Bone-edge electroacupuncture relieves pain by regulating expression of GRK5, β-arrestin 2 and PKCα proteins in locus coeruleus in bone cancer pain rats with morphine tolerance / 针刺研究

OBJECTIVE: To observe the effect of bone-edge electroacupuncture (EA) intervention on mechanical pain threshold (PT) and expression of G protein-coupled receptor kinase (GRK5), β-arrestin 2, total and phosphorylated PKC alpha (p-PKCα) proteins in the locus coeruleus (LC) of rats with bone cancer pain induced morphine tolerance, so as to reveal its partial central mechanisms underlying pain relief. METHODS: Forty SD rats were randomly divided into 5 groups, namely sham bone cancer, bone cancer pain, morphine tolerance, bone-edge EA, and sham EA (n＝ 8 rats in each group). The bone cancer with morphine tolerance model was established by intramedullary injection of MRMT-1 cells into the tibial cavity, and then intraperitoneal injection of morphine hydrochloride injection. After successful establishment of morphine tolerance model, the bone-edge EA (2 Hz/100 Hz，0.5－1.5 mA) was applied to bilateral "Zusanli" (ST36) and "Kunlun" (BL60) for 30 min, once a day for 7 days, after inserting the needle-tip to the tibial bone surface. The ipsilateral mechanical paw withdrawal thresholds (PWTs) were detected dynamically. The expression levels of GRK5, β-arrestin 2, PKCα and p-PKCα in the LC area were measured by Western blot. RESULTS: The PWTs of bone cancer pain rats were decreased on day 10 after inoculation of cancer cells (P0.05). The PWTs were significantly increased in the bone-edge EA intervention group (P0.05). In comparison with the sham bone cancer group, the expression of GRK5 protein in morphine tolerance group was significantly decreased (P<0.01); compared with morphine tolerance group, the expression of GRK5 protein in bone-edge EA group was increased(P<0.01). In comparison with the sham bone cancer group, the expression of β-arrestin 2 and p-PKCα in bone cancer group significantly increased (P<0.01). After the intervention, the increased β-arrestin 2 and p-PKCα expressions were reversed in the bone-edge EA group (P<0.01); compared with morphine tolerance group and sham EA group, the expression of PKCα protein was decreased(P<0.01). CONCLUSION: Bone-edge EA can effectively relieve morphine tolerance in bone cancer pain rats, which may be related to its functions in up-regulating GRK5 protein and down-regulating β-arrestin 2, PKCα and p-PKCα proteins in LC.　.

PLCE1 Promotes the Invasion and Migration of Esophageal Cancer Cells by Up-Regulating the PKCα/NF-κB Pathway

PURPOSE: To investigate the effect and mechanism of phospholipase C epsilon gene 1 (PLCE1) expression on esophageal cancer cell lines. MATERIALS AND METHODS: The esophageal carcinoma cell lines Eca109 and EC9706 and normal esophageal epithelial cell line HEEC were cultured. The expression of PLCE1, protein kinase C alpha (PKCα), and nuclear factor kappa B (NF-κB) p50/p65 homodimer in cells were comparatively analyzed. The esophageal cancer cells were divided into si-PLCE1, control siRNA (scramble), and mock groups that were transfected with specific siRNA for PLCE1, control siRNA, and blank controls, respectively. Expression of PLCE1, PKCα, p50, and p65 was detected by Western blotting. Transwell assay was used to detect migration and invasion of Eca109 and EC9706 cells. RESULTS: Compared with HEEC, the expression of PLCE1, PKCα, p50, and p65 was increased in Eca109 and EC9706 cells. The expression of PLCE1 was positively correlated with the expression of PKCα and p50 (PKCα: r=0.6328, p=0.032; p50: r=0.6754, p=0.041). PKCα expression had a positive correlation with the expression of p50 and p65 (p50: r=0.9127, p=0.000; p65: r=0.9256, p=0.000). Down-regulation of PLCE1 significantly decreased the expression of PKCα and NF-κB-related proteins (p65: p=0.002, p=0.004; p50: p=0.005, p=0.009) and inhibited the migration and invasion of Eca109 and EC9706 cells. CONCLUSION: PLCE1 activated NF-κB signaling by up-regulating PKCα, which could promote invasion and migration of esophageal cancer cells.

PLCε regulates invasion and migration of human bladder cancer cells T 24 through PKCα/β/TBX3 pathway / 基础医学与临床

Objective To investigate the molecular mechanisms of PLCεin regulating the invasion and migration of human bladder cancer cells in vitro.Methods After cells treated with recombinant adenovirus , the migratory/in-vasive abilities of T24 cells were explored by wound healing and Transwell chamber cell migration and invasion as -say;RT-PCR was used to detect the mRNA levels of PLCε;The protein levels of PLCε,PKCα,PKCβ, TBX3 and E-cadherin were determined by Western blot;QRT-PCR was used to detect the mRNA levels of TBX3 and E-cad-herin.Results It was confirmed by digesting and sequencing that the recombinant adenovirus had been constructed successfully .The expression of PLCε mRNA and PLCε protein were both decreased after the infection of Ad-shPLCε.Wound healing and Transwell chamber cell migration/invasion assay showed that Ad-shPLCε treatment could inhibit the migratory and invasive activity of bladder cancer cells(P<0.05).The results of Western blot indicated that the expression of PKCα/βin membrane decreased ( P<0.05 ) , and phosphorylation level of PKCαand PKCβwas reduced .QRT-PCR and Western blot analysis demonstrated that the expression level of TBX 3 de-creased , but the expression level of E-cadherin increased .Conclusions PLCε shRNA can inhibit migratory and invasive ability of bladder cancer cells through PKCα/β/TBX3/E-cadherin pathway .

PC-PLC/PKC-α pathway involves in TNF-α-induced IP3R1 expression in human mesangial cells / 中华急诊医学杂志

Objective To explore the effects of TNF-α on the expression of IP33R1 mRNA and protein in human mesangial cells (HMCs) and elucidate the mechnism of TNF-α indnces the IP3R1 expression in the occurrence of hepatorenal syndrome (HRS).Methods HMCs was stimulated by tumor (TNF-α) with 100 ng/mL for different hours (2,4,8,24 hours).The expression change of IP3R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblot assay.Several inhibitors including D609,U73122,PP1,Safingol,Rottlerin and non-radioactive PKC assay to examine the mechanism of signal transduction of TNF-α-regulated IP3R1 in HMCs.Results The levels of IP3R1 mRNA at 2 h post-TNF-α exposure were significantly enhanced and reached peak at 8 h in HMCs (P ＜ 0.01),then descened at 24 h (P ＜ 0.01).The levels of IP3R1 protein at 4 h post-TNF-α exposure were obviously increased and reached peak at 24 h post-TNF-α exposure (P ＜ 0.01).Compared with the control group,safingol (PKC-α inhibitor) and D609 (PC-PLC inhibitor) each significantly suppressed TNF-α-induced expression of IP3R1 mRNA (3.30 ± 0.81) vs.(1.95 ± 0.130,P ＜ 0.05 ; (2.10 ± 0.49),P ＜ 0.01 andIP3R1 protein (3.09±0.13) vs.(1.86+0.39),P＜0.01; (1.98±0.02),P＜0.01.TNF-αpromoted autophosphorylation,and hence the activation,of PKC-α with maximal phosphorylation that occurred 8 h post-stimulation measured by non-radioactive PKC assay,and the effect was marked attenuated by pretreated with D609 or safingol.Conclusions TNF-α increased the expression of IP3R1 and this was mediated,at least in part,through the PC-PLC/PKC-α signaling pathways in HMCs.

The study of miR-216a induces cell apoptosis by targeting PKCαin gastric cancer / 重庆医学

Objective To confirm whether miR-216a suppresses cell proliferation and induces cell apoptosis by targeting PKCα, thus to reveal molecular mechanism that miR-216a functions as a tumor suppressor in gastric cancer .Methods PKCα3′untranslat-ed region(UTR)-luciferase vector was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-216a on luciferase activity .MGC-803 cells were transfected with miR-216a mimics ,and next Western blotting was performed to detect the expression of PKCαprotein .The effects of PKCαdownregulation on cell proliferation and apoptosis were observed after PKCαsiRNA were transfected into MGC-803 cells .MGC-803 cell proliferation assays were performed when cotransfected with miR-216a mimics .Results The result demonstrated miR-216a could bind to the 3′UTR of PKCαand inhibited the luciferase activi-ty ,cut the 41% .PKCαprotein expressions were significantly down-regulated when miR-216a was overexpressed in MGC-803 .siR-NA-mediated downregulation of PKCα could suppress the potentials of cell proliferation and induce apoptosis .Conclusion miR-216a suppresses cell proliferation and induces apoptosis by targeting PKCαmRNA 3′UTR in gastric cancer .

Effects of PKC on TNF-α stimulated IP3 R1 protein expression in human mesangial cells / 中华微生物学和免疫学杂志

Objective To explore the effects of TNF-α on the expression of IP3 R1 mRNA and protein in human mesangial cells (HMCs) and elucidate the role of protein kinase C (PKC) in this signal pathway.Methods Quantitative real-time polymerase chain reaction and immunoblot assay were used to examine the effects of TNF-α on IP3R1 mRNA and protein expression.Depletion PKC,the selective inhibitor of PKCα Safingol and inhibitor of PKCδ Rottlerin,overexpression of dominant negative mutant of PKC to examine the mechanism of signal transduction of TNF-α-regulated IP3 R1 in HMCs.PKCα activation was assayed by Western blot.Results TNF-α increased IP3R1 mRNA and protein expression in HMCs,effects that were blocked by prolonged incubted chronic PMA,Safingol and also by domain negative PKCα construct.TNF-α promoted PKCα activation with maximal PKCα phosphorylation that occurred 8 h post-stimulation.Conclusion TNF-α increased the expression of IP3 R1 and this was mediated through the PKCα activation signaling pathways in HMCs.

Changes of Protein Kinase Cα and Cyclin D1 Expressions in Pulmonary Arteries from Smokers with and without Chronic Obstructive Pulmonary Disease / 华中科技大学学报（医学）（英德文版）

The purpose of this study was to investigate the changes of protein kinase Cα(PKCα)and cyclin D1 expressions in pulmonary arteries from smokers with normal lung function and smokers with mild to moderate chronic obstructive pulmonary disease(COPD).The peripheral lung tissues were obtained from 10 non-smokers with normal lung function(non-smoker group),14 smokers with normal lung function(smoker group),11 smokers with mild to moderate COPD(COPD group).The morphological changes of pulmonary arteries were observed by HE-staining.The expressions of α-smooth muscle actin(α-SMA),proliferating cell nuclear antigen(PCNA),PKCα and cyclin D1 proteins in pulmonary artery smooth muscle cells(PASMCs)were immunohistochemically determined.The percentages of PCNA-positive cells were taken as the smooth muscle cells proliferation index(PI).The mRNA expressions of PKCα and cyclin D1 in PASMCs were evaluated by real-time fluorescence PCR.Morphometrical analysis showed that the ratio of pulmonary artery wall area to total area(WA%)in smoker group and COPD group was significantly greater than that in non-smoker group(P<0.01).The PASMCs proliferation index in smoker group and COPD group was significantly higher than that in nonsmoker group(P<0.01).The protein levels of PKCα and cyclin D1 in PASMCs were significantly increased in smoker group and COPD group as compared with non-smoker group(P<0.01).The mRNA expressions of PKCa and cyclin D1 in PASMCs were significantly elevated in smoker group and COPD group as compared with non-smoker group(P<0.01).Significant correlations were found between PKCα protein and WA% or PI(P<0.01).Correlations between cyclin D1 protein and WA% or PI also existed(P<0.01).The expression of PKCα was positively correlated with the expression of cyclin D1 at both protein and mRNA levels(P<0.01).In conclusion,increased expressions of PKCα and cyclin D1 might be involved in the pathogenesis of abnormal proliferation of PASMCs in smokers with normal lung function and smokers with mild to moderate COPD.

Interaction between fragile histamine triad and protein kinase C alpha in human non-small cell lung cancer tissues / 西安交通大学学报·英文版

Objective: To investigate the interaction between fragile histamine triad (FHIT) and protein kinase C alpha (PKCα) in human non-small cell lung cancer tissues. Methods: FHIT and PKCα double positive samples were screened by immunohistochemical staining from 13 human non-small cell lung cancer tissues. Co-immunoprecipitation was performed by using anti-FHIT and anti-PKCα. The immune precipitate was analyzed by SDS-PAGE and Western blot. Results: Immune precipitate staining detection showed that 3 samples out of the 13 cases were double positive for FHIT and PKCα. FHIT protein was present in the immune precipitate of anti-PKCα while there was PKCα in the immune precipitate of anti-FHITmAb. Conclusion: FHIT and PKCα exist as a complex in human non-small cell lung cancer tissues, which will provide a new route for studying the pathogenesis and immunotherapy of human non-small cell lung cancer.