RESUMEN
To investigate the effect of jianpi-jiedu (JPJD) prescription-contained serum on colorectal cancer SW48 cell proliferation and the underlying mechanisms. Methods: Crude extract from JPJD was made by water extract method and the main components of crude extract from JPJD were analyzed by ultra-performance liquid phase high resolution time of flight mass spectrometry (UPLC-Q-TOF/MS). The low, medium, and high-concentration of JPJD-contained serum were prepared by the serum pharmacological method. The effect of serum containing JPJD on SW48 cell proliferation was determined by MTT assay. The cell cycle was detected by flow cytometric method. The protein levels of mammalian target of rapamycin (mTOR), phospho-mTOR, P-P53, and -P21, and the mRNA level of mTOR were examined by Western blot and RT-PCR, respectively. Results: Seven compounds including calycosin-7-glucoside, astragaloside, ginsenoside-Re, ginsenoside-Rb1, glycyrrhizinic acid, apigenin, atractylenolide-II were identified. MTT assays demonstrated that the SW48 cell proliferation was inhibited by medium and high concentration of JPJD-contained serum and the percentages of cells at G1 phase in SW48 cell cultured in the medium and high concentration of JPJD serum group were significantly higher than those in the control group (P<0.05). Meanwhile, the levels of mTOR mRNA and phospho-mTOR protein in the medium and high concentration of JPJD serum groups were substantially lower than those in the control group (P<0.05). Conversely, the expressions of phospho-P53 and P21 protein were significantly increased in the medium and high concentration of JPJD serum group compared with those in the control group. Conclusion: JPJD prescription-contained serum can inhibit SW48 cell proliferation, which may be related to mTOR-P53-P21 signaling pathways.
Asunto(s)
Animales , Humanos , Apigenina , Western Blotting , Ciclo Celular , División Celular , Proliferación Celular , Genética , Neoplasias Colorrectales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Medicamentos Herbarios Chinos , Farmacología , Citometría de Flujo , Ginsenósidos , Ácido Glicirrínico , Lactonas , Fosforilación , Genética , ARN Mensajero , Saponinas , Sesquiterpenos , Transducción de Señal , Serina-Treonina Quinasas TOR , Triterpenos , Proteína p53 Supresora de TumorRESUMEN
To investigate the effect of the jianpi-jiedu formula (JPJD) on the expression of angiogenesis-relevant genes in colon cancer. Methods: Crude extract was obtained from JPJD by water extract method. The effect of JPJD crude extract on colon cancer cell proliferation capacity was determined by MTT assays. The IC50 value was calculated by GraphPad Prism5 software. Affymetrix gene expression profiling chip was used to detect significant differences in expressions of genes after JPJD intervention, and pathway enrichment analysis was performed to analyze the differentially expressed genes. Ingenuity Pathway Analysis software was applied to analyze differentially expressed genes relevant to tumor angiogenesis based on mammalian target of rapamycin (mTOR) signaling pathway and then the network diagram was built. Western blot was used to verify the protein levels of key genes related to tumor angiogenesis. Results: JPJD crud extract inhibited the proliferation capacity in colon cancer cells. The IC50 values in 24, 48, and 72 hours after treatment were 13.060, 9.646 and 8.448 mg/mL, respectively. The results of chip showed that 218 genes significantly upgraded, and 252 genes significantly downgraded after JPJD treatment. Most of the genes were related to the function of biosynthesis, metabolism, cell apoptosis, antigen extraction, angiogenesis and so on. There were 12 differentially expressed angiogenesis genes. IPA software analysis showed that the JPJD downregulated expression of sphingomyelin phosphodiesterase 3 (SMPD3), VEGF, vascular endothelial growth factor A (VEGFA), integrin subunit alpha 1 (ITGA1), cathepsin B (CTSB), and cathepsin S (CTSS) genes, while upregulated expressions of GAB2 and plasminogen activator, urokinase receptor (PLAUR) genes in the colorectal cancer cell. Western blot results demonstrated that JPJD obviously downregulated expressions of phospho-mTOR (P-mTOR), signal transducer and activator of transcription 3 (STAT3), hypoxia inducible factor-1α (HIF-1α), and VEGF proteins, while obviously upregulated the level of phospho-P53 (P-P53) protein. Conclusion: JPJD may inhibit colorectal tumor angiogenesis through regulation of the mTOR-HIF-1α-VEGF signal pathway.