Apoptosis of gastric cancer MGC-803 cells induced by rosmarinic acid analogue-11 by inhibiting ERK/MAPK pathway / 中国药理学通报

Aim: To explore the apoptosis and mechanisms of human gastric cancer cells MGC-803 induced by RAA-11. Methods: MTT and colony assays were used to detect the survival of MGC-803 cells treated with RAA-11. The effect of RAA-11 on the apoptotic morphology of MGC-803 cells was observed by Hoechst 33258 staining. The effect of RAA-11 on the apoptosis rate of MGC-803 cells was detected with flow cytometry. The effects of RAA-11 on apoptosis related protein expression of caspase-3, Bcl-2 and Bax as well as pathway proteins ERK and p-ERK in MGC-803 cells were observed by Western blot. Results: MTT assay showed that the proliferation of MGC-803 cells was effectively inhibited in a time- and concentration-dependent manner (P <0. 01). The result of colony suggested that RAA-11 could inhibit the proliferation of MGC-803 cells. Hoechst 33258 staining indicated that the nucleus of MGC-803 cells had a typical apoptotic morphological change after intervention with RAA-11. The result of flow cytometry suggested that RAA-11 had a significant apoptotic effect on MGC-803 cells. The expressions of Bax and caspase-3 were significantly up-regulated (P <0. 01), and the expressions of Bcl-2, ERK and p-ERK were down-regulated (P <0. 01) in MGC- 803 cells treated with RAA-11. Conclusions: RAA-11 inhibits the proliferation and induces apoptosis in MGC-803 cells, which may be related to the inhibition of ERK/MAPK pathway.

Caffeoyl quinic acids in Chrysanthemum morifolium improve LPS-induced HUVEC vascular endothelial cell injury by regulating ERK/MAPK signaling pathway / 药学学报

To explore the effect of total extract of Chrysanthemum morifolium on lipopolysaccharide (LPS)-induced acute lung injury in mice, we studied the effects of three caffeoyl quinic acids isolated from Chrysanthemum morifolium on vascular endothelial cell injury and their mechanisms of action. All animal experiments were carried out strictly in accordance with the National Animal Welfare Ethics and Protection Regulations. A mouse model of acute lung injury was established by intranasal instillation of LPS. After 6 days of oral administration of chrysanthemum extract, the lung wet weight/dry weight ratio, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were measured in mouse bronchoalveolar lavage fluid. Human umbilical vein endothelial cells (HUVEC) were serum starved for 12 h and treated with 2.5 μg·mL-1 LPS for 24 h to establish the in vitro model of vascular endothelial cell injury. After 24 h of treatment of caffeoyl quinic acids from Chrysanthemum morifolium, the levels of TNF-α, IL-6, IL-1β, vascular cell adhesion molecule-1 (VCAM-1) and endothelin-1 (ET-1) were measured by ELISA in the cell culture supernatant, the malondialdehyde (MDA) level was detected by TBA method, the superoxide dismutase (SOD) level was determined by hydroxylamine method, and the nitric oxide (NO) level was assayed by a one-step method. The levels of p-MEK1/2, MEK1/2, p-ERK1/2, ERK1/2, p-JNK, JNK, p-P38 and P38 of mitogen-activated protein kinase (MAPK) signaling pathway were detected by Western blot. The total extract of Chrysanthemum morifolium can significantly reduce the wet weight/dry weight ratio of lung in mice and the levels of TNF-α, IL-6 and IL-1β in alveolar lavage fluid. The caffeoyl quinic acids from Chrysanthemum morifolium significantly increased the levels of SOD and NO, decreased the levels of TNF-α, IL-6, IL-1β, VCAM-1, ET-1 and MDA, and significantly reduced the levels of p-MEK1/2, p-ERK1/2. In conclusion, total extracts of Chrysanthemum morifolium exhibit certain protective effect on mice with acute lung injury, and three caffeoyl quinic acids from Chrysanthemum morifolium may improve LPS-induced vascular endothelial cell injury by inhibiting inflammatory cytokines and oxidative stress, and regulating inter-cellular adhesion molecule and vasomotor factors through ERK/MAPK signaling pathway.

Effects of Fritillariae Cirrhosae Bulbus on Airway Inflammation and ERK/MAPK Signal Pathway in Asthma Model Mice / 中国药房

OBJECTIVE: To investigate the effects of Fritillariae cirrhosae bulbus on airway inflammation and ERK/MAPK signal pathway of asthma model mice, and to explore its possible mechanism of the treatment of asthma. METHODS: The asthma model was induced by egg albumin. A total of 40 model mice were randomly divided into model group (0. 5％ carboxymethyl cellulose, intragastric administration), positive control group (0. 5 mg/kg dexamethasone, intraperitoneal injection), Fritillariae cirrhosae bulbus low-dose and high-dose groups (9. 0, 18. 0 mg/kg, intragastric administration), with 10 mice in each group. Other 10 normal mice were included in normal group (0. 5％ carboxymethyl cellulose, intragastric administration). They were given medicine once a day for consecutive 28 d. After medication, the number of total cells and differential cells (neutrophils, macrophages, lymphocytes and eosinophils) in bronchoalveolar lavage fluid (BALF) of mice were counted. The pathological morphology of bronchial smooth muscle in mice was observed under light microscope, and the inflammatory score was scored; the activities of ERK, phosphorylated ERK (p-ERK), p38 MAPK and phosphorylated p38 MAPK (p-p38 MAPK) were measured by ELISA. The protein expression of ERK, p-ERK, p38 MAPK and p-p38 MAPK in lung tissue were determined by Western blot assay. mRNA expression of ERK and p38 MAPK were determined by real time fluorescent quantitative PCR. RESULTS: Compared with normal group, the number of total cells and differential cells in BALF of mice, inflammation score, the activities of p-ERK and p-p38 MAPK in lung tissues were increased significantly of mice in model group (P<0. 01); the protein expression of p-ERK and p-p38 MAPK, mRNA expression of ERK and p38 MAPK were increased significantly in lung tissue of mice in model group (P<0. 01). Compared with model group, above indexes of treatment groups were all improved significantly (P<0. 05 or P<0. 01). CONCLUSIONS: Fritillariae Cirrhosae Bulbus can improve airway inflammation in asthma model mice, the mechanisms of which may be related to inhibiting the activation of ERK/MAPK signal pathway.

Cantharidin suppressed breast cancer MDA-MB-231 cell growth and migration by inhibiting MAPK signaling pathway

As an active constituent of the beetle Mylabris used in traditional Chinese medicine, cantharidin is a potent and selective inhibitor of protein phosphatase 2A (PP2A) that plays a crucial role in cell cycle progression, apoptosis, and cell fate. The role and possible mechanisms exerted by cantharidin in cell growth and metastasis of breast cancer were investigated in this study. Cantharidin was found to inhibit cell viability and clonogenic potential in a time- and dose-dependent manner. Cell cycle analysis revealed that cell percentage in G2/M phase decreased, whereas cells in S and G1 phases progressively accumulated with the increasing doses of cantharidin treatment. In a xenograft model of breast cancer, cantharidin inhibited tumor growth in a dose-dependent manner. Moreover, high doses of cantharidin treatment inhibited cell migration in wound and healing assay and downregulated protein levels of major matrix metalloproteinases (MMP)-2 and MMP-9. MDA-MB-231 cell migration and invasion were dose-dependently inhibited by cantharidin treatment. Interestingly, the members of the mitogen-activated protein kinase (MAPK) signaling family were less phosphorylated as the cantharidin dose increased. Cantharidin was hypothesized to exert its anticancer effect through the MAPK signaling pathway. The data of this study also highlighted the possibility of using PP2A as a therapeutic target for breast cancer treatment.

OTX1 Contributes to Hepatocellular Carcinoma Progression by Regulation of ERK/MAPK Pathway

Orthodenticlehomeobox 1 (OTX1) overexpression had previously been associated with the progression of several tumors. The present study aimed to determine the expression and role of OTX1 in human hepatocellular carcinoma (HCC). The expression level of OTX1 was examined by quantitative real-time PCR (qRT-PCR) in 10 samples of HCC and paired adjacent non-cancerous tissues, and by immunohistochemistry (IHC) analysis in 128 HCC samples and matched controls. The relationship between OTX1 expression and the clinicopathological features werealso analyzed. Furthermore, the effects of OTX1 knockdown on cell proliferation and migration were determined in HCC cell lines. Axenograft mouse model was also established to investigate the role of OTX1 in HCC tumor growth. TheqRT-PCR and IHC analyses revealed that OTX1 was significantly elevated in HCC tissues compared with the paired non-cancerous controls. Expression of OTX1 was positively correlated with nodal metastasis status (P = 0.009) and TNM staging (P = 0.001) in HCC tissues. In addition, knockdown of OTX1 by shRNA significantly inhibited the proliferation and migration, and induced cell cycle arrest in S phase in vitro. Tumor growth was markedly inhibited by OTX1 silencing in the xenograft. Moreover, OTX1 silencing was causable for the decreased phosphorylation level of ERK/MAPK signaling. In conclusion, OTX1 contributes to HCC progression possibly by regulation of ERK/MAPK pathway. OTX1 may be a novel target for molecular therapy towards HCC.

Effect of osthole on ERK/MAPK signaling pathway and the expression of COX-2 mRNA in the spinal dorsal horn of rats with nucleus pulposus-induced inflammatory radicular pain / 中国药理学通报

Aim Toinvestigatetheanalgesiceffectsof epidural osthole application on the mechanical allodyn-ia and the ERK/MAPK signaling pathway and the expression of COX-2 mRNA in the spinal dorsal horn.Methods 125adultmaleSDratswererandomizedin-to five groups( n=25 each) :Blank, Sham, NP, Ost and vehicle. At postoperative day 6, 1mg/rat osthole 50 μl was injected epidurally into group Ost and the same volume of vehicle was given into group vehicle. The mechanical pain threshold was measured by 50%MWT at 1 day before operation and the 3 rd,6 th,7 th, 14 th,21 st day after operation. After the measurement of pain threshold on postoperative day 14 , the L4-6 segment of spinal dorsal horn was removed for determi-nation of the expression of ERK, pERK and COX-2 mRNAbyWesternblotandRT-PCR.Results Com-pared with blank group, the mechanical pain threshold was only down-regulated at day 1 after operation in sham group, the expression of pERK and COX-2 mR-NA in sham group showed no significant difference ( P>0. 05 ); the mechanical pain threshold was signifi-cantly down-regulated after operation in NP, Ost and vehicle groups( P0. 05). The correla-tion analysis on pERK1/2 and COX-2 mRNA revealed the Pearson correlation coefficient was 0 . 878 and 0 . 910 , suggesting a strong positive correlation between pERKandCOX-2mRNA.Conclusions Ostholead-ministrated in the early stage after surgery can alleviate the nucleus pulposus-induced radicular inflammatory pain probably by inhibiting the expression of pERK and COX-2 mRNA in spinal dorsal horn.

The effect of Shugan-Yishen formula on HER2-ERK/MAPK-ERα Pathway of TAM-resistant breast cancer cell lines / 国际中医中药杂志

Objective To investigate the effect of Shugan-Yishen formula on Tamoxifen-resistant MCF-7 cell line(LCC9) through HER2-ERK/MAPK-ERα pathway. Methods Four serums were prepared with the method of serum pharmacology:the serum of Shugan-Yishen formula, Shugan-Yishen formula plus Tamoxifen(TAM), blank and TAM alone. After LCC-9 cells were affected in the above-mentioed serums in vitro, the inhibition rate and the level of mRNA as well as the protein expression of HER2, ERα, ERK/MAPK, p-ERK/MAPK were detected. Results MTT showed that TAM alone group、Shugan-Yishen formula group and the Shugan-Yishen formula plus TAM group had higher inhibition rates at 48th hour, the inhibition rates was(6.61±0.129)%, (47.43±2.24)%, (54.19±3.364)%, compared to the blank group, Shugan-Yishen formula group and the Shugan-Yishen formula plus TAM group had a stronger inhibition on LCC9 at 48th hour (P0.05). RT-PCR result showed that compared to the blank group, the expression levels of ESR1 mRNA of group of Shugan Yishen formula plus TAM werehigher by and large at 48th hour (P0.05). Conclusions ①The combination of Shugan-Yishen formula with tamoxifen can inhibit the LCC9 cells. The inhibition effect reaches the peak at 48th hour. ②The mechanism of Shugan-Yishen formula plus TAM combination effects on human breast cancer TAM-resistant cell line LCC9 may improve the sensitivity of ERαby inhibiting the expression of HER2, ERK/MAPK.

The Role of Protein Phosphorylation in the Gustatory Cortex and Amygdala During Taste Learning

Protein phosphorylation and dephosphorylation form a major post-translation mechanism that enables a given cell to respond to ever-changing internal and external environments. Neurons, similarly to any other cells, use protein phosphorylation/dephosphorylation to maintain an internal homeostasis, but they also use it for updating the state of synaptic and intrinsic properties, following activation by neurotransmitters and growth factors. In the present review we focus on the roles of several families of kinases, phosphatases, and other synaptic-plasticity-related proteins, which activate membrane receptors and various intracellular signals to promote transcription, translation and protein degradation, and to regulate the appropriate cellular proteomes required for taste memory acquisition, consolidation and maintenance. Attention is especially focused on the protein phosphorylation state in two forebrain areas that are necessary for taste-memory learning and retrieval: the insular cortex and the amygdala. The various temporal phases of taste learning require the activation of appropriate waves of biochemical signals. These include: extracellular signal regulated kinase I and II (ERKI/II) signal transduction pathways; Ca(2+)-dependent pathways; tyrosine kinase/phosphatase-dependent pathways; brain-derived neurotrophicfactor (BDNF)-dependent pathways; cAMP-responsive element bindingprotein (CREB); and translation-regulation factors, such as initiation and elongation factors, and the mammalian target of rapamycin (mTOR). Interestingly, coding of hedonic and aversive taste information in the forebrain requires activation of different signal transduction pathways.

Helicobacter pylori cytotoxin associated protein CagA up-regulating gastrin gene expression / 中华微生物学和免疫学杂志

Objective To study whether Helicobacter pylori CagA protein can control gastrin gene expression and the detailed mechanism. Methods First, pcDNA3. 1ZEO (-)/cagA7 was transfected into gastric cancer cell lines AGS and SGC-7901 cells. At the same time, culturing the Helicobacter pylori NCTC11637 and infecting AGS and SGC-7901 cells with it. Next, in the infected and transfecled AGS and SGC-7901 cells, respectively adding the JAK2 signaling pathway inhibitor AG490 and the ERK signaling pathway inhibitor U0126 to inhibit the two signaling pathway. Untreated gastric cancer cells and empty vector transfected cells as the control. Using real-time fluorescence quantitative PCR to detect the levels of gastrin mRNA in transfected and infected cells. Results After AGS and SGC-7901 cells were transfected with pcDNA3. lZE0(-)/cagA7 and infected with NCTC11637, the results showed that the expression of gastrin mRNA increased significantly (P ＜ 0. 05) in transfected and infected cells as compared with the control group, but after adding the inhibitor AG490 and U0126 respectively, the expression of gastrin mRNA decreased significantly(P＜0.05). Conclution These results suggest that CagA may up-regulate the expression of the gastrin gene, and CagA is one of the important proteins in regulating gastrin gene expression. The ERK/MAPK and JAK/STAT signaling pathways may be involved in controlling of gastrin gene expression by CagA.

Significance of ERK/MAPK signal transduction pathway in the targeting treatment of gastrointestinal cancer / 中国临床药理学与治疗学

Extracellular signal-regulated kinase/mitogen-activated protein kinase(ERK/MAPK) signaling pathway plays an important role in gastrointestinal tumorigenesis and development.ERK/MAPK signal transduction pathway has three important molecular targets: the small G protein Ras,Raf kinase and MEK1/2 and ERK1/2.At present,there are three kinds of approach which can inhibit ERK / MAPK signal transduction pathway:⑴ destroying the structure and(or) function of the target protein,⑵adopting the deficit strategy,⑶damaging the interaction between proteins.These approaches can provide new ideas for the treatment of gastrointestinal cancer.