Objective The aim of this study was to investigate the effect of p38 on stem cell maintenance of pancreatic cancer. Methods Human pancreatic cancer cells PANC-1 were treated with different concentrations of 5-fluorouracil(5-FU)(0.5×IC₅₀, IC₅₀, and 2×IC₅₀) for 24 hours, and VX-702 (p38 phosphorylation inhibitor) was added, and the cells were inoculated in 6-well culture dishes with ultra-low adhesion to observe the changes of sphere tumors. The expression levels of cyclin-dependent kinase 2(CDK2), cyclin B1 and D1, Octamer-binding transcription factor 4(OCT4), SRY-box transcription factor 2(SOX2), Nanog and p38 were measured by Western blot. The mRNA expression levels of p38, OCT4, Nanog and SOX2 were tested by RT-PCR. Cell cycle, apoptosis, and the proportion of CD44⁺CD133⁺PANC-1 cells were evaluated by flow cytometry. Results The results showed that 5-FU inhibited the formation of tumor spheres in PANC-1 cells, increased CD44⁺CD133⁺cell fragments, down-regulated the expression of OCT4, Nanog and SOX2, and inhibited the stemness maintenance of PANC-1 tumor stem cells. Phosphorylation of PANC-1 cells was inhibited by a highly selective p38 MAPK inhibitor, VX-702(p38 mitogen-activated protein kinase inhibitor), which had the same effect as 5-FU treatment. When VX-702 combined with 5-FU was used to treat PANC-1 cells, the therapeutic effect was enhanced. Conclusion p38 inhibitors decreased PANC-1 cell activity and increased cell apoptosis. p38 inhibitors inhibit the stemness maintenance of pancreatic cancer stem cells.
Humans ; Phosphorylation/drug effects* ; Cell Line, Tumor ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors* ; Neoplastic Stem Cells/metabolism* ; Drug Resistance, Neoplasm/drug effects* ; Fluorouracil/pharmacology* ; Pancreatic Neoplasms/pathology* ; Apoptosis/drug effects* ; SOXB1 Transcription Factors/genetics* ; Octamer Transcription Factor-3/genetics*

OBJECTIVES: Oxaliplatin (OXA) and 5-fluorouracil (5-FU) are 2 commonly used chemotherapeutic agents for colorectal cancer (CRC). MicroRNAs (miRNAs, miRs) play crucial roles in the development of chemoresistance in various cancers. However, the role and mechanism of miR-224-5p in regulating CRC chemoresistance remain unclear. This study aims to investigate the function of miR-224-5p in chemoresistant CRC cells and the underlying mechanisms. METHODS: CRC datasets GSE28702 and GSE69657 were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs between drug-sensitive and resistant groups (OXA or 5-FU) were analyzed, and miR-224-5p was identified as the target miRNA. Chemoresistant cell lines HCT15-OXR, HCT15-5-FU, SW480-OXR, and SW480-5-FU were established. Transient transfections were performed using miR-224-5p mimics, inhibitors, and their respective negative controls (control mimic, control inhibitor) in these cell lines. Cells were treated with different concentrations of OXA or 5-FU post-transfection, and the half-maximal inhibitory concentration (IC₅₀) was determined using the cell counting kit-8 (CCK-8) assay. Cell proliferation was assessed by CCK-8 and colony formation assays. The expression levels of miR-224-5p, LC3, and P62 were measured by real-time polymerase chain reaction (real-time PCR) and/or Western blotting. Autophagic flux was assessed using a tandem fluorescent-tagged LC3 reporter assay. TargetScan 8.0, miRTarBase, miRPathDB, and HADb were used to predict B-cell lymphoma-2 (Bcl-2) as a potential miR-244-5p target, which was further validated by dual-luciferase reporter assays. RESULTS: Chemoresistant CRC cells exhibited down-regulated miR-224-5p expression, whereas up-regulation of miR-224-5p enhanced chemotherapy sensitivity. Exposure to OXA or 5-FU significantly increased autophagic activity in chemoresistant CRC cells, which was reversed by miR-224-5p overexpression. Dual-luciferase assays verified Bcl-2 as a direct target of miR-224-5p. CONCLUSIONS MiR-224-5p regulates chemoresistance in CRC by modulating autophagy through direct targeting of Bcl-2.
Humans ; MicroRNAs/physiology* ; Colorectal Neoplasms/drug therapy* ; Drug Resistance, Neoplasm/genetics* ; Autophagy/drug effects* ; Fluorouracil/pharmacology* ; Oxaliplatin ; Cell Line, Tumor ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Gene Expression Regulation, Neoplastic

In recent years, the clinical treatment of colorectal cancer(CRC) has made great progress, but chemoresistance is still one of the main reasons for reducing the survival rate of patients with colorectal cancer. Therefore, ameliorating chemotherapy resis-tance is an urgent problem to be solved. The purpose of this study was to investigate the regulatory role and related molecular mechanisms of hydroxysafflor yellow A(HSYA) in colorectal cancer cell proliferation, migration, and 5-fluorouracil(5-FU) chemoresistance. In this study, HCT116 and HT-29 cells were used as research subjects. Firstly, methyl thiazolyl tetrazolium(MTT) assay and colony formation assay were used to detect and analyze the effect of HSYA on the proliferation of CRC cells. Secondly, the effect of HSYA on the cell cycle in CRC cells was analyzed by cell cycle assay. Furthermore, the effect of HSYA on the migration of CRC cells was analyzed by wound-healing assay and Transwell assay. Based on the above, the influences of HSYA on 5-FU chemoresistance of CRC cells and related molecular mechanisms were explored and analyzed. The results showed that HSYA significantly inhibited the proliferation and migration of CRC cells, and arrested the cell cycle in G_0/G_1 phase. In addition, HSYA significantly ameliorated the chemoresistance of CRC cells to 5-FU. The results of acridine orange staining and Western blot showed that the autophagy activity of CRC cells in the HSYA and 5-FU combined treatment group was significantly higher than that in the 5-FU single drug treatment group. As compared with the 5-FU single drug treatment group, the phosphorylation levels of protein kinase B(Akt) and mammalian target of rapamycin(mTOR) in the HSYA and 5-FU combined treatment group were significantly reduced, indicating that the Akt/mTOR signaling pathway in the combined treatment group was down-regulated in CRC cells. In conclusion, HSYA may upregulate autophagy activity through the Akt/mTOR signaling pathway, thereby inhibiting the proliferation and migration of CRC cells and ameliorating the chemoresistance to 5-FU.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Drug Resistance, Neoplasm ; Cell Line, Tumor ; TOR Serine-Threonine Kinases/metabolism* ; Fluorouracil/pharmacology* ; Cell Proliferation ; Autophagy ; Colorectal Neoplasms/drug therapy*

This study aims to investigate the regulatory effects of Astragalus polysaccharide(APS) and APS combined with 5-fluorouracil(5-FU) on indoleamine-2,3-dioxygenase(IDO1) in the colon tumor microenvironment. Sixty Balb/c mice were randomized into a blank group, a model group, an APS group, an APS + 5-FU group, an APS + low-dose 5-FU group, and a 5-FU group. A tumor model was established by subcutaneous transplantation with CT-26 mouse colon cancer cells in other groups except the blank group. After successful modeling, each group was treated with corresponding drugs for 7 days. The general condition, body weight, and tumor volume of the mice were observed and measured daily during the treatment period. The mice were sacrificed at the end of treatment, and the tumor suppression rate and spleen index of the mice were calculated. Western blot and fluorescence quantitative PCR were employed to determine the protein and mRNA levels, respectively, of IDO1 in the tumor tissue of mice. High performance liquid chromatography was employed to measure the levels of tryptophan(Trp) and kynurenine(Kyn) in the tumor tissue of mice. Hematoxylin-eosin(HE) staining was performed to observe the histological changes of the tumor tissue, and immunohistochemistry to detect the changes of CD4 and CD8 expression in the tumor tissue. Compared with that in the model group, the tumor volume of mice in each treatment group significantly reduced. The body weights of mice in APS + 5-FU group and 5-FU group significantly reduced from day 4 to day 7 of treatment. In addition, the APS + 5-FU group and 5-FU group showed significantly decreased spleen index. The protein and mRNA levels of IDO1 were significantly down-regulated in the APS, APS + 5-FU, and APS + low-dose 5-FU groups. The drug interventions significantly increased the Trp content and decreased the Kyn content. The APS + 5-FU group showed significantly reduced infiltration of CD4~+ T lymphocytes and increased infiltration of CD8~+ T lymphocytes. APS inhibited the expression of IDO1 in the colon tumor microenvironment to increase CD8~+ T lymphocyte infiltration, and the combination of APS with 5-FU demonstrated better effect.
Mice ; Animals ; Tumor Microenvironment ; Colonic Neoplasms/genetics* ; Fluorouracil/pharmacology* ; Polysaccharides/pharmacology* ; CD8-Positive T-Lymphocytes/metabolism* ; RNA, Messenger/metabolism*

Astragali Radix-Curcumae Rhizoma(AR-CR) is a combination commonly used in the clinical treatment of tumors. Based on the T helper 17(Th17)/regulatory T cell(Treg) balance, the present study explored the possible mechanism of AR-CR combined with 5-fluorouracil(5-FU) on the tumor growth of orthotopic xenograft model mice of colorectal carcinoma. Ninety male BALB/c mice were randomly divided into nine groups, i.e., a blank group, a model group, a 5-FU group, high-, medium-, and low-dose AR-CR(2∶1) groups, and high-, medium-, and low-dose AR-CR+5-FU groups, with 10 mice in each group. The orthotopic xenograft model of CT26.WT colorectal carcinoma was induced in mice except those in the blank group. Twenty-four hours after the ope-ration, mice in the blank group and the model group received normal saline by gavage(10 mL·kg~(-1), once per day), and those in the 5-FU group received 5-FU by intraperitoneal injection(25 mg·kg~(-1), once every other day). Mice in the AR-CR groups received AR and CR decoctions by gavage(12, 6, and 3 g·kg~(-1), once a day) and those in the combination groups received AR and CR decoctions and 5-FU(doses and administration methods were the same as above). After intervention for three weeks, all mice were sacrificed and tumor tissues were collected. The tumor mass was weighed and the average tumor weight was calculated. The changing trend of Th17/Treg(%) in the CD4~+T lymphocytes of the spleen tissues of the mice in each group was detected. The mRNA expression in the blood and protein expression in the tumor tissues of transforming growth factor-β(TGF-β), tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ), Smad4, N-cadherin, matrix metalloproteinase-7(MMP-7) were detected. The experimental results revealed that compared with the model group, the groups with drug intervention showed reduced tumor mass(P<0.01), decreased CD4~+IL-17~+ in the spleen tissues to varying degrees(P<0.001), and increased proportion of CD4~+Foxp3~+(P<0.001 or P<0.05), indicating that Th17/Treg maintained dynamic balance, and the effect of the combination groups was predominant. Additionally, the mRNA expression in the blood and protein expression in the tumor tissues of TGF-β, TNF-α, IFN-γ, Smad4, N-cadherin, and MMP-7 declined to varying degrees in a dose-dependent manner(P<0.01 or P<0.001). The AR-CR combined with 5-FU can inhibit the tumor growth of orthotopic xenograft model mice of CT26.WT colorectal carcinoma. The mechanism may be related to maintenance of Th17/Treg dynamic balance in the body and down-regulation of TGF-β, TNF-α, IFN-γ, Smad4, N-cadherin, and MMP-7 expression.
Animals ; Colorectal Neoplasms/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Fluorouracil/pharmacology* ; Heterografts ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger/metabolism* ; T-Lymphocytes, Regulatory ; Th17 Cells

Currently, chemoresistance seriously attenuates the curative outcome of liver cancer. The purpose of our work was to investigate the influence of 6-shogaol on the inhibition of 5-fluorouracil (5-FU) in liver cancer. The cell viability of cancer cells was determined by MTT assay. Liver cancer cell apoptosis and the cell cycle were examined utilizing flow cytometry. Moreover, qRT-PCR and western blotting was used to analyse the mRNA and protein expression levels, respectively. Immunohistochemistry assays were used to examine multidrug resistance protein 1 (MRP1) expression in tumour tissues. In liver cancer cells, we found that 6-shogaol-5-FU combination treatment inhibited cell viability, facilitated G0/G1 cell cycle arrest, and accelerated apoptosis compared with 6-shogaol or 5-FU treatment alone. In cancer cells cotreated with 6-shogaol and 5-FU, AKT/mTOR pathway- and cell cycle-related protein expression levels were inhibited, and MRP1 expression was downregulated. AKT activation or MRP1 increase reversed the influence of combination treatment on liver cancer cell viability, apoptosis and cell cycle arrest. The inhibition of AKT activation to the anticancer effect of 6-shogaol-5-FU could be reversed by MRP1 silencing. Moreover, our results showed that 6-shogaol-5-FU combination treatment notably inhibited tumour growth in vivo. In summary, our data demonstrated that 6-shogaol contributed to the curative outcome of 5-FU in liver cancer by inhibiting the AKT/mTOR/MRP1 signalling pathway.
ATP Binding Cassette Transporter, Subfamily B, Member 1 ; Apoptosis ; Catechols ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Fluorouracil/pharmacology* ; Humans ; Liver Neoplasms/genetics* ; Multidrug Resistance-Associated Proteins ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/metabolism*

OBJECTIVES: Fluorouracil chemotherapeutic drugs are the classic treatment drugs of gastric cancer. But the problem of drug resistance severely limits their clinical application. This study aims to investigate whether hypoxia microenvironment affects gastric cancer resistance to 5-fluorouracil (5-FU) and discuss the changes of gene and proteins directly related to drug resistance under hypoxia condition. METHODS: Gastric cancer cells were treated with 5-FU in hypoxia/normoxic environment, and were divided into a Normoxic+5-FU group and a Hypoxia+5-FU group. The apoptosis assay was conducted by flow cytometry Annexin V/PI double staining. The real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression level of hypoxia inducible factor-1α (HIF-1α), multidrug resistance (MDR1) gene, P-glycoprotein (P-gp), and vascular endothelial growth factor (VEGF) which were related to 5-FU drug-resistance. We analyzed the effect of hypoxia on the treatment of gastric cancer with 5-FU. RESULTS: Compared with the Normoxic+5-FU group, the apoptosis of gastric cancer cells treated with 5-FU in the Hypoxia+5-FU group was significantly reduced (P<0.05), and the expression of apoptosis promoter protein caspase 8 was also decreased. Compared with the the Normoxic+5-FU group, HIF-1α mRNA expression in the Hypoxia+5-FU group was significantly increased (P<0.05), and the mRNA and protein expression levels of MDR1, P-gp and VEGF were also significantly increased (all P<0.05). The increased expression of MDR1, P-gp and VEGF had the same trend with the expression of HIF-1α. CONCLUSIONS Hypoxia is a direct influencing factor in gastric cancer resistance to 5-FU chemotherapy. Improvement of the local hypoxia microenvironment of gastric cancer may be a new idea for overcoming the resistance to 5-FU in gastric cancer.
Humans ; Fluorouracil/therapeutic use* ; Vascular Endothelial Growth Factor A/metabolism* ; Stomach Neoplasms/drug therapy* ; Drug Resistance, Multiple ; Vascular Endothelial Growth Factors/metabolism* ; Hypoxia ; ATP Binding Cassette Transporter, Subfamily B/genetics* ; ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics* ; Cell Line, Tumor ; Cell Hypoxia ; RNA, Messenger/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Tumor Microenvironment

OBJECTIVE: To evaluate the protective function of Babao Dan (BBD) on 5-flurouracil (5-FU)-induced intestinal mucositis (IM) and uncover the underlying mechanism. METHODS: A total of 18 male mice were randomly divided into 3 groups by a random number table, including control, 5-FU and 5-FU combined BBD groups, 6 mice in each group. A single intraperitoneal injection of 5-FU (150 mg/kg) was performed in 5-FU and 5-FU combined BBD groups on day 0. Mice in 5-FU combined BBD group were gavaged with BBD (250 mg/kg) daily from day 1 to 6. Mice in the control group were gavaged with saline solution for 6 days. The body weight and diarrhea index of mice were recorded daily. On the 7th day, the blood from the heart of mice was collected to analyze the proportional changes of immunological cells, and the mice were subsequently euthanized by mild anesthesia with 2% pentobarbital sodium. Colorectal lengths and villus heights were measured. Intestinal-cellular apoptosis and proliferation were evaluated by Tunel assay and immunohistochemical staining of proliferating cell nuclear antigen, respectively. Immunohistochemistry and Western blot were performed to investigate the expressions of components in Wnt/β-catenin pathway (Wnt3, LRP5, β-catenin, c-Myc, LRG5 and CD44). RESULTS: BBD obviously alleviated 5-FU-induced body weight loss and diarrhea, and reversed the decrease in the number of white blood cells, including monocyte, granulocyte and lymphocyte, and platelet (P<0.01). The shortening of colon caused by 5-FU was also reversed by BBD (P<0.01). Moreover, BBD inhibited apoptosis and promoted proliferation in jejunum tissues so as to reduce the intestinal mucosal damage and improve the integrity of villus and crypts. Mechanically, the expression levels of Wnt/β -catenin mediators such as Wnt3, LRP5, β-catenin were upregulated by BBD, activating the transcription of c-Myc, LRG5 and CD44 (P<0.01). CONCLUSIONS BBD attenuates the adverse effects induced by 5-FU via Wnt/β-catenin pathway, suggesting it may act as a potential agent against chemotherapy-induced intestinal mucositis.
Animals ; Male ; Mice ; Antineoplastic Agents/therapeutic use* ; beta Catenin/metabolism* ; Diarrhea/drug therapy* ; Fluorouracil/pharmacology* ; Intestinal Mucosa ; Mucositis/metabolism* ; Pentobarbital/therapeutic use* ; Proliferating Cell Nuclear Antigen/metabolism* ; Saline Solution

This study aims to investigate the potential mechanism of curcumin in mediating interleukin-6(IL-6)/signal transducer and activator of transcription 3(STAT3) signaling pathway to repair intestinal mucosal injury induced by 5-fluorouracil(5-FU) chemotherapy for colon cancer. SD rats were intraperitoneally injected with 60 mg·kg~(-1)·d~(-1) 5-FU for 4 days to establish a model of intestinal mucosal injury. Then the rats were randomly divided into model group(equal volume of normal saline), curcumin low, medium and high dose groups(50, 100, 200 mg·kg~(-1)), and normal SD rats were used as control group(equal volume of normal saline). Each group received gavage administration for 4 consecutive days, and the changes of body weight and feces were recorded every day. After administration, blood was collected from the heart, and jejunum tissues were collected. The levels of serum interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were detected by ELISA, and at the same time, the concentration of Evans blue(EB) in jejunum was measured. Hematoxylin-eosin(HE) staining was used to observe the pathological state of jejunum, and the length of jejunum villi and the depth of crypt were measured. The positive expression levels of claudin, occludin and ZO-1 were detected by immunohistochemistry. Western blot was used to detect the protein expression of IL-6, p-STAT3, E-cadherin, vimentin and N-cadherin in jejunum tissues. The results showed that, curcumin significantly increased body weight and fecal weight(P<0.05 or P<0.01), decreased fecal score, EB concentration, IL-1β and TNF-α levels(P<0.05 or P<0.01) in rats. In addition, curcumin maintained the integrity of mucosal surface and villi structure of jejunum to a large extent, and reduced pathological changes in a dose-dependent manner. Meanwhile, curcumin could increase the positive expression of occludin, claudin and ZO-1(P<0.05 or P<0.01), repair intestinal barrier function, downregulate the protein expression of IL-6, p-STAT3, vimentin and N-cadherin in jejunum tissues(P<0.05 or P<0.01), and upregulate the protein expression of E-cadherin(P<0.05). Therefore, curcumin could repair the intestinal mucosal injury induced by 5-FU chemotherapy for colon cancer, and the mechanism may be related to the inhibition of IL-6/STAT3 signal and the inhibition of epithelial-mesenchymal transition(EMT) process.
Animals ; Colonic Neoplasms/drug therapy* ; Curcumin ; Fluorouracil/toxicity* ; Interleukin-6/genetics* ; Intestinal Mucosa/metabolism* ; Rats ; Rats, Sprague-Dawley ; STAT3 Transcription Factor/metabolism* ; Signal Transduction

OBJECTIVE: To investigate the effects of ethanol extract of Patrinia scabiosaefolia (EEPS) on chemo-resistance of colorectal cancer cells (CRC) and explore the possible molecular mechanisms. METHODS: 5-fluorouracil (5-FU)-resistant human colorectal carcinoma cell line (HCT-8/5-FU) and its parental cells HCT-8 were treated with EEPS (0, 0.25, 0.50, 1 or 2 mg/mL), or 5-FU (0, 100, 200, 400, 800 or 1600 μmol/L). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the cell viability. Cell density was observed by phase-contrast microscope, cell counting and colony formation assay were used to determine the cell proliferation of HCT-8/5-FU cells treated with 0, 0.5, 1 or 2 mg/mL EEPS. Cell apoptosis was determined by Hoechst staining. Western-blot was performed to detect the phosphorylation of AKT as well as the protein expression level of B-cell CLL/lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax). RESULTS: Compared with HCT-8 cells, MTT assay results indicated that HCT-8/5-FU cells were resistant to 5-FU treatment (P<0.05), and sensitive to EEPS treatment (P>0.05). Moreover, compared with untreated HCT-8/5-FU cells, 1 and 2 mg/mL of EEPS treatment significantly reduced cell density, cell number, inhibited cell survival (P<0.05), and induced apoptosis in HCT-8/5-FU cells. Furthermore, 1 and 2 mg/mL of EEPS significantly decreased the phosphorylation level of p-AKT and Bcl-2 protein expression, and increased the expression of Bax protein (P<0.05). CONCLUSION EEPS is a promising therapeutic agent that may overcome chemo-resistance in cancer cells, likely through suppression of the AKT pathway and promotion of cancer cell apoptosis.
Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Colorectal Neoplasms ; drug therapy ; pathology ; Drug Resistance, Neoplasm ; drug effects ; Fluorouracil ; pharmacology ; therapeutic use ; Humans ; Patrinia ; chemistry ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; drug effects ; Tumor Stem Cell Assay ; bcl-2-Associated X Protein ; metabolism