Lenvatinib down-regulates IGF1R/Mek/Erk signaling pathway in the treatment of regorafenib-resistant hepatocellular carcinoma / 中华肿瘤杂志

Objective: To investigate the therapeutic effect and mechanism of lenvatinib on regorafenib-resistant hepatocellular carcinoma cells. Methods: CCK-8 and clone formation assay were used to observe the inhibitory effect of lenvatinib on the growth of hepatocellular carcinoma cells. Flow cytometry was used to detect the apoptosis of regorafenib-resistant hepatocellular carcinoma cells treated with lenvatinib. The expression levels of related proteins were detected by western blot and immunohistochemical staining. The inhibitory effect of lenvatinib on the tumor formation ability of regorafenib-resistant hepatocellular carcinoma cells in vivo was observed by subcutaneous tumor formation experiment in mice. Results: CCK-8 and clone formation assay showed that lenvatinib could inhibit the proliferation of regorafenib-resistant hepatocellular carcinoma cells. The number of clones of HepG2, SMMC7721 and regorafenib-resistant HepG2, SMMC7721 cells in lenvatinib group (120.67±11.06, 53.00±11.14, 55.00±9.54, 78.67±14.64) were all lower than those in control group (478.00±24.52, 566.00±27.87, 333.67±7.02, 210.00±12.77, all P<0.05). Flow cytometry showed that lenvatinib could promote apoptosis of regorafenib-resistant hepatocellular carcinoma cells, the apoptosis rates of HepG2, SMMC7721 and regorafenib-resistant HepG2, SMMC7721 cells in lenvatinib group [(12.30±0.70)%, (9.83±0.38)%, (15.90±1.32)%, (10.60±0.00)%] were all higher than those in control group [(7.50±0.87)%, (5.00±1.21)%, (8.10±1.61)%, (7.05±0.78)%, all P<0.05]. The apoptosis-related protein levels suggested that apoptosis was increased in the treatment of lenvatinib. The animal study showed that lenvatinib can inhibit the growth of regorafenib-resistant cells in vivo. Immunohistochemistry and western blot results showed that lenvatinib could down-regulate the abnormally activated IGF1R/Mek/Erk signaling pathway in regorafenib-resistant cells. Conclusion: Lenvatinib can reverse regorafenib resistance in hepatocellular carcinoma, possibly by down-regulating IGF1R/Mek/Erk signaling pathway.

Effect of Doxycycline on Intrinsic Apoptosis of Myeloma Cell Line H929 and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the mechanism of the in vitro toxicity of doxycycline to myeloma cell line H929 and also the possible pathway involved its toxicity.@*METHODS@#Myeloma cell line H929 was treated with DOX, MEK inhibitor U0126 or RAS agonist ML-098, either alone or in combination. Then, the expression of p-MEK, caspase-3, caspase-9 and c-Jun in H929 were used to detected by Western blot; the cells proliferation and apoptosis were detected by CCK-8 assay and flow cytometry, respectively.@*RESULTS@#DOX significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK in H929 (P<0.05). MEK antagonist U0126 significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK (P<0.05). After Dox combined with ML-098 treatment of H929 cells, the apoptosis rate of H929 cells was lower than that of DOX alone treatment group(P<0.05). Compared with DOX alone treatment group, the expressions of p-MEK and p-ERK1/2 in DOX+ML-098 combined treatment group were increased, and the levels of cleaved caspase-3,9 in H929 cells were decreased (P<0.05). The levels of c-Jun mRNA and protein increased in H929 when treated by DOX alone (P<0.05).@*CONCLUSION@#DOX can induce apoptosis of H929 via intrinsic apoptosis pathway, and MEK/ERK pathway and c-Jun possibly play a role in this process.

Combination therapy with miR34a and doxorubicin synergistically inhibits Dox-resistant breast cancer progression

Resistance to breast cancer (BCa) chemotherapy severely hampers the patient's prognosis. MicroRNAs provide a potential therapeutic prospect for BCa. In this study, the reversal function of microRNA34a (miR34a) on doxorubicin (Dox) resistance of BCa and the possible mechanism was investigated. We found that the relative level of miR34a was significantly decreased in Dox-resistant breast cancer cell MCF-7 (MCF-7/A) compared with Dox-sensitive MCF-7 cells. Transfection with miR34a significantly suppressed the invasion, migration, adhesion of MCF-7/A cells without inhibiting their growth obviously. The combination of miR34a and Dox could significantly inhibit the proliferation, migration, invasion and induce the apoptosis of MCF-7/A cells. The synergistic effect of this combination on resistant MCF-7/A cells has no obvious relation with the expressions of classical drug-resistant proteins P-GP, MRP and GST-

Regulatory effect of annexin A5 on glioma cell invasion andmigration and its mechanisms / 西安交通大学学报(医学版)

【Objective】 To investigate the regulatory effect of annexin A5 on glioma cell invasion and migration and its mechanism. 【Methods】 The expression of annexin A5 in 100 cases of glioma tissues and 20 cases of normal brain tissues was detected by immunohistochemistry. The expression of annexin A5 was downregulated by transfection with siRNA targeting annexin A5 (si-Annexin A5) in human glioma cell line (U251). The expression of annexin A5 was confirmed by RT-PCR and Western blot analysis. The proliferation ability of U251 cells was detected by MTT test and colony formation test, the apoptosis of U251 cells was detected by flow cytometry and Hoechst 33258 staining, and the migration and invasion ability of U251 cells was examined by wound healing test and Matrigel Transwell invasion test. The expressions of Raf, p-Raf, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, c-Myc and E-Cadherin in U251 cells were analyzed by Western blot. 【Results】 Compared with those of normal brain tissues, the mRNA and protein expression levels of Annexin A5 in glioma tissues increased by 2.45 times and 2.87 times, respectively (P<0.05). Pearson correlation analysis showed that with the increase of tumor grade, the positive rate of Annexin A5 gradually increased, and the tumor grade and positive rate were significantly positively correlated (r=1.000, P=0.000). The cell viability of U251 cells in the si-Annexin A5 group after 48 h and 72 h of culture was significantly reduced by 29.46% and 40.43%, respectively, compared with that in the control group (P<0.05). Compared with the control group, in the si-Annexin A5 group the colony formation rate was reduced by 68.58%, while the apoptosis rate was increased by 24.41 times (P<0.05); the cell migration rate and invasion rate were reduced by 65.35% and 68.80% (P<0.05). The protein expression of p-Raf, p-MEK1/2, p-ERK1/2 and c-Myc in the si-Annexin A5 group were significantly reduced by 54.67%, 70.37%, 60.26% and 54.95%, respectively, and that of E-Cadherin was increased by 3.58 times (P<0.05). 【Conclusion】 Downregulation of Annexin A5 inhibits the growth and motility of glioma cells and induces cell apoptosis by inhibiting the Raf/MEK/ERK signaling pathway.

Baicalein restrains proliferation, migration, and invasion of human malignant melanoma cells by down-regulating colon cancer associated transcript-1

Baicalein (BAI) is an acknowledged flavonoids compound, which is regarded as a useful therapeutic pharmaceutical for numerous cancers. However, its involvement in melanoma is largely unknown. This study aimed to examine the anti-melanoma function of BAI and unraveled the regulatory mechanism involved. A375 and SK-MEL-28 were treated with BAI for 24 h. Then, CCK-8 assay, flow cytometry, and transwell assay were carried out to investigate cell growth, migration, and invasion. RT-qPCR was applied to detect the expression of colon cancer associated transcript-1 (CCAT1) in melanoma tissues and cells. The functions of CCAT1 in melanoma cells were also evaluated. Western blot was utilized to appraise Wnt/β-catenin or MEK/ERK pathways. BAI restrained cell proliferation and stimulated cell apoptotic capability of melanoma by suppressing cleaved-caspase-3 and cleaved-PARP. Cell migratory and invasive abilities were restrained by BAI via inhibiting MMP-2 and vimentin. CCAT1 was over-expressed in melanoma tissues and down-regulated by BAI in melanoma cells. Overexpressed CCAT1 reversed the BAI-induced anti-growth, anti-migratory, and anti-invasive effects. Furthermore, BAI inhibited Wnt/β-catenin and MEK/ERK pathways-axis via regulating CCAT1. Our study indicated that BAI blocked Wnt/β-catenin and MEK/ERK pathways via regulating CCAT1, thereby inhibiting melanoma cell proliferation, migration, and invasion.

Polydatin protects H9c2 cells from hypoxia-induced injury via up-regulating long non-coding RNA DGCR5

Polydatin (PD), a monocrystalline polyphenolic drug mainly found in the roots of Polygonum cuspidatum, has various pharmacological activities. Long non-coding RNAs (lncRNA) DiGeorge syndrome critical region gene 5 (DGCR5) was found to participate in the suppression of multiple cancers. Here, we proposed to study the effect of PD on myocardial infarction (MI) by inducing DGCR5. CCK-8 assay was performed to detect the viability of H9c2 cells. Flow cytometry was utilized to test apoptosis of H9c2 cells. These results determined the optimal concentration and effect time of hypoxia as well as PD. Si-DGCR5 was transfected into cells and the expression level was determined by qRT-PCR. Western blot was utilized to evaluate the expression of apoptosis-related proteins, Bcl-2, Bax, and cleaved-caspase-3, as well as autophagy-associated proteins including Beclin-1, p62, and LC3-II/LC3-I. As a result, PD efficiently attenuated hypoxia-induced apoptosis and autophagy in H9c2 cells. The expression of DGCR5 was down-regulated by hypoxia and up-regulated by PD. Besides, knocking-down the expression of DGCR5 inhibited the protection of PD in H9c2 cells. In addition, PD up-regulated the accumulation of DGCR5, DGCR5 decreased the expression of Bcl-2 and p62, raised the expression of Bax and cleaved-caspase-3, and the proportion of LC3-II/LC3-I. PD stimulated the PI3K/AKT/mTOR and MEK/ERK signaling pathways via up-regulating the expression of DGCR5. Our data demonstrated that PD reduced cell apoptosis and autophagy induced by hypoxia in cardiomyocytes. Moreover, PD activated PI3K/AKT/mTOR and MEK/ERK signaling pathways by up-regulating the expression of DGCR5.

Screening of monomers from traditional Chinese medicine for specifically inhibiting brafV600E CT26 cells / 中国药理学通报

Aim: To screen BRAFV600E CT26 cell inhibitors from monomers of traditional Chinese medicine (TCM). Methods CT26 cell line was constructed with lentivirus plasmid to stably express BRAFV6C0E. The proliferation, migration and expression of related proteins in MEK/ERK signaling pathway were detected. The monomers of TCM were detected for biological activities as potential BRAF inhibitors by Discovery Studio 4. 0, and further evaluated by MTT assay. Results: The proliferation and migration of BRAFV6C0E CT26 cells were obviously strengthened compared with wild type control. The expressions of proteins in MEK/ ERK pathway were also activated in BRAFV6C0E CT26 cells. Compared with wild type control, Aloin, Angoroside C and Cyasterone exhibited the potent effect against BRAFV600E in CT26 cells (P <0. 05), and could down-regulate the expression of BRAFV600E. Conclusion: Aloin, Angoroside C, Cyasterone might be the potent inhibitors against BRAF for colon treatment.

Anti-tumor activity and mechanisms of isorhamnetin in combination with sorafenib for renal cancer / 药学学报

This study aimed to explore the anti-tumor activity and mechanisms of action of isorhamnetin, a compound isolated from Astragalus membranaceus, in combination with sorafenib for treatment of renal cell carcinoma (RCC). The anti-tumor activity of isorhamnetin in combination with sorafenib was detected by MTT assay with cells in culture or Renca xenograft model in mice. Western blot was used to study the mechanisms of isorhamnetin in combination with sorafenib. Lymphocyte proliferation assay was also used to investigate the effects of the two drugs in combination. The results indicated that isorhamnetin inhibited the proliferation of RCC cells, with IC50 for A498, 786-O and Renca cell lines with being 31.7, 28.8 and 106.0 μmol·L-1, respectively. Isorhamnetin in combination with sorafenib improved the anti-lymphocyte proliferation activity of sorafenib with the IC50 down to 12.0 μmol·L-1. Isorhamnetin inhibited the growth of RCC in mice slightly with the inhibition efficiency at 26.9%. With 50.0 mg·kg-1 isorhamnetin in combination with 20.0 mg·kg-1 sorafenib, the anti-tumor activity of sorafenib was enhanced, with inhibition of growth rate increased to 60.7%. Meanwhile, isorhamnetin in combination with sorafenib could promote the lymphocytes proliferation in Renca xenograft model. Western blot results showed that combination of isorhamnetin and sorafenib could inhibit c-Raf/MEK/ERK and AKT/mTOR signaling pathways. In conclusion, the combination of isorhamnetin with sorafenib could increase the anti-tumor activity of sorafenib in RCC in vitro and in vivo. The mechanisms may be related to the inhibition of c-Raf/MEK/ERK and AKT/mTOR signaling pathways. Procedures for animal study were performed with approval of the Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College.

Anti-tumor effect of Hinesol on liver cancer via downregulating MEK/ERK and NF-κB pathway in SMMC-7721 and LM3cells / 中国药理学与毒理学杂志

OBJECTIVE Liver cancer is one of the most common causes of cancer related deaths worldwide, specially, in China. Hinesol, extracted from Atractylodeslance a(Thunb.) DC. has been proved that has anti-cancer effect in leukemia in vitro and in vivo.However,it has been not well under-stood in liver cancer cells.METHODS Cell proliferation,apoptosis,cell cycle and invasion were performed to investigate the anti-liver cancer effect of hinesol in SMMC-7721 and LM3 by MTT assay,flow cytometry and scratch assay.Western blot was used to research the potential mechanism.RESULTS We revealed that hinesol suppresses cell proliferation and invasion,prompts population of G1 phase,induces apop-tosis in dose-dependent manner in SMMC-7721 and LM3 cells.Western blot data showed that hinesol could inhibits the expression of cyclin-D1, Bcl-2 and Bax, and inhibited phosphorylation of MEK and ERK, down-regulated the expressions of NF-κB p65 and phosphor-p65 in nucleus. The results indicated that hinesol reduces cell proliferation via arresting cell cycle at G1 phase and induces apoptosis.Further-more,western blot showed that hinesol inhibited phosphorylation of MEK and ERK,down-regulated the expressions of NF-κB p65 and phosphor-p65 in nucleus.CONCLUSION Our results demonstrate that hinesolreduces cell proliferation via arresting cell cycle at G1 phase and induces apoptosis, it has potent anti-cancer effect against liver cancer cells via down-regulation of MEK/ERK and NF-κB pathway,and indicate that hinesol is a potential liver cancer drug for further research.

An integrative pharmacological-based study on network mechanism of Xinkeshu Tablets intervention in atherosclerosis / 中草药

Objective: To explore the mechanism of the intervention of Xinkeshu Tablets (XKST) on atherosclerosis (AS) and provide reference for the secondary development and clinical application of XKST. Methods: The integrated pharmacology platform was used to predict the key targets and pathways of the intervention of XKST on AS and its molecular mechanism was also explored. Results: In the integrative analysis of heterogeneous network of “TCM-component-target-pathway”, 80 relevant effective ingredients were found, including B4GALT4, B4GALT2, PRKCD, GCK, GNB1, and other key targets; Endocrine system, thyroid hormone signaling pathway, nervous system, estrogen signaling pathway, and chemokine signaling pathway were key pathways related with its anti-atherosclerosis. Conclusion: According to the analysis and prediction of the enrichment information, the effect of XKST on common regulating PI3K/Akt/eNOS and Raf/MEK/ERK signaling pathway and protecting vascular endothelial cells is first prompted, thus achieving the intervention in AS.

Seneciphylline induced the autophagy of cervical cancer cells via MEK/ERK1/2 regulation / 中国药科大学学报

@#The purpose of this study was to explore the effects of seneciphylline on the proliferation and autophagy of cervical cancer HeLa and Caski cells and the possible mechanisms of autophagy. MTT assay was used to evaluate the effect of seneciphylline on the proliferation of cervical cancer cells. Immunofluorescence assay was applied to investigate the formation of autophagosomes in GFP-LC3/HeLa and GFP-LC3/Caski cells. The effect of seneciphylline on the expression of autophagy-related proteins was checked by Western blotting. In addition, fluorescence colocation assay was used to detect the fusion of autophagosomes and lysosomes. Human cervical cancer subcutaneous xenografts in nude mice were used to evaluate the effect of seneciphylline on the growth of the tumor in vivo. Results showed that HeLa cells proliferation was inhibited by seneciphylline in a dose- and time- dependent manner. Seneciphylline could induce formation of autophagosomes, increase the expression of LC3-II and decrease the expression of P62, suggesting that seneciphylline induced autophagy in HeLa and Caski cells. Compared with seneciphylline alone, seneciphylline combined with later-stage autophagy inhibitor chloroquine significantly increased the expression of LC3-II and P62. Moreover, and fluorescence colocation assay showed that autophagosomes induced by seneciphylline could colocate with lysosomes, indicating that seneciphylline could induce the complete autophagy flux. Compared with seneciphylline alone, seneciphylline combined with earlier-stage autophagy inhibitor 3MA significantly increased the expression of LC3-II and significantly decreased HeLa and Caski cells viability, suggesting that seneciphylline induced protective autophagy. Compared with seneciphylline alone, seneciphylline combined with MEK inhibitor significantly decreased the expression of P-ERK1/2 and formation of autophagosomes, suggesting that autophagy induced by seneciphylline activated MEK/ERK1/2 signal pathway. In addition, seneciphylline showed a significant inhibitory effect on growth of human cervical cancer cells subcutaneous xenografts.

The inhibitory effect of isoquercitrin onRaf/MEK/ERK signaling pathway in HepG2 cells / 中国药理学通报

Aim To study the inhibitory effect of isoquercitrin on Raf/MEK/ERK signaling pathway in HepG2 cells.Methods MTT was used to detect the proliferation of human liver cancer HepG2 cells after the treatment of isoquercitrin.The morphology and growth of cells were observed under inverted microscope after the different concentrations of isoquercitrin(0, 40, 80, 160, 320 μmol·L-1) to treat HepG2 cells for 24 and 48 h.Cell cycle was assessed by flow cytometry.Ras, Raf, MEK, ERK expression was assayed by Western blot, and mRNA expression was detected by quantitative fluorescence PCR.Results Isoquercitrin could inhibit the growth of HepG2 cells in a concentration-and time-dependent manner.Typical morphological changes of apoptosis were observed by inverted microscopy after HepG2 cells were treated with different concentrations of of isoquercitrin for 24 h or 48 h.The cell cycle assay showed that with the increasing concentration of isoquerditrin, the number of cells that was arrested in G1 phase gradually increased.Compared with the blank group, the expressions of Ras, Raf, MEK, ERK mRNA were down-regulated, and related proteins expression were also down-regulated(P<0.05), and these results had statistical significance.Conclusion Isoquercitrin can induce the apoptosis of HepG2 cells, which may be related to the Raf/MEK/ERK signaling pathway.

Effect of Amarogentin on the Induction of Liver Cancer Cell Line Huh-7 Apoptosis and the Regulation of PKA/C / 中国药学杂志

OBJECTIVE: To explore the effect of amarogentin on the induction of liver cancer cell line Huh-7 apoptosis and the regulation of PKA/C. METHODS: Liver cancer cell line Huh-7 were divided into 4 groups, control, amarogentin, amarogentin+H89 and amarogentin+H7 group (n=8). The cells were treated with amarogentin (30 mmolL-1) for 6 h besides control group. The amarogentin+H89 and amarogentin+H7 group cells were treated with corresponding compounds at the last 3 h (H89 at 10 mmolL-1 and H7 10 mmolL-1). The apoptotic proteins and MEK/ERK signaling pathway related proteins were detected by Western blotting. The Caspase 3 and Caspase 9 were also be assayed by immune-cytochemistry. At the meaning time, the apoptosis state was assayed by DAPI. RESULTS: The results showed that the Bax, Caspase 3 and Caspase 9 were increased (P>0.05) while the Bcl2 were decreased (P>0.05) expressed greatly after the medication of amarogentin when compared with control. At the same time, the expression of Ras, Rsf, MEK and ERK1/2 were increased (P>0.05) greatly after the medication of amarogentin when compared with control. Those abnormalities were normalized greatly by the medication of H89 (P>0.05) but not H7(P>0.05). CONCLUSION: Amarogentin could promote the apoptosis of liver cancer cell line Huh-7 which is mediated by PKA.

Advances in research on the modulation of autophagy by Ras/Raf/MEK/ERK signaling pathway / 中国药科大学学报

@#Autophagy is a conserved self-defense mechanism of organism, degrading the necrotic organelles and excess protein into small molecules for recycling. Autophagy plays a role in both physiological and pathological condition, influencing the expression of intracellular substance through multiple signaling pathways. Although it has been demonstrated that Ras/Raf/MEK/ERK signaling pathway was not only extensively involved in the regulation of cell growth, proliferation, differentiation and apoptosis, but was also implicated in autophagy and autophagic cell death, though its detailed mechanisms involved in regulation of autophagy has not been fully elucidated yet. This review focused on the advances of autophagy induced by Ras/Raf/MEK/ERK signaling pathway, to better understand the role of Ras/Raf/MEK/ERK signaling pathway in regulation of autophagy.

EdCC reduces myocardial ischemia-reperfusion injury via MEK-ERK signaling pathway / 中国病理生理杂志

AIM:To investigate the myocardial protective effect of endometrial stem cell ( EnSC)-derived cyto-kine cocktail ( EdCC) on myocardial ischemic reperfusion injury and the MEK-ERK signaling pathway.METHODS: A mouse model of myocardial ischemic reperfusion injury was established.Infarct area, cell apoptosis, and expression of cleaved caspase-3 and phosphorylatied ERK1/2 were determined by TTC/Evans blue staining, TUNEL assay and Western blot, respectively.RESULTS:The mesenchymal characteristics were observed in the EnSCs with expressing CD90 and in absence of CD34 and CD45.EdCC contained (6 811 ±312) ng/g epidermal growth factor (EGF) protein.The phospho-rylation of ERK1/2 markedly increased after injection of EdCC, but was abolished by MEK1 inhibitor PD98059 ( 5 mg/kg) .EdCC decreased the infarct area and apoptotic cell number in the border zone and inhibited caspase-3 activation. However, the effects were abolished by MEK1 specific inhibitor PD98059.EGF did not decrease the infarct area, but the EGF receptor antagonist AG-1487 (6 mg/kg) partly abolished the myocardial protective effect of EdCC.CONCLUSION:EdCC protects the myocardium from ischemic reperfusion injury via activating MEK1-ERK signaling pathway, indicating an essential role in the transmission of stem cell therapy from the cell transplantation to cytokine based strategy.

Effect and Mechanism of Propranolol on the Myocardial Abnormal Electrophysiology Station in Diabetic Model Rats / 中国药房

OBJECTIVE:To study the effects and mechanism of propranolol on the myocardial abnormal electrophysiology sta-tion in diabetic model rats. METHODS:SD rats were randomly divided into normal control(normal saline)group,diabetic(nor-mal saline)group,PD98059(ERK inhibitor,10 mg/kg)group and propranolol low-dose,medium-dose and high-dose(1,20,50 mg/kg)groups,with 8 rats in each group. Except for normal control group,rats were given alloxan(20 mg/kg)intravenously via tail vein to induce diabetic model. They were given relevant medicine intragastrically,once a day,for consecutive 42 days. The car-diac index,electrocardiogram and action potential durations (APD) of rats were analyzed;the expression of TNF-α,IL-2,IL-6 and IL-10 protein in serum were detected,and the expression of Ras,Raf,ERK kinase(MEK)and ERK1/2 in myocardial tissue were detected. RESULTS:Compared with normal control group,cardiac index increased in diabetes group;heart rate decreased;QT interval and APD were prolonged;the relative expression of TNF-α,IL-2,IL-6,IL-10,Ras,Raf,MEK and ERK1/2 protein increased (P<0.01). Compared with diabetes group,cardiac index decreased in propranolol medium-dose and high-dose groups and PD98059 group,heart rate increased,QT interval and APD were shortened;the relative expression of TNF-α,IL-2,IL-6, IL-10,Ras,Raf,MEK and ERK1/2 protein decreased(P<0.05 or P<0.01). CONCLUSIONS:Propranolol can improve myocar-dial abnormal electrophysiology station of diabetic model rats by down-regulating inflammatory reactions in serum and inhibiting the activation of MEK/ERK signaling pathway.

MEK/ERK signaling pathway in apoptosis of SW620 cell line and inhibition effect of resveratrol

Objective: To study the involvement of MAPK MEK/ERK signaling transduction pathway in the apoptosis process of SW620 tumor cell line and the inhibition effect of resveratrol. Methods: SW620 cell lines were divided into 5 groups, namely, control group, PD98059 group, low-dose resveratrol group, mid-dose resveratrol group and high-dose resveratrol group. The inhibition rate of cell proliferation was detected by MTT method. The expression of apoptotic molecules and MEK/ERK signaling pathway related proteins were assayed by real-time PCR and Western blotting. Results: Compared with control group, the proliferation of cells treated with resveratrol was significantly inhibited. In the case of apoptotic molecules, the expression of Bax, Caspase 3 and Caspase 9 was increased significantly while the expression of anti-apoptotic molecule Bcl2 was decreased significantly in resveratrol groups with a dose-dependent manner. In the case of molecules in MEK/ERK signaling pathway, the expression of Ras, Raf, MEK and ERK1/2 was decreased significantly in resveratrol groups with a dose-dependent manner. Conclusions: PD98059 and resveratrol can effectively inhibit the proliferation of SW620 through inhibiting the MEK/ERK signaling pathway.

MEK/ERK signaling pathway in apoptosis of SW620 cell line and inhibition effect of resveratrol

OBJECTIVE@#To study the involvement of MAPK MEK/ERK signaling transduction pathway in the apoptosis process of SW620 tumor cell line and the inhibition effect of resveratrol.@*METHODS@#SW620 cell lines were divided into 5 groups, namely, control group, PD98059 group, low-dose resveratrol group, mid-dose resveratrol group and high-dose resveratrol group. The inhibition rate of cell proliferation was detected by MTT method. The expression of apoptotic molecules and MEK/ERK signaling pathway related proteins were assayed by real-time PCR and Western blotting.@*RESULTS@#Compared with control group, the proliferation of cells treated with resveratrol was significantly inhibited. In the case of apoptotic molecules, the expression of Bax, Caspase 3 and Caspase 9 was increased significantly while the expression of anti-apoptotic molecule Bcl2 was decreased significantly in resveratrol groups with a dose-dependent manner. In the case of molecules in MEK/ERK signaling pathway, the expression of Ras, Raf, MEK and ERK1/2 was decreased significantly in resveratrol groups with a dose-dependent manner.@*CONCLUSIONS@#PD98059 and resveratrol can effectively inhibit the proliferation of SW620 through inhibiting the MEK/ERK signaling pathway.

Glucosylceramide synthase upregulates apoptosis-related gene bcl-2 ex-pression via MEK/ERK signaling pathway in leukemia multidrug-resis-tant cell line / 中国病理生理杂志

AIM: To investigate whether glucosylceramide synthase (GCS) regulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway , thus enhancing drug resistance of K 562/A02 human leukemia multidrug resistant cell line.METHODS:siRNA targeting GCS was transfected into K562/A02 cells.Bcl-2, p-ERK and total ERK expression at mRNA and protein levels after GCS knockdown were detected by real-time PCR and Western blotting .After exposed to MEK-ERK pathway inhibitor U0126, the expression of Bcl-2 at mRNA and protein levels also was analyzed by real-time PCR and Western blotting , respectively.The viability of the cells was evaluated by CCK-8 assay.RESULTS:The expression of GCS and Bcl-2, as well as MEK/ERK signaling were significantly inhibited in K 562/A02 cells by GCS siRNA transfection compared with negative control group .Inactivation of MEK/ERK signaling due to U0126 treatment de-creased Bcl-2 mRNA and protein levels in a concentration-dependent manner , and sensitized K562/A02 cells to adriamy-cin.CONCLUSION:GCS may affect the expression of apoptosis-related gene bcl-2 by MEK/ERK signaling pathway , thus regulating multidrug resistance of human leukemia K 562/A02 cells.

Study on the Mechanism of Autophagy of HeLa Cells in Human Cervical Cancer Induced by Fucoxanthin / 中国药房

OBJECTIVE:To study the mechanism of autophagy of HeLa cell in human cervical cancer induced by fucoxanthin. METHODS:MTT was adopted to determine the cell activity and calculate the inhibition rate after HeLa cells were cultured by 1, 10,20,40 and 80 μmol/L of fucoxanthin for 48 h. Flow cytometry was used to determine the cell cycle and apoptosis rate after HeLa cells were cultured by 0(blank control),10,20 and 40 μmol/L of fucoxanthin for 48 h;acridine orange staining,LysoTrack-er Red staining,HeLa-GFP-LC3 method and fluorescence microscope were used to observe the autophagy state;Western blot was used to determine the expressions of proteins related to autophagy. RESULTS:The cells had obvious inhibition effect on the cell growth after being cultured by 0,10,20,40 and 80 μmol/L of fucoxanthin. The cell was blocked in G0/G1 stage after being cul-tured by 10,20 and 40μmol/L of fucoxanthin,and had no obvious effect on the apoptosis rate;autophagy degree was increased af-ter the cells were cultured by 40 μmol/L fucoxanthin for 48 h. Compared with blank control,40 μmol/L fucoxanthin could promote LC3Ⅰ transferring into LC3Ⅱ and the expressions of Beclin-1,PTEN,p21;and inhibit the phosphorylation of p-Akt,p-p70S6K and p-mTOR. The pre-treatment by autophagy inhibitor 3-methyladenine(5 mmol/L)could reverse the autophagy of HeLa cells in-duced by fucoxanthin;U0126 could partly reverse the autophagy of HeLa cells induced by fucoxanthin. CONCLUSIONS:Fucoxan-thin can induce the authphagy of HeLa cells by inhibiting Akt signaling pathway and activating MEK/ERK signaling pathway.