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Objective:To observe the effect of Jianpi Xiaoai prescription on long non-coding RNA Hox transcript antisense intergenic RNA （lncRNA HOTAIR）/Janus kinase 2 （JAK2） /signal transducer and activator of transcription 3 （STAT3） signaling pathway and to explore the potential mechanism of Jianpi Xiaoai prescription in suppressing the metastasis of colon cancer. Method:The expression of lncRNA HOTAIR in different cells was analyzed. Following the treatment of HCT116 cells with 10%，15%，and 20% Jianpi Xiaoai prescription -containing serum， the invasive ability of Jianpi Xiaoai prescription on HCT116 cells was assessed by transwell assay. The mRNA expression levels of lncRNA HOTAIR，JAK2，and STAT3 were measured by Real-time polymerase chain reaction （Real-time PCR）， and the protein expression levels of JAK2， phosphorylated STAT3 （p-STAT3） and STAT3 by Western blot. Result:The highest expression of lncRNA HOTAIR was detected in HCT116 cells. Compared with the blank group， each Jianpi Xiaoai prescription group exhibited a decreased number of invasive cells （<italic>P</italic><0.05， <italic>P</italic><0.01）. The relative JAK2 mRNA expression in the middle-dose Jianpi Xiaoai prescription group was down-regulated （<italic>P</italic><0.05）， and the relative lncRNA HOTAIR mRNA expression in the middle- and high-dose Jianpi Xiaoai prescription groups and the relative JAK2 mRNA expression in the high-dose Jianpi Xiaoai prescription group were remarkably down-regulated （<italic>P</italic><0.01）. Compared with the blank group，the relative p-STAT3 protein expression was down-regulated in the middle-dose Jianpi Xiaoai prescription group （<italic>P</italic><0.05）， and the relative JAK2 protein expression in the middle- and high-dose Jianpi Xiaoai prescription groups and the relative p-STAT3 protein expression in the high-dose Jianpi Xiaoai prescription group were remarkably down-regulated （<italic>P</italic><0.01）. Conclusion:Jianpi Xiaoai prescription effectively inhibits the metastasis of colon cancer cells， which may be related to the inhibition of lncRNA HOTAIR/JAK2/STAT3 signaling pathway.
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Objective::To investigate the effect of drug-containing serum of Jianpi Xiaoai prescription on protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in colorectal cells HCT116. Method::The HTC116 cells were treated by 15%concentration of drug-contained serum, and then the cell migration and invasion were detected by Transwell assay, the protein expression levels of Akt, phosphorylated protein kinase B (p-Akt), mTOR, phosphorylated mammalian target of rapamycin (p-mTOR), ribosomal protein S6 kinase, polypeptide1(S6K1), phosphorylated ribosomal protein S6 kinase, polypeptide1 (p-S6K1), 4E-binding protein1(4EBP1), and phosphorylated 4E-binding protein1(p-4EBP1) in HCT116 cells were detected by Western blot. The control group was treated by untreated serum (15%), and 10%fetal bovine serum(FBS). Result::As compared with the control group, the number of migration and invasion cells was significantly reduced in drug-contained serum group (P<0.01), the expression of Akt had no obvious decrease, p-Akt protein expression was significantly lowered in the drug-contained serum group (P<0.01), the expression of mTOR had no obvious decrease, but p-mTOR protein expression was significantly lowered in drug-contained serum group (P<0.01), the expression of S6K1 had no obvious decrease, but p-S6K1 protein expression was significantly lowered in the drug-contained serum group (P<0.01), the protein expression of 4EBP1 had no obvious decrease, but p-4EBP1 protein expression was significantly lowered in the drug-contained serum group (P<0.01). Conclusion::The anti-tumor mechanism and transfer of Jianpi Xiaoai prescription may be related to inhibiting the activation of Akt/mTOR signaling pathways in colorectal cancer.
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Objective:To observe the effect of polyphyllin Ⅰ on the expressions of forkhead box Q1（FOXQ1）and epithelial-mesenchymal transition (EMT)-related factors, in order to explore the possible mechanism of polyphyllin Ⅰ in inhibiting the metastasis of colon cancer. Method:After the treatment with 1.25，2.50 μmol·L-1 polyphyllin Ⅰ on HCT116 cells, Western blot and Real-time PCR were used to detect the expressions of FOXQ1，E-cadherin，Vimentin protein and mRNA. Result:Compared with the blank group, relative expressions of FOXQ1 protein and mRNA in low-concentration polyphyllin Ⅰ group were decreased, while relative expression of E-cadherin mRNA was increased, the differences were not statistically significant, and relative expressions of Vimentin protein and mRNA in low-concentration polyphyllin Ⅰ group were decreased (P<0.05)， and relative expression of E-cadherin protein in low-concentration polyphyllin Ⅰ group was increased (P<0.01). Compared with the blank group, relative expressions of FOXQ1, Vimentin protein and mRNA in high-concentration polyphyllin Ⅰ group were decreased，while relative expressions of E-cadherin protein and mRNA were increased (P<0.05, P<0.01). Compared with low-concentration polyphyllin Ⅰ group, relative expressions of Vimentin protein and mRNA in high-concentration polyphyllin Ⅰ group were decreased, but the difference was not statistically significant, relative expressions of E-cadherin protein and mRNA in high-concentration polyphyllin Ⅰ group were increased, whereas relative expressions of FOXQ1 protein and mRNA were decreased (P<0.05, P<0.01). Conclusion:Mechanism of polyphyllin Ⅰ inhibiting the metastasis of colon cancer may be related to the decrease of FOXQ1 and Vimentin expressions, and the up-regulation of E-cadherin.
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Objective:To observe the effect of Jianpi Xiaoai prescription on the activation of normal human embryonic lung fibroblasts （HFL1） into tumor-associated fibroblasts （CAFs） induced by human colon cancer cells （HCT116） derived exosomes. Method:SD rats were gavaged with 13.1 g·kg-1 of Jianpi Xiaoai prescription to prepare drug-containing serum， and HCT116 cell exosomes-containing 10% exosomes-free serum and 20% Jianpi Xiaoai prescription drug serum were isolated by ultra-high speed centrifugation. The particle size distribution of exosomes were detected by Nanoparticle tracking analyzer （Zetaview）， and the exosomes' marker proteins apoptotic transfer gene 2 interaction protein X （Alix）， heat shock protein 70 （HSP70）， and tumor-susceptibility gene 101 （TSG101） were identified by Western blot， and the uptake of exosomes labeled with cell membrane staining kit （PKH67） by HFL1 was observed by fluorescence microscope. HFL1 cells were divided into six groups： the blank group， the transforming growth factor-β1 （TGF-β1） group， the TGF-β1 combined with HCT116 exosomes of 2 mg·L-1 group， the TGF-β1 combined with HCT116 exosomes of 4 mg·L-1 group， the TGF-β1 combined with Jianpi Xiaoai prescription exosomes of 2 mg·L-1 group， and the TGF-β1 combined with Jianpi Xiaoai prescription exosomes of 4 mg·L-1 group， and all groups were cultivated for 48 h. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to determine the protein and mRNA expressions of α-smooth muscle actin （α-SMA）. Result:The particle size distribution detected by Zetaview was mainly between 50-100 nm， and the exosomes were verified based on the expressions of marker proteins Alix， HSP70 and TSG101. After co-incubation of HFL1 cells with exosomes， a large number of exosomes were absorbed by HFL1 cells under fluorescence microscope. Compared with the blank control group， the protein and mRNA expressions of α-SMA in the TGF-β1 group and TGF-β1 combined with HCT116 exosome groups were increased （P<0.01）. Compared with the TGF-β1 combined with HCT116 exosome groups， the protein and mRNA expressions of α-SMA were decreased in the TGF-β1 combined with Jianpi Xiaoai prescription exosome groups （P<0.01）. Conclusion:Human colon cancer cell exosomes combined with TGF-β1 can induce the activation of HFL1 into CAFs， and Jianpi Xiaoai prescription can reduce the activation of HFL1 by affecting the expressions of α-SMA， thus antagonizing the lung metastasis of colon cancer.
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OBJECTIVE@#To evaluate the association between Chinese medicine (CM) therapy and disease-free survival (DFS) outcomes in postoperative patients with non-small cell lung cancer (NSCLC).@*METHODS@#This multiple-center prospective cohort study was conducted in 13 medical centers in China. Patients with stage I, II, or IIIA NSCLC who had undergone radical resection and received conventional postoperative treatment according to the National Comprehensive Cancer Network (NCCN) guidelines were recruited. The recruited patients were divided into a CM treatment group and a control group according to their wishes. Patients in the CM treatment group received continuous CM therapy for more than 6 months or until disease progression. Patients in the control group received CM therapy for less than 1 month. Follow-up was conducted over 3 years. The primary outcome was DFS, with recurrence/metastasis rates as a secondary outcome.@*RESULTS@#Between May 2013 and August 2016, 503 patients were enrolled into the cohort; 266 were classified in the CM treatment group and 237 in the control group. Adjusting for covariates, high exposure to CM was associated with better DFS [hazard ratio (HR) = 0.417, 95% confidential interval (CI): 0.307-0.567)]. A longer duration of CM therapy (6-12 months, 12-18 months, >24 months) was associated with lower recurrence and metastasis rates (HR = 0.225, 0.119 and 0.083, respectively). In a subgroup exploratory analysis, CM therapy was also a protective factor of cancer recurrence and metastasis in both stage I-IIIA (HR=0.50, 95% CI: 0.37-0.67) and stage IIIA NSCLC postoperative patients (HR = 0.48, 95% CI: 0.33-0.71), DFS was even longer among CM treatment group patients.@*CONCLUSIONS@#Longer duration of CM therapy could be considered a protective factor of cancer recurrence and metastasis. CM treatment is associated with improving survival outcomes of postoperative NSCLC patients in China. (Registration No. ChiCTR-OOC-14005398).
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Objective To study the effects of Jianpi Xiaoai Prescription on cell cycle and apoptosis of human colon cancer HCT116 cells and related factors of Wnt/β-catenin signaling pathway; To investigate its mechanisms of anti-metastasis and recurrence of colorectal cancer. Methods The logarithmic growth phase HCT116 cells were divided into blank group, saline group, 5-Fu group, and Jianpi Xiaoai Prescription low-, medium-, and high-dose groups. After intervention for 48 h, the cells were harvested, and the cell cycle and apoptosis were detected by flow cytometry. The protein expression of β-catenin in the nucleus was detected by Western blot, and the expression of c-myc and cyclinD1 mRNA was detected by PCR. Results Compared with the blank group and saline group, the ratio of HCT116 cells apoptosis of Jianpi Xiaoai Prescription low-, medium-, and high-dose groups increased; the proportion of cells in phase G1 increased and the proportion of S cells decreased; the expression of β-catenin protein in the nucleus and the expression of c-myc,cyclinD1 mRNA decreased,especially in the Jianpi Xiaoai Prescription high-dose group,with statistical significance(P<0.05,P<0.01).Conclusion Jianpi Xiaoai Prescription can promote apoptosis of human colon cancer HCT116 cells and block the cell cycle, and its mechanism may be related to regulating Wnt/β-catenin signaling pathway.
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Objective To observe the effects of Jianpi Xiaoai Prescription on epithelial-mesenchymal transition of colorectal cancer cell SW620 induced by TGF-β1; To discuss its possible mechanism of action for prevention and treatment of colorectal cancer. Methods Colorectal cancer cells SW620 were cultured in vitro. By conducting CCK-8 and Transwell experiments, the proliferation, invasion and migration of colorectal cancer cell (SW620) were detected. qRT-PCR and Western blot experiments were applied to verify the mRNA and protein expression level of E-cadherin and Vimentin. Results Jianpi Xiaoai Prescription showed in vitro inhibitory effect on the proliferation, invasion and migration of colorectal cancer cell SW620. Compared with blank group, the expression of E-cadherin in TGF-β1 induced group was reduced and Vimentin was increased (P<0.05); Meanwhile, the expression of E-cadherin in Jianpi Xiaoai Prescription group was increased and Vimentin was decreased when compared with TGF-β1 induced group (P<0.05). Conclusion Jianpi Xiaoai Prescription can inhibit the proliferation, invasion and migration of colorectal cancer cell by reverting the epithelial-mesenchymal transition of colorectal cancer cell induced by TGF-β1, with a purpose to achieve the goal of preventing and treating the recurring and migration of colorectal cancer.