/c-Myc pathway in chemoresistant cells to regulate metastasis induced by chemoresistance in colorectal cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-03 15:51:18","doi":"10.21203/rs.3.rs-2536022/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43ee1818-9cd8-4056-bc64-d624126a301d","owner":[],"postedDate":"February 3rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-03-27T16:59:28+00:00","versionOfRecord":[],"versionCreatedAt":"2023-02-03 15:51:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2536022","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2536022","identity":"rs-2536022","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}