Inhibitory Effect of the Multi-target TKI, Anlotinib, in 5-FU Resistant Colorectal Cancer HCT-8/15 Cells: Down Regulation of Drug Resistance-associated Protein Expression.

Inhibitory Effect of the Multi-target TKI, Anlotinib, in 5-FU Resistant Colorectal Cancer HCT-8/15 Cells: Down Regulation of Drug Resistance-associated Protein Expression. | 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 Inhibitory Effect of the Multi-target TKI, Anlotinib, in 5-FU Resistant Colorectal Cancer HCT-8/15 Cells: Down Regulation of Drug Resistance-associated Protein Expression. JUAN LIU, HAOLIN SUN, XIXI ZHENG, NINA MA, Xiaoling Liu, Ruizhen Cao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4450056/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 Purpose Colorectal cancer is the most prevalent gastrointestinal malignancy. Treatment usually includes 5-fluorouracil (5-FU), oxaliplatin, and irinotecan, with 5-FU usually being the first choice. 5-FU treatment failure occurs when cancer cells acquire resistance. Therefore, it is crucial to identify compounds effective against 5-FU-resistant tumors. Herein, we determined the efficacy and mechanism of anlotinib in 5-FU-resistant colon cancer cells. Materials and methods Human colon cancer cells (HCT-8/5-FU and HCT-15/5-FU) resistant to 5-FU were subjected to treatment with anlotinib, 5-FU, or both. Cell proliferation was assessed via MTS and clone formation assays. Cell cycle progression was studied using flow cytometry. Through immunoblotting, we evaluated changes in the protein levels of p-AKT and multidrug resistance 1. Results MTS assays indicated that HCT-8/5-FU and HCT-15/5-FU cells were sensitive to anlotinib and resistant to 5-FU. At 48 h, HCT-8/5-FU had an IC50 of 2246.5 ± 204.5 µM, while HCT-15/5-FU had an IC50 of 18.49 ± 3.23 mM for 5-FU. The IC50 of anlotinib for HCT-8/5-FU cells was 53.69 ± 8.10µM at 24 h and 17.39 ± 1.98µM at 48 h. The IC50 values for HCT-15/5-FU at 24 and 48 h were 55.03 ± 3.44µM and 8.83 ± 3.02µM, respectively. Anlotinib enhanced 5-FU sensitivity in resistant cells, with low concentrations (IC10) considerably enhancing the antiproliferative effects of 5-FU. Further, anlotinib significantly increased the number of cells in the G0-G1 phase dose-dependently, while the proportion of cells entering S phase decreased. MDR1 and AKT expression decreased with increasing anlotinib concentration. Conclusion Anlotinib suppressed the proliferation of 5-FU-resistant colon cancer cells by preventing entry into S phase, thus sensitizing cells to 5-FU. Moreover, anlotinib may reverse the effect of 5-FU on drug-resistant cells by down-regulating the expression of multidrug-resistant proteins, in which the AKT signaling pathway may play an important role. colorectal cancer multi-target TKI anlotinib 5-fluorouracil drug resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Colorectal cancer is the most common gastrointestinal malignancy, ranking second and fourth in terms of mortality and morbidity, respectively, and thus posing a heavy burden on public health. 1 Chemotherapy remains the standard of care, even though other therapeutic approaches, including targeted therapy, immunotherapy, and combination therapy, are available. 2 The three most widely used chemotherapy drugs for colorectal cancer are 5-fluorouracil (5-FU), oxaliplatin, and irinotecan. The first-line treatment is 5-FU combined with oxaliplatin or irinotecan. However, cancer cells quickly develop resistance to 5-FU, which leads to treatment failure. Therefore, the identification of therapeutics effective against 5-FU-resistant colon cancer cells is crucial. Multidrug resistance (MDR) is the most common cause of chemotherapy failure, with ATP-binding cassette (ABC) drug transporters playing an important role in tumor MDR. The three most important ABC transporter family members are ABCB1 (P-gp/MDR1), ABCC1 (MRP1), and ABCG2 (BCRP). Moreover, various studies have shown that MDR1 overexpression is the main cause of MDR. Anlotinib is a novel oral tyrosine kinase inhibitor with several targets. It strongly inhibits the vascular endothelial factor receptor (VEGFR2/3), fibroblast growth factor receptor (FGFR1-4), and platelet-derived growth factor receptor (PDGFR). Kinases PDGFR and c-kit are two receptor tyrosine kinases linked to angiogenesis and tumor growth, for which antitumor properties have also been reported. 3 – 5 AKT is the most commonly studied signaling pathway in research on anlotinib in cancer. Anlotinib has been approved for treating advanced non-small-cell lung cancer as well as soft tissue sarcoma. Its efficacy and safety against metastatic colorectal cancer (mCRC) were discussed at the 2018 Chinese Society of Clinical Oncology (CSCO) meeting. Anlotinib monotherapy had an objective response rate (ORR; primary endpoint) of 6.45% and a disease control rate (DCR) of 87.1%. The ALTER0703 trial (NCT02332499) confirmed that anlotinib significantly prolongs progression-free survival (PFS) as a third-line or subsequent treatment in Chinese patients with mCRC. 6 In 2022, results from the NCT04080843 trial where anlotinib was combined with chemotherapy as first-line treatment of mCRC with wild-type RAS/BRAF showed a considerable improvement in ORR, DCR, PFS, and duration of response (DOR). Many trials evaluating anlotinib against advanced colorectal cancer are currently ongoing. Although anlotinib efficacy has been confirmed in clinical trials, its specific mechanism of action against colorectal cancer cells has been poorly studied. TKI is thought to perform its role by competitively binding to ATP binding sites. It can attenuate downstream signaling pathways associated with tumor proliferation, invasion, migration, and angiogenesis. Anlotinib blocks numerous signaling pathways, and little is known regarding its effects in drug-resistant colon cancer cells. Herein, we sought to determine whether anlotinib is effective against 5-FU-resistant colorectal cancer cells and whether it might reverse resistance by suppressing the MDR1 and AKT pathways. Methods Cell culture and reagents HCT-8 cells were purchased from ATCC and cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). The cells were cultured at 5% CO 2 and 37°C. We obtained HCT-15 and HCT-15/5-FU (5-FU-resistant human colon cancer cells) from China Shanghai Yihe Biotechnology. HCT-8/5-FU cells were acquired from China Beijing Being Biotechnology. 5-FU was added to HCT-8/5-FU cells at a concentration of 1 g/mL and to HCT-15/5-FU cells at 3.2 g/mL. Anlotinib was purchased from China Chia Tai Tianqing Pharmaceutical Group Co., Ltd., (Nanjing, China). MTS reagent was purchased from Promega Corporation (Madison, WI, USA). Propidium iodide was purchased from Sigma-Aldrich (Saint Louis, MO, USA). MTS assay Drug cytotoxicity and resistance were assessed using the MTS test. Briefly, 5,000 cells at the logarithmic growth phase were seeded in 96-well plates and were grown for 24h at 37°C. Cells were then treated with various 5-FU or anlotinib doses for 24h and 48h, followed by a 2h incubation period after the addition of 20µL MTS reagent (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega Corporation, USA) to each well. Absorbance at 490 nm was obtained using a Bio-Rad Microplate Reader Model 680(Spectramax M3; Molecular Devices, USA). The assays were conducted in triplicate. Colony formation assays One thousand logarithmic growth cells were seeded in 6-well plates, followed by incubation for 24 h. Thereafter, cells were re-incubated and cultivated for a further 24h in medium with drugs at various doses. Following a further 12 days of cultivation in drug-free medium, the cells were fixed in 10% formaldehyde, stained with Giemsa, rinsed with phosphate-buffered saline (PBS), and imaged. This was performed in triplicate. Cell cycle analysis To examine the effect of anlotinib on 5-FU-resistant colon cell lines, we determined cell cycle distribution in HCT-8/FU and HCT-15/FU. HCT-8/FU and HCT-15/FU were seeded in 6-well plates. Cells were separated into four groups and supplied with different amounts of anlotinib for 24h once they reached 70–80% confluence. Prior to trypsinization, cells were centrifuged at 1,000 rpm for 3min, followed by two rounds of washing in ice-cold PBS. The cells were then fixed in 3mL of 70% cold ethanol at 4°C overnight. The fixed cells were centrifuged, washed with PBS, and treated with 250µL propidium iodide at room temperature for 15min. Finally, cell cycle distribution was analyzed using a FACScan (BD FACSCalibur™, USA). Immunoblotting Immunoblotting was used to assess phosphorylation and protein levels. HCT-8/Fu and HCT-15/Fu cells were seeded in 6-well plates and grown to 70–80% confluence. Following respective treatments (different drug concentrations for 24h), cells were lysed in HEPES 50µM, NaCl 150µM, EDTA 1mM, 1% Triton, and 10% glycerol supplemented with protease inhibitors. The lysates were centrifuged at 4°C at 12,000 rpm for 15min.A BCA protein assay kit (Merck, Darmstadt, Germany) was used to measure total protein concentration. After separation on a 10% SDS-PAGE gel, proteins were transferred to a PVDF membrane (Millipore) and blocked in 5% non-fat milk. Membranes were incubated with primary antibodies overnight at 4°C and secondary antibodies for 2h at room temperature. TBST buffer was used to wash off the unbound antibodies. Protein bands were visualized on an ECL plus system (Beyotime). Image J software(USA) was used for image analysis. The following specific primary antibodies were used for western blot analysis: MRP1/ABCC1 rabbit antibody (1:1000, 72202S, CST); MDR1/ABCB1 rabbit antibody(1:1000, 13342, CST); GAPDH mouse monoclonal antibody (1:5000, Cat. No:60004-1-Ig, Proteintech); p-AKT rabbit monoclonal antibody (1:1000, 4060S, CST); and AKT rabbit polyclonal antibody (1:1000, Cat. No:10176-2-AP, Proteintech). Statistical analysis GraphPad Prism 5.0 software was used for statistical analysis. Data are presented as the mean ± SD. To evaluate statistical significance, we employed Shapiro-Wilk tests for normality of distribution and Barlett tests for homogeneity of variance. For data with normal distribution and homogenous variance, we employed one-way analysis of variance with Tukey post-hoc test for multiple comparisons. Nemenyi test were used for multiple comparisons if not satisfied using Kruskal-Wallis tests. Differences were deemed significant at a p-value of 0.05. Results Inhibitory effects of anlotinib and 5-FU on colon cancer cell proliferation We exposed HCT-8/5-FU and HCT-15/5-FU cells to various concentrations of anlotinib and 5-FU for 24h and 48h in order to examine the impact on cell growth. In both lines, anlotinib and 5-FU inhibited proliferation, which was dependent on time and dose (Fig. 1 ). At 48h, the half-maximal inhibitory concentration (IC50) of 5-FU was 2246.5 ± 204.5 µM in HCT-8/5-FU cells and 18.49 ± 3.23 mM in HCT-15/5-FU cells. IC50 values could not be obtained at 24h. To assess the inhibitory activity of anlotinib, we employed a range of concentrations. The observed effects depended on both time and dose. The IC50 for anlotinib in HCT-8/5-FU cells was 53.69 ± 8.10 µM at 24 h and 17.39 ± 1.98 µM at 48 h. At 24h, the IC50 in HCT-15/5-FU cells was 55.03 ± 3.44 µM, compared to 8.83 ± 3.02 µM at 48h. Anlotinib promotes 5-FU sensitivity in resistant colon cancer cells We treated HCT-8/5-FU and HCT-15/5-FU cells with anlotinib at a non-lethal dose along with increasing 5-FU concentrations for 24 h, in order to determine if anlotinib would sensitize cells to 5-FU. In resistant cell lines, doses below the IC10 are typically regarded as safe amounts. We determined the IC5 and IC10 doses of anlotinib in HCT-8/5-FU (3.15 ± 2.30 µM and 6.27 ± 3.74 µM, respectively) and HCT-15/5-FU cells (12.01 ± 3.16µM and 17.09 ± 3.95µM, respectively). We used the IC5 and IC10 dosages of anlotinib to evaluate sensitivity in 5-FU-resistant cell lines. Notably, the proliferation of HCT-8/5-FU and HCT-15/5-FU cells was synergistically inhibited by anlotinib and 5-FU. In comparison to single drugs, the combination induced a significantly higher inhibition rate. Colony formation assays confirmed this synergistic impact to an even greater extent. The number of colonies in the drug combination group was much less than that in single drug groups (Fig. 2 – 3 ). Effects of anlotinib on cell cycle progression in drug-resistant colorectal cancer cells We explored the effects of anlotinib on cell cycle progression in light of its anti-proliferative properties. We treated HCT-15/5-FU cells with 10 µM, 20 µM, and 40 µM doses for cell cycle analysis, as no suppression of proliferation had been observed at 5 µM based on the MTS results. Anlotinib increased the percentage of cells in the G0/G1 phase, with fewer cells entering S phase (Fig. 4 ). This effect was most pronounced at 20 µM in HCT-15/5-FU cells and at 10 µM in HCT-8/5-FU cells. Apoptosis was not enhanced under any of the treatment conditions. Anlotinib inhibits drug resistance-related protein expression and AKT pathway activation Drug resistance proteins are frequently upregulated in drug-resistant tumor cells. We assessed the expression of resistance-related proteins MRP1 and MDR1 to further interrogate the mechanism of action of anlotinib in colorectal cancer cells. High concentrations were chosen due to the low inhibition rate at lower concentrations, as per MTS results of anlotinib on drug-resistant colon cancer cells. MDR1 and MRP1 were strongly expressed in HCT-8/5-FU and HCT-15/5-FU cells, as determined via immunoblotting. Increasing anlotinib concentration suppressed their expression. We then assessed the levels of total and phosphorylated AKT (Fig. 5 ). Increasing anlotinib concentration suppressed p-AKT levels. Discussion According to the World Health Organization, colorectal cancer is the third most prevalent malignancy among both men and women, being the second most common cause of cancer-related death worldwide. Colorectal cancer treatment has considerably improved with chemotherapy, targeted therapy, and immunotherapy. However, therapy resistance is common, particularly 5-FU resistance, which results in treatment failure. It is, therefore, crucial to identify novel therapeutics to be employed for overcoming drug resistance. 7 The novel oral tyrosine kinase inhibitor anlotinib, a small-molecule compound with several targets, has been approved for the treatment of esophageal cancer, advanced lung cancer, and periodontal sarcoma. Further, it has been successful in the treatment of various solid tumors, including colorectal, stomach, and liver. 3 There is an increasing body of fundamental research into the effects of anlotinib. In the small-cell lung H446 cell line, Tang et al. 8 discovered that anlotinib suppressed cell invasion, migration, and proliferation by inhibiting ERK1/2 and c-met signaling. Anlotinib was also found to downregulate lung adenocarcinoma lymphangiogenesis and lymphatic metastasis. 9 Anlotinib partially blocks the MEK/ERK pathway to prevent the development of KRAS-mutant lung cancer cells. 10 Several studies have also demonstrated the effectiveness of anlotinib in digestive tract tumors. Results of the ALTER0703 trial (NCT04080843) revealed that anlotinib was more effective than chemotherapy for treating advanced colorectal cancer. 11 miR-940 was shown to improve the inhibitory effect of anlotinib on colorectal cancer cell growth and invasion. 12 Yang et al. 13 reported that anlotinib suppresses colorectal cancer cell proliferation as well as angiogenesis. Anlotinib and dihydroartemisinin were shown to exhibit synergistic anticancer effects in patients with gastric cancer. 14 In addition, anlotinib kills liver cancer cells as well as pancreatic cancer cells, in addition to reversing drug resistance in lung cancer and modulating the tumor microenvironment. 15 – 23 Herein, we used two 5-FU-resistant human colorectal cancer cell lines (HCT-8/5-FU and HCT-15/5-FU) to assess the impact of anlotinib on 5-FU resistance. Treatment with anlotinib drastically reduced cell growth, suggesting that it may be conducive for overcoming chemoresistance. However, anlotinib did not enhance the apoptosis of resistant cancer cells. Tumor cells exhibit either primary or acquired resistance to chemotherapy. The former may arise from multiple origins, including the tumor microenvironment, a low tumor cell growth rate, the difficulty of drug entry into G0 phase cells, and compromised drug activity upon cell entry. The upregulation of resistance-associated proteins is the primary cause of acquired resistance. 24 At present, there are 48 members of the ABC transporter family, which can be divided into seven groups (subfamily A-G). They include nine MDR-related drug transporters, ABCB1 (P-gp/MDR1), ABCB4 (MDR3), ABCB11 (BSEP/SPGP), ABCC1 (MRP1), ABCC2 (MRP2), ABCC3 (MRP3), ABCC4 (MRP4), ABCC5 (MRP5) and ABCG2 (BCRP), among which the three most important ones are ABCB1 (P-gp/MDR1), ABCC1 (MRP1), and ABCG2 (BCRP). In particular, MDR1 overexpression is the primary cause of MDR, as described in various studies. 25 As an energy-dependent drug efflux pump, MDR1 can influence the efficacy of different chemotherapeutics. 26 MDR1 may also act against therapy resistance by upregulating apoptosis-suppressing proteins and inhibiting enzymatic activity. To better understand its mechanism of action in treatment-resistant cells, we examined the effects of anlotinib on cell cycle progression and MDR protein levels. Anlotinib suppressed cancer cell proliferation, preventing entry into S phase. Furthermore, anlotinib suppressed MRP1 and MDR1 expression in a concentration-dependent manner. The PI3K-AKT-mTOR pathway is among the common signaling cascades dysregulated in cancer. AKT/ERK signaling is a mediator of anlotinib's impact on colorectal and liver cancer cell growth. Herein, we observed that anlotinib decreased p-AKT levels in a concentration-dependent manner. Conclusion In the present study, we show that anlotinib can suppress the proliferation of 5-FU-resistant colon cancer cells, preventing their entry into the S phase. Anlotinib dose-dependently suppressed AKT signaling and drug resistance-associated protein expression, which may be the key to reversing resistance. Our findings on the efficacy of anlotinib against drug-resistant colorectal cancer cells are yet to be validated in animal studies. Further, the impact on other signaling pathways remains to be explored. The mutation status of factors such as RAS/BRAF genes, microsatellite status, and HER-2 positivity should also be considered. Declarations Competing Interests There are the authors declare no competing interests mentioned in the work. Funding This work was supported by Grants No. 82173056 from the Natural Science Foundation of China (to Bangwei Cao) and Beijing Municipal Natural Science Foundation (7222032 to Bangwei Cao) . Author Contribution J.L. conducted most of the experiments, analyzed data, and wrote the manuscript; L.H., X.Z., N.M., X.L., and R.C. analyzed the data and wrote the original paper; B.C. and M.Y. designed and supervised the project; B.C. takes responsibility for data in this project; and all authors reviewed the manuscript. Data Availability declaration All data supporting the results of this study can be found in the paper. References Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68(6):394–424. 10.3322/caac.21492 Stamou K, Gouvas N, Pechlivanides G, Xynos E (2018) Primary curative surgery and preemptive or adjuvant hyperthermic peritoneal chemotherapy in colorectal cancer patients at high risk to develop peritoneal carcinomatosis. A systematic review. J BUON 23(5):1249–1261 Sun Y, Niu W, Du F et al (2016) Safety, pharmacokinetics, and antitumor properties of anlotinib, an oral multi-target tyrosine kinase inhibitor, in patients with advanced refractory solid tumors. J Hematol Oncol 9(1):105. 10.1186/s13045-016-0332-8 Syed YY (2018) Anlotinib: first global approval. Drugs 78(10):1057–1062. 10.1007/s40265-018-0939-x Shen G, Zheng F, Ren D et al (2018) Anlotinib: a novel multi-targeting tyrosine kinase inhibitor in clinical development. J Hematol Oncol 11(1):120. 10.1186/s13045-018-0664-7 Chi Y, Shu Y, Ba Y et al (2021) Anlotinib monotherapy for refractory metastatic colorectal cancer: a double-blinded, placebo-controlled, randomized phase iii trial(Alter0703). Oncologist 26(10):e1693–e1703. 10.1002/onco.13857 Siddik ZH (2003) Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22(47):7265–7279. 10.1038/sj.onc.1206933 Tang X, Zheng Y, Jiao D et al (2021) Anlotinib inhibits cell proliferation, migration and invasion via suppression of c-met pathway and activation of erk1/2 pathway in h446 cells. Anticancer Agents Med Chem 21(6):747–755. 10.2174/1871520620666200718235748 Qin T, Liu Z, Wang J et al (2020) Anlotinib suppresses lymphangiogenesis and lymphatic metastasis in lung adenocarcinoma through a process potentially involving VEGFR-3 signaling. Cancer Biol Med 17(3):753–767. 10.20892/j.issn.2095-3941.2020.0024 Hu H, Liu Y, Tan S et al (2020) Anlotinib exerts anti-cancer effects on kras-mutated lung cancer cell through suppressing the mek/erk pathway. Cancer Manag Res 12:3579–3587. 10.2147/CMAR.S243660 Cheng Y, Du FC, Fang FQ, Duan ZJ, Lei W, Shi KG (2021) Third-line treatment for metastatic colorectal cancer: anlotinib is superior to chemotherapy and similar to fruquintinib or regorafenib. Neoplasma 67(06):1384–1390. 10.4149/neo_2020_191125N1212 Wang Y, Zhao M, Zhao H, Cheng S, Bai R, Song M (2019) MicroRNA-940 restricts the expression of metastasis-associated gene MACC1 and enhances the antitumor effect of Anlotinib on colorectal cancer. Onco Targets Ther 12:2809–2822. 10.2147/OTT.S195364 Yang Q, Ni L, Imani S et al (2020) Anlotinib suppresses colorectal cancer proliferation and angiogenesis via inhibition of akt/erk signaling cascade. Cancer Manag Res 12:4937–4948. 10.2147/CMAR.S252181 Luo Q, Zhang S, Zhang D et al (2021) Effects and mechanisms of anlotinib and dihydroartemisinin combination therapy in ameliorating malignant biological behavior of gastric cancer cells. Curr Pharm Biotechnol 22(4):523–533. 10.2174/1389201021666200623132803 Wang J, Wu DX, Meng L, Ji G (2020) Anlotinib combined with SOX regimen (S1 (tegafur, gimeracil and oteracil porassium capsules) + oxaliplatin) in treating stage IV gastric cancer: study protocol for a single-armed and single-centred clinical trial. BMJ Open 10(6):e034685. 10.1136/bmjopen-2019-034685 He C, Wu T, Hao Y (2018) Anlotinib induces hepatocellular carcinoma apoptosis and inhibits proliferation via Erk and Akt pathway. Biochem Biophys Res Commun 503(4):3093–3099. 10.1016/j.bbrc.2018.08.098 Song F, Hu B, Cheng JW et al (2020) Anlotinib suppresses tumor progression via blocking the VEGFR2/PI3K/AKT cascade in intrahepatic cholangiocarcinoma. Cell Death Dis 11(7):573. 10.1038/s41419-020-02749-7 Yang L, Zhou X, Sun J et al (2020) Reactive oxygen species mediate anlotinib-induced apoptosis via activation of endoplasmic reticulum stress in pancreatic cancer. Cell Death Dis 11(9):766. 10.1038/s41419-020-02938-4 Lian Z, Du W, Zhang Y et al (2020) Anlotinib can overcome acquired resistance to EGFR-TKIs via FGFR1 signaling in non‐small cell lung cancer without harboring EGFR T790M mutation. Thorac Cancer 11(7):1934–1943. 10.1111/1759-7714.13485 Yan LH, Zhang D, Mo SS, Yuan H, Mo XW, Zhao JM (2021) Anlotinib suppresses metastasis and multidrug resistance via dual blockade of MET/ABCB1 in colorectal carcinoma cells. J Cancer 12(7):2092–2104. 10.7150/jca.45618 Lan W, Zhao J, Chen W, Shang H, Peng J, Lin J (2021) Anlotinib overcomes multiple drug resistant colorectal cancer cells via inactivating pi3k/akt pathway. Anticancer Agents Med Chem 21(15):1987–1995. 10.2174/1871520621666210112113852 Liu S, Qin T, Liu Z et al (2020) Anlotinib alters tumor immune microenvironment by downregulating pd-l1 expression on vascular endothelial cells. Cell Death Dis 11(5):309. 10.1038/s41419-020-2511-3 Yang Y, Li L, Jiang Z, Wang B, Pan Z (2020) Anlotinib optimizes anti-tumor innate immunity to potentiate the therapeutic effect of PD-1 blockade in lung cancer. Cancer Immunol Immunother 69(12):2523–2532. 10.1007/s00262-020-02641-5 Rivera G, Wakelee HA (2016) Resistance to therapy. In: Reckamp KL, ed. Lung Cancer . Vol 170. Springer International Publishing; :183–202. 10.1007/978-3-319-40389-2_9 Kim ES (2016) Chemotherapy resistance in lung cancer. In: Ahmad A, Gadgeel S, eds. Lung Cancer and Personalized Medicine . Vol 893. Springer International Publishing; :189–209. 10.1007/978-3-319-24223-1_10 Mealey KL, Fidel J (2015) P-glycoprotein mediated drug interactions in animals and humans with cancer. J Vet Intern Med 29(1):1–6. 10.1111/jvim.12525 Additional Declarations No competing interests reported. Supplementary Files ThefulluncroppedGelsandBlotsimages.rar Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4450056","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":308366425,"identity":"821071cd-81a3-4998-913d-cbf725c48663","order_by":0,"name":"JUAN LIU","email":"","orcid":"","institution":"Beijing Friendship Hospital","correspondingAuthor":false,"prefix":"","firstName":"JUAN","middleName":"","lastName":"LIU","suffix":""},{"id":308366426,"identity":"d58f6967-d4bc-4686-ba1c-116088c41a88","order_by":1,"name":"HAOLIN SUN","email":"","orcid":"","institution":"Beijing Friendship Hospital","correspondingAuthor":false,"prefix":"","firstName":"HAOLIN","middleName":"","lastName":"SUN","suffix":""},{"id":308366427,"identity":"2f7ea8b7-6c8c-481f-be82-8f767f2f8aa3","order_by":2,"name":"XIXI ZHENG","email":"","orcid":"","institution":"Beijing Friendship Hospital","correspondingAuthor":false,"prefix":"","firstName":"XIXI","middleName":"","lastName":"ZHENG","suffix":""},{"id":308366428,"identity":"2c600f2f-f257-4678-8e68-7b38e8f975e1","order_by":3,"name":"NINA MA","email":"","orcid":"","institution":"Beijing Friendship Hospital","correspondingAuthor":false,"prefix":"","firstName":"NINA","middleName":"","lastName":"MA","suffix":""},{"id":308366429,"identity":"f5325363-0567-4505-8ffc-869bd9cfb651","order_by":4,"name":"Xiaoling Liu","email":"","orcid":"","institution":"Shanxi Cancer Hospital Affiliated to Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Liu","suffix":""},{"id":308366430,"identity":"a90d9fc6-5495-4070-b49c-540e510433aa","order_by":5,"name":"Ruizhen Cao","email":"","orcid":"","institution":"Ordos Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ruizhen","middleName":"","lastName":"Cao","suffix":""},{"id":308366431,"identity":"9293b82f-e06d-4dc1-a5a6-8303cbc36d17","order_by":6,"name":"Bangwei Cao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYLCCB0DMD2UzNhClJQGIJRtI1mJwgFgt/NLNDx8kVNQmbj5++OlmHgYb2Q0HmJ89wKdFcs4xY4OEM8cTt51JM7vNw5BmvOEAm7kBPi0GN3LYJBLbjiVuu8HDBtRyOHHDAR42CaK0bJ4B1vKfaC01iRskwFoOENYiOSMN5JcDxjOAfrk5xyDZeOZhNjO8Wvglkh8++FBRJ9vffvjZjTcVdrJ9x5uf4dUCBYcdGyDuBGJmItQDQZ09cepGwSgYBaNgRAIALj1MBwgyKScAAAAASUVORK5CYII=","orcid":"","institution":"Beijing Friendship Hospital","correspondingAuthor":true,"prefix":"","firstName":"Bangwei","middleName":"","lastName":"Cao","suffix":""},{"id":308366432,"identity":"f380d57d-a70a-4e5b-b15d-628007f51344","order_by":7,"name":"Mudan Yang","email":"","orcid":"","institution":"Shanxi Cancer Hospital Affiliated to Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mudan","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-05-20 15:27:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4450056/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4450056/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57791756,"identity":"1e21a273-b6ab-49d6-b47d-725b78170220","added_by":"auto","created_at":"2024-06-05 17:41:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67500,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of anlotinib and 5-fluorouracil (5-FU) on colon cancer cell proliferation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) HCT-8/5-FU cells treated with 5-FU for 24 h and 48 h. IC50 (48 h) = 2246.5±204.5 μM. (B) HCT-8/5-FU cells were treated with anlotinib for 24 h and 48 h. IC50 (24 h) = 53.69±8.10 μM; IC50 (48 h) = 17.39±1.98 μM. (C) HCT-15/5-FU cells treated with 5-FU for 24h and 48h. IC50 (48 h) = 18.49±3.23 mM. (D) HCT-15/5-FU cells treated with anlotinib for 24 h and 48 h. IC50 (24 h) = 55.03±3.44 μM; IC50 (48 h) = 28.83±3.02 µΜ. Data are presented as the mean±SD obtained from three independent experiments.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4450056/v1/e3b551cb23b46f95ec2a6294.png"},{"id":57792039,"identity":"6713dbb7-a0ee-4a3f-a60d-8a3ff27491b8","added_by":"auto","created_at":"2024-06-05 17:49:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":177574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnlotinib reverses of 5-FU resistance in HCT-8/5-FU colon cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) HCT-8/5-FU cells were treated with anlotinib (at IC5 and IC10) and 5-FU at specified concentrations for 24 h. Cell viability was evaluated using the MTS assay. Data are presented as the mean±SD. Doses below the IC10 are typically regarded as safe. In order to avoid cytotoxicity attributed to high doses rather than sensitization, we used IC10 in this experiment. (B) Colonies formed in three independent experiments were calculated from (C). (C) Representative photos of HCT-8/5-FU colonies in the control, 6 µM anlotinib (IC10), 2 mM 5-FU (IC10), and combined treatment group. Following treatment with the indicated reagents, the number of colonies formed per dish was assessed. *p\u0026lt;0.05 represents the comparison between the experimental and the control group; #p\u0026lt;0.05 represents the comparison between the combination and single drug groups. Data represent three independent experiments.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4450056/v1/8901010a9a93bdbd27ccbb31.png"},{"id":57792038,"identity":"d5c99a8a-720f-4f80-9fd7-956b7b6337fa","added_by":"auto","created_at":"2024-06-05 17:49:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":194488,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnlotinib reverses drug resistance in HCT-15/5-FU colon cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) HCT-15/5-FU cells treated with anlotinib (IC5 and IC10) together with indicated 5-FU concentrations for 24h. We used MTS assays to determine cell viability. Data are presented as the mean±SD of three independent experiments. (B) Number of colonies formed under the indicated treatment was calculated from (C). (C) Representative photos of HCT-15/5-FU colonies under control, 6 μM anlotinib (IC10), 2 mM 5-FU (IC10), and combination treatment. Colonies per dish after indicated treatment. Three experiments were conducted independently.*p\u0026lt;0.05 represents the comparison between the experimental and control groups; #p\u0026lt;0.05 represents the comparison between the drug combination group and single drug groups. Data represent three independent experiments.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4450056/v1/6c6d9d568e909386a72d46c3.png"},{"id":57791759,"identity":"953c537b-d0be-43d2-8ac6-dfdba51f5bed","added_by":"auto","created_at":"2024-06-05 17:41:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":165859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of anlotinib on the cellcycle.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) The effect of different anlotinib concentrations (0, 5, 10, 20 µM, in a–d, respectively) on HCT-8/5-FU cells. (C, D) HCT-15/5-FU cells assayed as in (A) and (B), e–h. Anlotinib reduced the number of cells in the S phase while increasing those in the G0/G1 phase (p\u0026lt;0.05). Concentrations of 20 µM and 10 µM had the greatest effect in HCT-15/5-FU and HCT-8/5-FU cells, respectively. Data are presented as the mean±SD of three experiments. *p\u0026lt;0.05 represents the comparison between the experimental and control groups. Anlotinib suppressed the viability of HCT-8/FU cells even at 5 µM, while no effect was observed in HCT-15/FU cells. Therefore, 5 µM was not included as a treatment for HCT-15/FU cells. Meanwhile, 80 µM had the strongest cytotoxic effect. To avoid excess cell death, this dose was not selected.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4450056/v1/b7ed75d0800051d8cab2aeb8.png"},{"id":57791758,"identity":"7ad88393-0605-4b02-96b7-ff6b6ff1014f","added_by":"auto","created_at":"2024-06-05 17:41:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of anlotinib on drug resistance-associated protein expressionand AKT signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunoblotting analysis of MDR1, MRP1, p-AKT, and AKT in HCT-8/5-FU and HCT-15/5-FU cells. Cells were treated with anlotinib at the indicated doses for 24 h. MDR1 and MRP1 were highly expressed in both HCT-8/5-FU and HCT-15/5-FU cells. Increasing anlotinib suppressed phospho-AKT levels. As per MTS results, we selected high concentrations of anlotinib due to the low inhibition rate at lower concentrations. Data are presented as the mean±SD of three independent experiments.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4450056/v1/86861e52bd1fb59257ff9480.png"},{"id":60103335,"identity":"7c4209e3-838f-4af3-96d1-afc9f29565d0","added_by":"auto","created_at":"2024-07-11 20:46:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1221875,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4450056/v1/76725b0c-4ab8-4f98-bf9c-fc228e025beb.pdf"},{"id":57791760,"identity":"a333abf9-a034-4e16-87f1-6d7a93c07493","added_by":"auto","created_at":"2024-06-05 17:41:28","extension":"rar","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7551107,"visible":true,"origin":"","legend":"","description":"","filename":"ThefulluncroppedGelsandBlotsimages.rar","url":"https://assets-eu.researchsquare.com/files/rs-4450056/v1/ce5ee81d870b30286e45552a.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhibitory Effect of the Multi-target TKI, Anlotinib, in 5-FU Resistant Colorectal Cancer HCT-8/15 Cells: Down Regulation of Drug Resistance-associated Protein Expression.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer is the most common gastrointestinal malignancy, ranking second and fourth in terms of mortality and morbidity, respectively, and thus posing a heavy burden on public health.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Chemotherapy remains the standard of care, even though other therapeutic approaches, including targeted therapy, immunotherapy, and combination therapy, are available.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e The three most widely used chemotherapy drugs for colorectal cancer are 5-fluorouracil (5-FU), oxaliplatin, and irinotecan. The first-line treatment is 5-FU combined with oxaliplatin or irinotecan. However, cancer cells quickly develop resistance to 5-FU, which leads to treatment failure. Therefore, the identification of therapeutics effective against 5-FU-resistant colon cancer cells is crucial. Multidrug resistance (MDR) is the most common cause of chemotherapy failure, with ATP-binding cassette (ABC) drug transporters playing an important role in tumor MDR. The three most important ABC transporter family members are ABCB1 (P-gp/MDR1), ABCC1 (MRP1), and ABCG2 (BCRP). Moreover, various studies have shown that MDR1 overexpression is the main cause of MDR.\u003c/p\u003e \u003cp\u003eAnlotinib is a novel oral tyrosine kinase inhibitor with several targets. It strongly inhibits the vascular endothelial factor receptor (VEGFR2/3), fibroblast growth factor receptor (FGFR1-4), and platelet-derived growth factor receptor (PDGFR). Kinases PDGFR and c-kit are two receptor tyrosine kinases linked to angiogenesis and tumor growth, for which antitumor properties have also been reported.\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e AKT is the most commonly studied signaling pathway in research on anlotinib in cancer. Anlotinib has been approved for treating advanced non-small-cell lung cancer as well as soft tissue sarcoma. Its efficacy and safety against metastatic colorectal cancer (mCRC) were discussed at the 2018 Chinese Society of Clinical Oncology (CSCO) meeting. Anlotinib monotherapy had an objective response rate (ORR; primary endpoint) of 6.45% and a disease control rate (DCR) of 87.1%. The ALTER0703 trial (NCT02332499) confirmed that anlotinib significantly prolongs progression-free survival (PFS) as a third-line or subsequent treatment in Chinese patients with mCRC.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e In 2022, results from the NCT04080843 trial where anlotinib was combined with chemotherapy as first-line treatment of mCRC with wild-type RAS/BRAF showed a considerable improvement in ORR, DCR, PFS, and duration of response (DOR). Many trials evaluating anlotinib against advanced colorectal cancer are currently ongoing. Although anlotinib efficacy has been confirmed in clinical trials, its specific mechanism of action against colorectal cancer cells has been poorly studied. TKI is thought to perform its role by competitively binding to ATP binding sites. It can attenuate downstream signaling pathways associated with tumor proliferation, invasion, migration, and angiogenesis.\u003c/p\u003e \u003cp\u003eAnlotinib blocks numerous signaling pathways, and little is known regarding its effects in drug-resistant colon cancer cells. Herein, we sought to determine whether anlotinib is effective against 5-FU-resistant colorectal cancer cells and whether it might reverse resistance by suppressing the MDR1 and AKT pathways.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and reagents\u003c/h2\u003e \u003cp\u003eHCT-8 cells were purchased from ATCC and cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). The cells were cultured at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37\u0026deg;C. We obtained HCT-15 and HCT-15/5-FU (5-FU-resistant human colon cancer cells) from China Shanghai Yihe Biotechnology. HCT-8/5-FU cells were acquired from China Beijing Being Biotechnology. 5-FU was added to HCT-8/5-FU cells at a concentration of 1 g/mL and to HCT-15/5-FU cells at 3.2 g/mL. Anlotinib was purchased from China Chia Tai Tianqing Pharmaceutical Group Co., Ltd., (Nanjing, China). MTS reagent was purchased from Promega Corporation (Madison, WI, USA). Propidium iodide was purchased from Sigma-Aldrich (Saint Louis, MO, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMTS assay\u003c/h2\u003e \u003cp\u003eDrug cytotoxicity and resistance were assessed using the MTS test. Briefly, 5,000 cells at the logarithmic growth phase were seeded in 96-well plates and were grown for 24h at 37\u0026deg;C. Cells were then treated with various 5-FU or anlotinib doses for 24h and 48h, followed by a 2h incubation period after the addition of 20\u0026micro;L MTS reagent (CellTiter 96\u0026reg; AQueous One Solution Cell Proliferation Assay, Promega Corporation, USA) to each well. Absorbance at 490 nm was obtained using a Bio-Rad Microplate Reader Model 680(Spectramax M3; Molecular Devices, USA). The assays were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assays\u003c/h2\u003e \u003cp\u003eOne thousand logarithmic growth cells were seeded in 6-well plates, followed by incubation for 24 h. Thereafter, cells were re-incubated and cultivated for a further 24h in medium with drugs at various doses. Following a further 12 days of cultivation in drug-free medium, the cells were fixed in 10% formaldehyde, stained with Giemsa, rinsed with phosphate-buffered saline (PBS), and imaged. This was performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis\u003c/h2\u003e \u003cp\u003eTo examine the effect of anlotinib on 5-FU-resistant colon cell lines, we determined cell cycle distribution in HCT-8/FU and HCT-15/FU. HCT-8/FU and HCT-15/FU were seeded in 6-well plates. Cells were separated into four groups and supplied with different amounts of anlotinib for 24h once they reached 70\u0026ndash;80% confluence. Prior to trypsinization, cells were centrifuged at 1,000 rpm for 3min, followed by two rounds of washing in ice-cold PBS. The cells were then fixed in 3mL of 70% cold ethanol at 4\u0026deg;C overnight. The fixed cells were centrifuged, washed with PBS, and treated with 250\u0026micro;L propidium iodide at room temperature for 15min. Finally, cell cycle distribution was analyzed using a FACScan (BD FACSCalibur\u0026trade;, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eImmunoblotting was used to assess phosphorylation and protein levels. HCT-8/Fu and HCT-15/Fu cells were seeded in 6-well plates and grown to 70\u0026ndash;80% confluence. Following respective treatments (different drug concentrations for 24h), cells were lysed in HEPES 50\u0026micro;M, NaCl 150\u0026micro;M, EDTA 1mM, 1% Triton, and 10% glycerol supplemented with protease inhibitors. The lysates were centrifuged at 4\u0026deg;C at 12,000 rpm for 15min.A BCA protein assay kit (Merck, Darmstadt, Germany) was used to measure total protein concentration. After separation on a 10% SDS-PAGE gel, proteins were transferred to a PVDF membrane (Millipore) and blocked in 5% non-fat milk. Membranes were incubated with primary antibodies overnight at 4\u0026deg;C and secondary antibodies for 2h at room temperature. TBST buffer was used to wash off the unbound antibodies. Protein bands were visualized on an ECL plus system (Beyotime). Image J software(USA) was used for image analysis. The following specific primary antibodies were used for western blot analysis: MRP1/ABCC1 rabbit antibody (1:1000, 72202S, CST); MDR1/ABCB1 rabbit antibody(1:1000, 13342, CST); GAPDH mouse monoclonal antibody (1:5000, Cat. No:60004-1-Ig, Proteintech); p-AKT rabbit monoclonal antibody (1:1000, 4060S, CST); and AKT rabbit polyclonal antibody (1:1000, Cat. No:10176-2-AP, Proteintech).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 5.0 software was used for statistical analysis. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. To evaluate statistical significance, we employed Shapiro-Wilk tests for normality of distribution and Barlett tests for homogeneity of variance. For data with normal distribution and homogenous variance, we employed one-way analysis of variance with Tukey post-hoc test for multiple comparisons. Nemenyi test were used for multiple comparisons if not satisfied using Kruskal-Wallis tests. Differences were deemed significant at a p-value of 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eInhibitory effects of anlotinib and 5-FU on colon cancer cell proliferation\u003c/h2\u003e\n \u003cp\u003eWe exposed HCT-8/5-FU and HCT-15/5-FU cells to various concentrations of anlotinib and 5-FU for 24h and 48h in order to examine the impact on cell growth. In both lines, anlotinib and 5-FU inhibited proliferation, which was dependent on time and dose (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). At 48h, the half-maximal inhibitory concentration (IC50) of 5-FU was 2246.5\u0026thinsp;\u0026plusmn;\u0026thinsp;204.5 \u0026micro;M in HCT-8/5-FU cells and 18.49\u0026thinsp;\u0026plusmn;\u0026thinsp;3.23 mM in HCT-15/5-FU cells. IC50 values could not be obtained at 24h. To assess the inhibitory activity of anlotinib, we employed a range of concentrations. The observed effects depended on both time and dose. The IC50 for anlotinib in HCT-8/5-FU cells was 53.69\u0026thinsp;\u0026plusmn;\u0026thinsp;8.10 \u0026micro;M at 24 h and 17.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98 \u0026micro;M at 48 h. At 24h, the IC50 in HCT-15/5-FU cells was 55.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44 \u0026micro;M, compared to 8.83\u0026thinsp;\u0026plusmn;\u0026thinsp;3.02 \u0026micro;M at 48h.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eAnlotinib promotes 5-FU sensitivity in resistant colon cancer cells\u003c/h2\u003e\n \u003cp\u003eWe treated HCT-8/5-FU and HCT-15/5-FU cells with anlotinib at a non-lethal dose along with increasing 5-FU concentrations for 24 h, in order to determine if anlotinib would sensitize cells to 5-FU. In resistant cell lines, doses below the IC10 are typically regarded as safe amounts. We determined the IC5 and IC10 doses of anlotinib in HCT-8/5-FU (3.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30 \u0026micro;M and 6.27\u0026thinsp;\u0026plusmn;\u0026thinsp;3.74 \u0026micro;M, respectively) and HCT-15/5-FU cells (12.01\u0026thinsp;\u0026plusmn;\u0026thinsp;3.16\u0026micro;M and 17.09\u0026thinsp;\u0026plusmn;\u0026thinsp;3.95\u0026micro;M, respectively). We used the IC5 and IC10 dosages of anlotinib to evaluate sensitivity in 5-FU-resistant cell lines. Notably, the proliferation of HCT-8/5-FU and HCT-15/5-FU cells was synergistically inhibited by anlotinib and 5-FU. In comparison to single drugs, the combination induced a significantly higher inhibition rate. Colony formation assays confirmed this synergistic impact to an even greater extent. The number of colonies in the drug combination group was much less than that in single drug groups (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eEffects of anlotinib on cell cycle progression in drug-resistant colorectal cancer cells\u003c/h2\u003e\n \u003cp\u003eWe explored the effects of anlotinib on cell cycle progression in light of its anti-proliferative properties. We treated HCT-15/5-FU cells with 10 \u0026micro;M, 20 \u0026micro;M, and 40 \u0026micro;M doses for cell cycle analysis, as no suppression of proliferation had been observed at 5 \u0026micro;M based on the MTS results. Anlotinib increased the percentage of cells in the G0/G1 phase, with fewer cells entering S phase (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). This effect was most pronounced at 20 \u0026micro;M in HCT-15/5-FU cells and at 10 \u0026micro;M in HCT-8/5-FU cells. Apoptosis was not enhanced under any of the treatment conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eAnlotinib inhibits drug resistance-related protein expression and AKT pathway activation\u003c/h2\u003e\n \u003cp\u003eDrug resistance proteins are frequently upregulated in drug-resistant tumor cells. We assessed the expression of resistance-related proteins MRP1 and MDR1 to further interrogate the mechanism of action of anlotinib in colorectal cancer cells. High concentrations were chosen due to the low inhibition rate at lower concentrations, as per MTS results of anlotinib on drug-resistant colon cancer cells. MDR1 and MRP1 were strongly expressed in HCT-8/5-FU and HCT-15/5-FU cells, as determined via immunoblotting. Increasing anlotinib concentration suppressed their expression. We then assessed the levels of total and phosphorylated AKT (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Increasing anlotinib concentration suppressed p-AKT levels.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAccording to the World Health Organization, colorectal cancer is the third most prevalent malignancy among both men and women, being the second most common cause of cancer-related death worldwide. Colorectal cancer treatment has considerably improved with chemotherapy, targeted therapy, and immunotherapy. However, therapy resistance is common, particularly 5-FU resistance, which results in treatment failure. It is, therefore, crucial to identify novel therapeutics to be employed for overcoming drug resistance.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe novel oral tyrosine kinase inhibitor anlotinib, a small-molecule compound with several targets, has been approved for the treatment of esophageal cancer, advanced lung cancer, and periodontal sarcoma. Further, it has been successful in the treatment of various solid tumors, including colorectal, stomach, and liver.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e There is an increasing body of fundamental research into the effects of anlotinib. In the small-cell lung H446 cell line, Tang et al.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e discovered that anlotinib suppressed cell invasion, migration, and proliferation by inhibiting ERK1/2 and c-met signaling. Anlotinib was also found to downregulate lung adenocarcinoma lymphangiogenesis and lymphatic metastasis.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Anlotinib partially blocks the MEK/ERK pathway to prevent the development of KRAS-mutant lung cancer cells.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Several studies have also demonstrated the effectiveness of anlotinib in digestive tract tumors. Results of the ALTER0703 trial (NCT04080843) revealed that anlotinib was more effective than chemotherapy for treating advanced colorectal cancer.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e miR-940 was shown to improve the inhibitory effect of anlotinib on colorectal cancer cell growth and invasion.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Yang et al.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e reported that anlotinib suppresses colorectal cancer cell proliferation as well as angiogenesis. Anlotinib and dihydroartemisinin were shown to exhibit synergistic anticancer effects in patients with gastric cancer.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e In addition, anlotinib kills liver cancer cells as well as pancreatic cancer cells, in addition to reversing drug resistance in lung cancer and modulating the tumor microenvironment.\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19 CR20 CR21 CR22\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHerein, we used two 5-FU-resistant human colorectal cancer cell lines (HCT-8/5-FU and HCT-15/5-FU) to assess the impact of anlotinib on 5-FU resistance. Treatment with anlotinib drastically reduced cell growth, suggesting that it may be conducive for overcoming chemoresistance. However, anlotinib did not enhance the apoptosis of resistant cancer cells.\u003c/p\u003e \u003cp\u003eTumor cells exhibit either primary or acquired resistance to chemotherapy. The former may arise from multiple origins, including the tumor microenvironment, a low tumor cell growth rate, the difficulty of drug entry into G0 phase cells, and compromised drug activity upon cell entry. The upregulation of resistance-associated proteins is the primary cause of acquired resistance.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e At present, there are 48 members of the ABC transporter family, which can be divided into seven groups (subfamily A-G). They include nine MDR-related drug transporters, ABCB1 (P-gp/MDR1), ABCB4 (MDR3), ABCB11 (BSEP/SPGP), ABCC1 (MRP1), ABCC2 (MRP2), ABCC3 (MRP3), ABCC4 (MRP4), ABCC5 (MRP5) and ABCG2 (BCRP), among which the three most important ones are ABCB1 (P-gp/MDR1), ABCC1 (MRP1), and ABCG2 (BCRP). In particular, MDR1 overexpression is the primary cause of MDR, as described in various studies.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e As an energy-dependent drug efflux pump, MDR1 can influence the efficacy of different chemotherapeutics.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e MDR1 may also act against therapy resistance by upregulating apoptosis-suppressing proteins and inhibiting enzymatic activity. To better understand its mechanism of action in treatment-resistant cells, we examined the effects of anlotinib on cell cycle progression and MDR protein levels. Anlotinib suppressed cancer cell proliferation, preventing entry into S phase. Furthermore, anlotinib suppressed MRP1 and MDR1 expression in a concentration-dependent manner.\u003c/p\u003e \u003cp\u003eThe PI3K-AKT-mTOR pathway is among the common signaling cascades dysregulated in cancer. AKT/ERK signaling is a mediator of anlotinib's impact on colorectal and liver cancer cell growth. Herein, we observed that anlotinib decreased p-AKT levels in a concentration-dependent manner.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present study, we show that anlotinib can suppress the proliferation of 5-FU-resistant colon cancer cells, preventing their entry into the S phase. Anlotinib dose-dependently suppressed AKT signaling and drug resistance-associated protein expression, which may be the key to reversing resistance. Our findings on the efficacy of anlotinib against drug-resistant colorectal cancer cells are yet to be validated in animal studies. Further, the impact on other signaling pathways remains to be explored. The mutation status of factors such as RAS/BRAF genes, microsatellite status, and HER-2 positivity should also be considered.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThere are the authors declare no competing interests mentioned in the work.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Grants No. 82173056 from the Natural Science Foundation of China (to Bangwei Cao) and Beijing Municipal Natural Science Foundation (7222032 to Bangwei Cao) .\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.L. conducted most of the experiments, analyzed data, and wrote the manuscript; L.H., X.Z., N.M., X.L., and R.C. analyzed the data and wrote the original paper; B.C. and M.Y. designed and supervised the project; B.C. takes responsibility for data in this project; and all authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability declaration\u003c/h2\u003e\u003cp\u003eAll data supporting the results of this study can be found in the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68(6):394\u0026ndash;424. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3322/caac.21492\u003c/span\u003e\u003cspan address=\"10.3322/caac.21492\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStamou K, Gouvas N, Pechlivanides G, Xynos E (2018) Primary curative surgery and preemptive or adjuvant hyperthermic peritoneal chemotherapy in colorectal cancer patients at high risk to develop peritoneal carcinomatosis. A systematic review. J BUON 23(5):1249\u0026ndash;1261\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Niu W, Du F et al (2016) Safety, pharmacokinetics, and antitumor properties of anlotinib, an oral multi-target tyrosine kinase inhibitor, in patients with advanced refractory solid tumors. J Hematol Oncol 9(1):105. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13045-016-0332-8\u003c/span\u003e\u003cspan address=\"10.1186/s13045-016-0332-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSyed YY (2018) Anlotinib: first global approval. Drugs 78(10):1057\u0026ndash;1062. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s40265-018-0939-x\u003c/span\u003e\u003cspan address=\"10.1007/s40265-018-0939-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen G, Zheng F, Ren D et al (2018) Anlotinib: a novel multi-targeting tyrosine kinase inhibitor in clinical development. J Hematol Oncol 11(1):120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13045-018-0664-7\u003c/span\u003e\u003cspan address=\"10.1186/s13045-018-0664-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChi Y, Shu Y, Ba Y et al (2021) Anlotinib monotherapy for refractory metastatic colorectal cancer: a double-blinded, placebo-controlled, randomized phase iii trial(Alter0703). Oncologist 26(10):e1693\u0026ndash;e1703. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/onco.13857\u003c/span\u003e\u003cspan address=\"10.1002/onco.13857\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiddik ZH (2003) Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22(47):7265\u0026ndash;7279. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/sj.onc.1206933\u003c/span\u003e\u003cspan address=\"10.1038/sj.onc.1206933\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang X, Zheng Y, Jiao D et al (2021) Anlotinib inhibits cell proliferation, migration and invasion via suppression of c-met pathway and activation of erk1/2 pathway in h446 cells. Anticancer Agents Med Chem 21(6):747\u0026ndash;755. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2174/1871520620666200718235748\u003c/span\u003e\u003cspan address=\"10.2174/1871520620666200718235748\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin T, Liu Z, Wang J et al (2020) Anlotinib suppresses lymphangiogenesis and lymphatic metastasis in lung adenocarcinoma through a process potentially involving VEGFR-3 signaling. Cancer Biol Med 17(3):753\u0026ndash;767. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.20892/j.issn.2095-3941.2020.0024\u003c/span\u003e\u003cspan address=\"10.20892/j.issn.2095-3941.2020.0024\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu H, Liu Y, Tan S et al (2020) Anlotinib exerts anti-cancer effects on kras-mutated lung cancer cell through suppressing the mek/erk pathway. Cancer Manag Res 12:3579\u0026ndash;3587. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/CMAR.S243660\u003c/span\u003e\u003cspan address=\"10.2147/CMAR.S243660\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng Y, Du FC, Fang FQ, Duan ZJ, Lei W, Shi KG (2021) Third-line treatment for metastatic colorectal cancer: anlotinib is superior to chemotherapy and similar to fruquintinib or regorafenib. Neoplasma 67(06):1384\u0026ndash;1390. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4149/neo_2020_191125N1212\u003c/span\u003e\u003cspan address=\"10.4149/neo_2020_191125N1212\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Zhao M, Zhao H, Cheng S, Bai R, Song M (2019) MicroRNA-940 restricts the expression of metastasis-associated gene MACC1 and enhances the antitumor effect of Anlotinib on colorectal cancer. Onco Targets Ther 12:2809\u0026ndash;2822. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/OTT.S195364\u003c/span\u003e\u003cspan address=\"10.2147/OTT.S195364\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Q, Ni L, Imani S et al (2020) Anlotinib suppresses colorectal cancer proliferation and angiogenesis via inhibition of akt/erk signaling cascade. Cancer Manag Res 12:4937\u0026ndash;4948. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/CMAR.S252181\u003c/span\u003e\u003cspan address=\"10.2147/CMAR.S252181\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo Q, Zhang S, Zhang D et al (2021) Effects and mechanisms of anlotinib and dihydroartemisinin combination therapy in ameliorating malignant biological behavior of gastric cancer cells. Curr Pharm Biotechnol 22(4):523\u0026ndash;533. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2174/1389201021666200623132803\u003c/span\u003e\u003cspan address=\"10.2174/1389201021666200623132803\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Wu DX, Meng L, Ji G (2020) Anlotinib combined with SOX regimen (S1 (tegafur, gimeracil and oteracil porassium capsules)\u0026thinsp;+\u0026thinsp;oxaliplatin) in treating stage IV gastric cancer: study protocol for a single-armed and single-centred clinical trial. BMJ Open 10(6):e034685. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1136/bmjopen-2019-034685\u003c/span\u003e\u003cspan address=\"10.1136/bmjopen-2019-034685\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe C, Wu T, Hao Y (2018) Anlotinib induces hepatocellular carcinoma apoptosis and inhibits proliferation via Erk and Akt pathway. Biochem Biophys Res Commun 503(4):3093\u0026ndash;3099. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbrc.2018.08.098\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2018.08.098\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong F, Hu B, Cheng JW et al (2020) Anlotinib suppresses tumor progression via blocking the VEGFR2/PI3K/AKT cascade in intrahepatic cholangiocarcinoma. Cell Death Dis 11(7):573. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41419-020-02749-7\u003c/span\u003e\u003cspan address=\"10.1038/s41419-020-02749-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L, Zhou X, Sun J et al (2020) Reactive oxygen species mediate anlotinib-induced apoptosis via activation of endoplasmic reticulum stress in pancreatic cancer. Cell Death Dis 11(9):766. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41419-020-02938-4\u003c/span\u003e\u003cspan address=\"10.1038/s41419-020-02938-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLian Z, Du W, Zhang Y et al (2020) Anlotinib can overcome acquired resistance to EGFR-TKIs via FGFR1 signaling in non‐small cell lung cancer without harboring EGFR T790M mutation. Thorac Cancer 11(7):1934\u0026ndash;1943. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/1759-7714.13485\u003c/span\u003e\u003cspan address=\"10.1111/1759-7714.13485\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan LH, Zhang D, Mo SS, Yuan H, Mo XW, Zhao JM (2021) Anlotinib suppresses metastasis and multidrug resistance \u003cem\u003evia\u003c/em\u003e dual blockade of MET/ABCB1 in colorectal carcinoma cells. J Cancer 12(7):2092\u0026ndash;2104. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7150/jca.45618\u003c/span\u003e\u003cspan address=\"10.7150/jca.45618\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLan W, Zhao J, Chen W, Shang H, Peng J, Lin J (2021) Anlotinib overcomes multiple drug resistant colorectal cancer cells via inactivating pi3k/akt pathway. Anticancer Agents Med Chem 21(15):1987\u0026ndash;1995. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2174/1871520621666210112113852\u003c/span\u003e\u003cspan address=\"10.2174/1871520621666210112113852\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu S, Qin T, Liu Z et al (2020) Anlotinib alters tumor immune microenvironment by downregulating pd-l1 expression on vascular endothelial cells. Cell Death Dis 11(5):309. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41419-020-2511-3\u003c/span\u003e\u003cspan address=\"10.1038/s41419-020-2511-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Li L, Jiang Z, Wang B, Pan Z (2020) Anlotinib optimizes anti-tumor innate immunity to potentiate the therapeutic effect of PD-1 blockade in lung cancer. Cancer Immunol Immunother 69(12):2523\u0026ndash;2532. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00262-020-02641-5\u003c/span\u003e\u003cspan address=\"10.1007/s00262-020-02641-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRivera G, Wakelee HA (2016) Resistance to therapy. In: Reckamp KL, ed. \u003cem\u003eLung Cancer\u003c/em\u003e. Vol 170. Springer International Publishing; :183\u0026ndash;202. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-319-40389-2_9\u003c/span\u003e\u003cspan address=\"10.1007/978-3-319-40389-2_9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim ES (2016) Chemotherapy resistance in lung cancer. In: Ahmad A, Gadgeel S, eds. \u003cem\u003eLung Cancer and Personalized Medicine\u003c/em\u003e. Vol 893. Springer International Publishing; :189\u0026ndash;209. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-319-24223-1_10\u003c/span\u003e\u003cspan address=\"10.1007/978-3-319-24223-1_10\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMealey KL, Fidel J (2015) P-glycoprotein mediated drug interactions in animals and humans with cancer. J Vet Intern Med 29(1):1\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/jvim.12525\u003c/span\u003e\u003cspan address=\"10.1111/jvim.12525\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"colorectal cancer, multi-target TKI, anlotinib, 5-fluorouracil, drug resistance","lastPublishedDoi":"10.21203/rs.3.rs-4450056/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4450056/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eColorectal cancer is the most prevalent gastrointestinal malignancy. Treatment usually includes 5-fluorouracil (5-FU), oxaliplatin, and irinotecan, with 5-FU usually being the first choice. 5-FU treatment failure occurs when cancer cells acquire resistance. Therefore, it is crucial to identify compounds effective against 5-FU-resistant tumors. Herein, we determined the efficacy and mechanism of anlotinib in 5-FU-resistant colon cancer cells.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003eHuman colon cancer cells (HCT-8/5-FU and HCT-15/5-FU) resistant to 5-FU were subjected to treatment with anlotinib, 5-FU, or both. Cell proliferation was assessed via MTS and clone formation assays. Cell cycle progression was studied using flow cytometry. Through immunoblotting, we evaluated changes in the protein levels of p-AKT and multidrug resistance 1.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMTS assays indicated that HCT-8/5-FU and HCT-15/5-FU cells were sensitive to anlotinib and resistant to 5-FU. At 48 h, HCT-8/5-FU had an IC50 of 2246.5\u0026thinsp;\u0026plusmn;\u0026thinsp;204.5 \u0026micro;M, while HCT-15/5-FU had an IC50 of 18.49\u0026thinsp;\u0026plusmn;\u0026thinsp;3.23 mM for 5-FU. The IC50 of anlotinib for HCT-8/5-FU cells was 53.69\u0026thinsp;\u0026plusmn;\u0026thinsp;8.10\u0026micro;M at 24 h and 17.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u0026micro;M at 48 h. The IC50 values for HCT-15/5-FU at 24 and 48 h were 55.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44\u0026micro;M and 8.83\u0026thinsp;\u0026plusmn;\u0026thinsp;3.02\u0026micro;M, respectively. Anlotinib enhanced 5-FU sensitivity in resistant cells, with low concentrations (IC10) considerably enhancing the antiproliferative effects of 5-FU. Further, anlotinib significantly increased the number of cells in the G0-G1 phase dose-dependently, while the proportion of cells entering S phase decreased. MDR1 and AKT expression decreased with increasing anlotinib concentration.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eAnlotinib suppressed the proliferation of 5-FU-resistant colon cancer cells by preventing entry into S phase, thus sensitizing cells to 5-FU. Moreover, anlotinib may reverse the effect of 5-FU on drug-resistant cells by down-regulating the expression of multidrug-resistant proteins, in which the AKT signaling pathway may play an important role.\u003c/p\u003e","manuscriptTitle":"Inhibitory Effect of the Multi-target TKI, Anlotinib, in 5-FU Resistant Colorectal Cancer HCT-8/15 Cells: Down Regulation of Drug Resistance-associated Protein Expression.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-05 17:41:23","doi":"10.21203/rs.3.rs-4450056/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":"e6071fc9-f824-41cf-a8d0-6e9743cda36b","owner":[],"postedDate":"June 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-11T20:38:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-05 17:41:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4450056","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4450056","identity":"rs-4450056","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}