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Objective:To observe the effect of berberine on leukemia drug-resistant cell strain K562/A02 to Adriamycin resistance and protein kinase C-alpha (PRKCA) and explore its possible mechanism.Methods:The leukemia K562 cells of human chronic myeloid and Adriamycin resistant strain K562/A02 were cultured in vitro with 2.5-50.0 μmol/L doxorubicin to treat thoese cells and drug resistance of K562 and K562/A02 to Adriamycin was detected, the 50% inhibitory concentration (IC 50) of the drug was calculatedthe resistance of K562 and K562/A02 to doxorubicin was detectd , and, K562/A02 cells were treated with doxorubicin solution at a final concentration of 5 μmol/L, and K562/A02 cells were divided into control group, inhibitor group (50 μmol/L PRKCA inhibitor), low dose berberine group, medium dose berberine group and high dose berberine group. Cell counting (CCK-8) method was used to detect the inhibition rate of cell proliferation, the apoptosis was detected by flow cytometry, real-time fluorescent quantitative PCR assay detects PRKCA, MRP, multidrug resistance related genes (MDR1) levels, and the protein expressions of protein kinase C-α (PRKCA), multidrug resistance related protein (MRP), P-glycoprotein (P-gp) were detected by Western blotting. Results:The IC 50 concentration of K562/A02 to Adriamycin was significantly higher than K562. Compared with the control group, the inhibition rate of cell proliferation and the apoptosis rate in the inhibitor group, low-dose berberine group, medium-dose berberine group, and high-dose berberine group were significantly increased ( P<0.05), the expression of PRKCA mRNA (0.45±0.08, 0.92±0.10, 0.57±0.05, 0.35±0.04 vs. 1.00±0.12), MDR1 gene (0.73±0.08, 0.87±0.09, 0.65±0.07, 0.41±0.05 vs. 1.00±0.11) and PRKCA (0.59±0.09, 0.78±0.12, 0.61±0.11, 0.42±0.07 vs. 0.96±0.14), MRP (0.62±0.08, 0.79±0.13, 0.62±0.10, 0.41±0.06 vs. 0.98±0.14), P-gp (0.55±0.08, 0.75±0.12, 0.59±0.09, 0.35±0.06 vs. 0.92±0.15) were significantly reduced ( P<0.05), and berberine was dose-dependent ( P<0.05); Overexpression of PRKCA can inhibit the effect of berberine on reversing the drug resistance of K562/A02 cells. Conclusion:Berberine may reverse the drug resistance of K562/A02 to Adriamycin by down-regulating PRKCA.
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Objective To evaluate the role of protein kinase Cα (PKCoα)/heme oxygenase-1 (HO-1) signaling pathway in lipopolysaccharide (LPS)-caused damage to type Ⅱ alveolar epithelial cells of rats and the relationship with mitochondrial fusion.Methods Type Ⅱ alveolar epithelial cells were seeded in 96-well plates at a density of 2× 105 cells/ml and divided into 5 groups (n =40 each) using a random number table method:control group (group C),Go6976 group (group G),LPS group (group L),LPS plus PKCα inhibitor Go6976 group (group LG) and LPS plus dimethyl sulfoxide (DMSO) group (group LD).Group LG and group LD were pretreated with 5 μmol/L Go6976 and the equal volume of 0.1％ DMSO,respectively,for 30 min,lipopolysaccharide (LPS) 10 μg/ml was then given to establish the model of type Ⅱ alveolar epithelial cell damage in L,LG and LD groups,Go6976 5 μmol/L was added in group G,and the equal volume of phosphate buffer solution was added in group C.The cells were collected after 24 h of incubation for measurement of malondialdehyde (MDA) and reactive oxygen species (ROS) contents,superoxide dismutase (SOD) activity and expression of PKCα,HO-1,mitochondrial fusion-related proteins 1 and 2 (Mfn1,Mfn2),optic atrophy 1 (OPA1) protein and mRNA (by fluorescent quantitative polymerase chain reaction or Western blot).Results Compared with group C,MDA and ROS contents were significantly increased,the SOD activity was decreased,the expression of PKCα and HO-1 protein and mRNA was up-regulated,and the expression of Mfn1,Mfn2 and OPA1 protein and mRNA was downregulated in L,LG and LD groups (P＜0.05),and no significant change was found in the parameters mentioned above in group G (P＞0.05).Compared with group L,MDA and ROS contents were significantly increased,the SOD activity was decreased,and the expression of PKCα,HO-1,Mfn1,Mfn2 and OPA1 protein and mRNA was down-regulated in group LG (P＜0.05),and no significant change was found in the parameters mentioned above in group LD (P＞0.05).Conclusion Activation of PKCα/HO-1 signaling pathway is the endogenous protective mechanism of LPS-caused damage to type Ⅱ alveolar epithelial cells,which may be related to promoting mitochondrial fusion in rats.
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Objective To investigate the effect of angiotensin Ⅱ on protein kinase Cε (PKCε) and protein kinase Cα (PKCα) expression in hepatic stellate cells.Methods Hepatic stellate cell (HSC)-T6 cells were treated with different concentrations of angiotensin Ⅱ and the proliferation of HSC-T6 cells was detected by methyl thiazolyl tetrazolium (MTT) assay.The expression of PKCε and PKCα was detected by immunofluorescence staining.PKCε and PKCα mRNA levels was detected by real time polymerase chain reaction (PCR).Results Angiotensin Ⅱ concentrated the proliferation of HSC-T6 cells and the level of hydroxyproline (F =25.321,13.283,P ＜ 0.001) and showed a dose-dependent effect.With the increase of angiotensin Ⅱ concentration,PKCε significantly increased and translocated in the cell membrane;PKCα increased significantly,especially in transplanted membrane and cytoplasm (F =21.387,19.431,P ＜0.01),and showed obvious dose effect.Meanwhile,Angiotensin Ⅱ increased the expression of PKCε and PKCα,and induced cell proliferation by up-regulating PKCε and PKCα mRNA levels (F =13.279,15.174,P ＜ 0.05).Conclusions Angiotensin Ⅱ can up-regulate the expression of collagen in hepatic stellate cells in a dose-dependent manner,increase the expression of protein kinase Cε and Cα,and promote the proliferation of hepatic stellate cells.
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Objective To evaluate the role of protein kinase Cα (PKCα)/heme oxygenase-1 (HO-1) signaling pathway in endotoxin-induced damage to alveolar macrophages and the relationship with mitofusin-1 (Mfn1) in rats.Methods Rat alveolar macrophages NR8383 cells cultured in vitro were seeded in 96-well plates at a density of 1 × 104 cells/ml.NR8383 cells were divided into 5 groups (n =15 each)using a random number table:control group (group C),endotoxin challenge model group (group E),PKCα inhibitor Go6976 group (group G),PKCα agonist PMA group (group P) and dimethyl sulfoxide group (group D).NR8383 cells were stimulated with 10 μg/ml lipopolysaccharide (LPS) to establish the model of endotoxin challenge in alveolar macrophages.In G,P and D groups,cells were pretreated with 5 μmol/L Go6976,100 nmol/L PMA and 0.1％ dimethyl sulfoxide,respectively,for 30 min starting from 30 min before stimulation with LPS,and 10 μg/ml LPS was then given.The cells were collected after 24 h of incubation for measurement of malondialdehyde (MDA) and reactive oxygen species (ROS) contents,superoxide dismutase (SOD) activity and expression of PKCα,HO-1 and Mfn1 protein and mRNA (by fluorescent quantitative polymerase chain reaction or Western blot).Results Compared with group C,MDA and ROS contents were significantly increased,the SOD activity was decreased,the expression of PKCα and HO-1 protein and mRNA was up-regulated,and the expression of Mfn1 protein and mRNA was downregulated in E,G,P and D groups (P＜0.05).Compared with group E,MDA and ROS contents were significantly increased,the SOD activity was decreased,and the expression of PKCα,HO-1 and Mfn1 protein and mRNA was down-regulated in group G,MDA and ROS contents were significantly decreased,the SOD activity was increased,and the expression of PKCα,HO-1 and Mfn1 protein and mRNA was upregulated in group P (P＜0.05),and no significant change was found in the parameters mentioned above in group D (P＞0.05).Conclusion Promotion of Mfn1 expression following PKCα/HO-1 signaling pathway activation is the endogenous protective mechanism of endotoxin-induced damage to alveolar macrophages of rats.
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Objective To evaluate the role of PICK1 in remifentanil-induced miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors in spinal dorsal horn neurons and in the expression of AMPA receptors in juvenile rats.Methods Thirty-six male Sprague-Dawley rats,aged 14-18 days,weighing 50-60 g,were used in the study.Eighteen rats were randomly selected,their lumbar segments of the spinal cord were immediately removed,and 108 spinal cord slices (400 μm thick,for wholecell patch-clamp recording) aud 108 spinal cord slices (5 μm thick,for immunofluorescence detection) were prepared.The 108 slices with two kinds of thickness were divided into 3 groups (n=36 each) using a random number table:blank control group (group C),remifentanil group (group R) and remifentanil plus PICK inhibitor group (group R+PlCKi).Spinal cord slices were incubated in artificial cerebrospinal fluid (ACSF) for 90 min in group C.Spinal cord slices were incubated for 90 min in ACSF containing remifentanil at the fiual concentration of 4 mnol/L in group R.Spinal cord slices were incubated for 90 min in ACSF containing remifentanil at the final concentration of 4 nmol/L and 50 μmol PICK inhibitor in group R+PICKi.The whole-cell patch-clamp technique was used to record the amplitude and time interval of AMPA receptors-mediated mEPSCs.The method of immunofluorescence was used to detect the expression of AMPA receptors.Results Compared with group C,the amplitude of mEPSCs was significantly increased,and the time interval of mEPSCs was shortened,the expression of GluR1 and GluR3 was up-regulated,and the expression of GluR2 was down-regulated in R and R+PICKi groups (P＜0.05).Compared with group R,the amplitude of mEPSCs was significantly decreased,and the time interval was prolonged,the expression of GluR3 was down-regulated,the expression of GluR2 was up-regulated (P＜0.05),and no significant change was found in GluR1 expression in group R+PICKi (P＞0.05).Conclusion PICK1 is involved in the process of remifentanil-induced mEPSCs mediated by AMPA receptors in spinal dorsal horn neurons and of expression of AMPA receptors in juvenile rats,which may be the mechanism underlying remifentanil-induced hyperalgesia.
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Objective To evaluate the effect of recombinant human annexin A5 on the expression of phosphorylated protein kinase C alpha (p-PKCα) and p120-catenin during endotoxin-induced damage to cardiomyocytes.Methods H9c2 cells cultured in vitro were randomly divided into 3 groups (n=18 each) using a random number table:control group (group C),endotoxin group (group L),and recombinant human annexin A5 group (group A).Recombinant human annexin A5 (final concentration 5 ng/ml) was added,and the cells were incubated for 2 h in group A,and then lipopolysaccharide (final concentration 1 μg/ml) was added,and the cells were incubated for 4 h in L and A groups.At 4 h of incubation,cell apoptosis was detected using the cell apoptosis detection kit,the intercellular space was measured using the confocal microscopy,and the expression of p-PKCα and p120-catenin was determined by Western blot.The apoptosis rate was calculated.Results Compared with group C,the apoptosis rate was significantly increased,the intercellular space was significantly widened,the expression of p120-catenin was significantly down-regulated,and the expression of p-PKCα was significantly up-regulated in group L (P＜0.05).Compared with group L,the apoptosis rate and intercellular space were significantly decreased,the expression of p120-catenin was significantly up-regulated,and the expression of p-PKCα was significantly down-regulated in group A (P＜0.05).Conclusion Recombinant human annexin A5 can inhibit phosphorylation of PKCα and up-regulate the expression of p120-catenin,thus attenuating endotoxin-induced damage to cardiomyocytes.
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Objective To evaluate regulatory effects of glutamate receptor antagonists on the proliferation and migration of WM451LU malignant melanoma cells, and to explore their related mechanisms. Methods WM451LU cells at exponential growth phase were classified into 3 groups to be treated with the glutamate receptor antagonist MK?801 at 100μmol/L(MK?801 group), the glutamate receptor antagonist CPCCOEt at 10μmol/L(CPCCOEt group), or culture medium(control group). After 24?hour treatment, methyl thiazolyl tetrazolium(MTT)assay was performed to determine cell proliferation rates, scratch assay to evaluate the migration activity of cells, and Western?blot analysis to measure expression levels of proliferating cell nuclear antigen (PCNA), protein kinase Cα(PKCα) both on cell membrane and in cytoplasm, and phosphorylated mitogen?activated protein kinase(p?MAPK). Results After 24?hour treatment, cell proliferation rates were significantly decreased in the MK?801 group and CPCCOEt group compared with the control group(63%± 3.1%and 60%± 2.4%vs. 100%± 1.1%, both P<0.05). The scratch assay showed that cell?free zones in the control group gradually narrowed over time, and the scratch wound tended to close. However, the cell?free zones in the MK?801 group and CPCCOEt group narrowed more slowly compared with the control group, and were still wide after 24?hour culture with no obvious closure of the scratch. The MK?801 group and CPCCOEt group both showed significantly decreased expressions of PCNA(77.0% ± 5.4% and 72.0% ± 4.2% respectively), PKCα on the cell membrane(0.12 ± 0.02 and 0.14 ± 0.02 respectively), and p?MAPK(0.48 ± 0.03 and 0.36 ± 0.04 respectively) compared with the control group(PCNA:100.0%± 1.3%;PKCα:0.38 ± 0.01;p?MAPK:1.00 ± 0.02;all P<0.05).Conclusion In vitro suppression of glutamate receptors can inhibit the proliferation and migration of WM451LU cells, likely through the mediation of the PKCα?MAPK signaling pathway.
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Objective To evaluate the changes in the expression of protein interacting with Cα kinase 1 (PICK1) in the spinal cord and dorsal root ganglion (DRG) neurons during remifentanil-induced hyperalgesia in rats with incisional pain.Methods Thirty-two male Sprague-Dawley rats, weighing 240-260 g, aged 42-49 days, were randomly divided into 4 groups (n =8 each) using a random number table: control group (group C) , incisional pain group (group Ⅰ) , remifentanil group (group R), and remifentanil + incisional pain group (group R + Ⅰ).In R and R+Ⅰ groups, remifentanil was infused intravenously for 60 min at the rate of 1.2 p,g · kg-1 · min-1.In C and Ⅰ groups, normal saline was infused intravenously for 60 min at the rate of 0.12 ml · kg-1 · min-1.In Ⅰ and R+Ⅰ groups, the model of incisional pain was established, and remifentanil and normal saline were infused intravenously, respectively, at the same time.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before normal saline or remifentanil infusion, and at 2, 6, 24, and 48 h after the end of normal saline or remifentanil infusion (T1-4).The rats were sacrificed after the last measurement of pain threshold.The lumbar segment (L4-6) of the spinal cord and left DRGs were removed for determination of the expression of PICKl mRNA (by quantitative real-time reverse transcriptase-polymerase chain reaction) and PICK1 protein (by Western blot).Results Compared with group C, the MWT was significantly decreased, the TWL was shortened, and the expression of PICK1 protein and mRNA was up-regulated in R and R+Ⅰ groups, and the MWT was significantly decreased, the TWL was shortened (P＜0.05) , and no significant change was found in the expression of PICK1 protein and mRNA in group Ⅰ (P＞0.05).Compared with group Ⅰ, the MWT was significantly decreased, the TWL was shortened, and the expression of PICK1 protein and mRNA was up-regulated in group R+Ⅰ (P＜0.05).Compared with group R, the MWT was significantly decreased, the TWL was shortened (P＜0.05) , and no significant change was found in the expression of PICK1 protein and mRNA in group R+ Ⅰ (P＞0.05).Conclusion The mechanism by which remifentanil induces hyperalgesia may be related to up-regulation of PICK1 expression in the spinal cord and DRG neurons of rats with incisional pain.
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Objective To evaluate the role of protein kinase Ca (PKCα) in electroacupuncture (EA)-induced reduction of acute kidney injury (AKI) induced by endotoxic shock,and the relationship with nuclear factor E2-related factor 2/heme oxygenase-1 Nrf2/HO-1 pathway in rabbits.Methods Eighty heahhy male New Zealand white rabbits,aged 2 months,weighing 1.5-2.0 kg,were randomly divided into 8 groups (n=10 each) using a random number table:sham operation group (group S),group AKI,specific PKCα inhibitor chelerythrine + AKI group (group CHA),chelerythrine group (group Che),dimethyl sulfoxide (DMSO) group (group D),EA at acupoints + AKI group (group EA),EA at non-acupoints + AKI group (group SEA),and EA at acupoints + chelerythrine + AKI group (group CEA).Bilateral 30 min EA (disperse-dense wave,wave length 0.2-0.6 ms,frequency 2/15 Hz,intensity 1-2 mA) stimulation of Zusanli and Shenshu acupoints was performed once a day for 4 days before establishment of the model and during the process of establishment of the model in EA and CEA groups.In group SEA,EA was performed at the points 0.5 cm lateral to the acupoints of Zusanli and Shenshu with the same parameters.The animals were anesthetized with iv 20％ urethane 5 ml/kg,tracheostomized and kept spontaneous breathing.Lipopolysaccharide 5 mg/kg (in 2 ml of normal saline) was injected via the auricular vein to establish the model of endotoxic shock-induced AKI in AKI,CHA,EA,SEA and CEA groups,while the equal volume of normal saline was given in S,Che and D groups.At 30 min before establishment of the model,chelerythrine 5 mg/kg (in 0.5 ml of 1％ DMSO) was injected intravenously in CHA and CEA groups,the equal volume of chelerythrine was given in Che group,while the equal volume of DMSO was given in group D.At 6 h after lipopolysaccharide or normal saline injection,blood samples were taken from the internal carotid artery for determination of serum urea nitrogen (BUN) and creatinine (Cr) concentrations.The rabbits were then sacrificed by exsanguinations.The kidney specimens were removed for microscopic examination of pathologic changes which were scored and for determination of superoxide dismutase (SOD) activities,malondialdehyde (MDA) contents,and expression of PKCα protein and HO-1 protein,and expression of Nrf2 in nucleoprotein and total protein.Results Compared with group S,the serum BUN and Cr concentrations were significantly increased,MDA contents were increased,the activities of SOD were decreased,the kidney injury scores were increased,and the expression of PKCα protein,HO-1 protein,and Nrf2 in nucleoprotein and total protein was up-regulated in AKI,CHA,EA,SEA and CEA groups.Compared with group AKI,the serum BUN and Cr concentrations were significantly decreased,MDA contents were decreased,the activities of SOD were increased,the kidney injury scores were decreased,and the expression of PKCα protein,HO-1 protein,and Nrf2 in nucleoprotein and total protein was up-regulated in group EA,and the serum BUN and Cr concentrations were significantly increased,MDA contents were increased,the activities of SOD were decreased,the kidney injury scores were increased,and the expression of PKCα protein,HO-1 protein,and Nrf2 in nucleoprotein and total protein was down-regulated in CHA and CEA groups.The serum BUN and Cr concentrations were significantly higher,MDA contents were higher,the activities of SOD were lower,the kidney injury scores were higher,and the expression of PKCα protein,HO-1 protein,and Nrf2 in nucleoprotein and total protein was lower in group CEA than in group EA,and in CHA group than in CEA group.Conclusion PKCα mediates reduction of endotoxic shock-induced AKI by EA of Zusanli and Shenshu acupoints in rabbits,and the mechanism may be related to activation of Nrf2/HO-1.
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Objective To investigate the signal mechanism of protein kinase C alpha(PKC-α)participated in enhanced expression of type Ⅰ inositol 1,4,5-trisphophate receptors (IP3 RI) induced by tumor necrosis factor alpha(TNFα),in order to delineate the mechanisms of decreased glomerular filtration rate (GFR) in hepatorenal syndrome caused by TNFα.Methods The glomerular mesangial cells (GMCs)line from rats was chosen as experimental material.GMCs were divided into control (D),TNFα-2 h,TNFα-4 h,TNFα-8 h,and TNFα-24 h groups.Moreover,another two groups were sanflngol-8h (S),TNFα + Sanfingol-8h(TS)groups.The effect of TNFα on the expression of IP3RI was detected by immunocytochemical staining,Western blotting,teal time-polymerase chain reaction (PCR) assays.Results Immunocytochemical staining demonstrated that IP3 RI was mainly distributed in cytoplasm of GMCs.Enhanced positive staining was determined in all TNFα-treated groups,especially in TNFα-8 h group.Western blotting demonstrated that the expression of IP3RI protein was significantly higher in TNFα-4 h,TNFα-8h and TNFα-24 h groups than control group(4 h:1.82 ± 0.63 ; 8 h:2.95 ± 0.66 ; 24 h:2.48 ± 0.72 ; D:1 ±0.02 ; F =9.24,P ＜ 0.05).The expression of IP3 RI protein was the highest in TNFα-8 h and TNFα-24 h groups(P ＜0.05).No difference was found among S,TS,and control groups(S:1.39 ±0.65; TS:1.35± 0.37 ; P ＞ 0.05).Real time-PCR found the expression of IP3 RImRNA was significantly higher in all TNFα-treated groups than control group(2 h:3.35 ± 1.97; 4 h:3.16 ± 1.35; 8 h:3.70 ± 1.76; 24 h:4.49±1.70; D:1 ±0.01; F =6.167,P ＜0.05).No difference was found among all TNFα-treated groups(P ＞0.05).No difference was found among S,TS,and control groups(S:1.53 ±0.79; TS:1.32 ± 0.38 ; P ＞ 0.05).Conclusions IP3 RI was mainly distributed in cytoplasm of GMCs.TNFa could enhance the expression of IP3 RI protein and IP3 RI mRNA,which could be blocked by sanfingol,a PKCα inhibitor.It might be an important signal in the mechanisms of GFR decrease caused by TNFα in hepatorenal syndrome.
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An eleven-year-old spayed female Yorkshire Terrier presented with a sublumbar mass and upon ultrasonographic examination, was revealed to have a mammary gland tumor. Black to reddish colored masses, located in the visceral peritoneum of the sublumbar region was observed on laparotomy with masectomy of the right side. In the laparotomy, we observed reddish masses multifocally located in the serosal membrane of the large intestine. Histopathologic examination of the intestinal and abdominal mass showed highly invasiveness into the muscle and metastasis of melanocytic tumor cells through the blood vessels. The mammary glands showed abnormal hyperplasia of melanocytes, destruction of the normal glands by tumor cells and infiltration of some lymphocytes in the pool of melanocytic cells. We have identified a malignant melanoma containing an angiotumoral complex in which tumor cells occupied a pericytic location along the microvessels with intravasation determined by immunohistochemistry for S100 protein and protein kinase C-alpha. Histologic findings in this dog lead to a diagnosis of an angiotropic metastatic malignant melanoma.
Subject(s)
Animals , Dogs , Female , Humans , Blood Vessels , Hyperplasia , Immunohistochemistry , Intestine, Large , Laparotomy , Lymphocytes , Mammary Glands, Human , Melanocytes , Melanoma , Membranes , Microvessels , Muscles , Neoplasm Metastasis , Peritoneum , Protein Kinase C-alphaABSTRACT
Epigallocatechin-3-gallate (EGCG) is the most potent antioxidant polyphenol in green tea. In the present study, we investigated whether EGCG plays a role in the expression of transforming growth factor-beta1 (TGF-beta1), protein kinase C (PKC) alpha/betaII, and nuclear factor-kappaB (NF-kappaB) in glomerular epithelial cells (GECs) against high-glucose injury. Treatment with high glucose (30 mM) increased reactive oxygen species (ROS)/lipid peroxidation (LPO) and decreased glutathione (GSH) in GECs. Pretreatment with 100 microM EGCG attenuated the increase in ROS/LPO and restored the levels of GSH, whereas ROS, LPO, and GSH levels were not affected by treatment with 30 mM mannitol as an osmotic control. Interestingly, high-glucose treatment affected 3 separate signal transduction pathways in GECs. It increased the expression of TGF-beta1, PKC alpha/betaII, and NF-kappaB in GECs, respectively. EGCG (1, 10, 100 microM) pretreatment significantly decreased the expression of TGF-beta1 induced by high glucose in a dose-dependent manner. In addition, EGCG (100 microM) inhibited the phosphorylation of PKC alpha/betaII caused by glucose at 30 mM. Moreover, EGCG (1, 10, 100 microM) pretreatment significantly decreased the transcriptional activity of NF-kappaB induced by high glucose in a dose-dependent manner. These data suggest that EGCG could be a useful factor in modulating the injury to GECs caused by high glucose.
Subject(s)
Catechin , Epithelial Cells , Glucose , Glutathione , Mannitol , NF-kappa B , Phosphorylation , Protein Kinase C , Reactive Oxygen Species , Signal Transduction , Tea , Transforming Growth Factor beta1ABSTRACT
Epigallocatechin-3-gallate (EGCG) is the most potent antioxidant polyphenol in green tea. In the present study, we investigated whether EGCG plays a role in the expression of transforming growth factor-beta1 (TGF-beta1), protein kinase C (PKC) alpha/betaII, and nuclear factor-kappaB (NF-kappaB) in glomerular epithelial cells (GECs) against high-glucose injury. Treatment with high glucose (30 mM) increased reactive oxygen species (ROS)/lipid peroxidation (LPO) and decreased glutathione (GSH) in GECs. Pretreatment with 100 microM EGCG attenuated the increase in ROS/LPO and restored the levels of GSH, whereas ROS, LPO, and GSH levels were not affected by treatment with 30 mM mannitol as an osmotic control. Interestingly, high-glucose treatment affected 3 separate signal transduction pathways in GECs. It increased the expression of TGF-beta1, PKC alpha/betaII, and NF-kappaB in GECs, respectively. EGCG (1, 10, 100 microM) pretreatment significantly decreased the expression of TGF-beta1 induced by high glucose in a dose-dependent manner. In addition, EGCG (100 microM) inhibited the phosphorylation of PKC alpha/betaII caused by glucose at 30 mM. Moreover, EGCG (1, 10, 100 microM) pretreatment significantly decreased the transcriptional activity of NF-kappaB induced by high glucose in a dose-dependent manner. These data suggest that EGCG could be a useful factor in modulating the injury to GECs caused by high glucose.
Subject(s)
Catechin , Epithelial Cells , Glucose , Glutathione , Mannitol , NF-kappa B , Phosphorylation , Protein Kinase C , Reactive Oxygen Species , Signal Transduction , Tea , Transforming Growth Factor beta1ABSTRACT
Cholangiocarcinoma (CC) is a chemoresistant intrahepatic bile duct carcinoma with a poor prognosis. The aims of this study were to identify molecular pathways that enhance sesquiterpene lactone parthenolide (PTL)-induced anticancer effects on CC cells. The effects of PTL on apoptosis and hemoxygenase-1 (HO-1) induction were examined in CC cell lines. The enhancement of PTL-mediated apoptosis by modulation of HO-1 expression and the mechanisms involved were also examined in an in vitro cell system. Low PTL concentrations (5 to 10 micrometer) led to Nrf2-dependent HO-1 induction, which attenuated the apoptogenic effect of PTL in Choi-CK and SCK cells. PTL-mediated apoptosis was enhanced by the protein kinase C-alpha inhibitor Ro317549 (Ro) through inhibition of expression and nuclear translocation of Nrf2, resulting in blockage of HO-1 expression. Finally, HO-1 silencing resulted in enhancement of apoptotic cell death in CC cells. The combination of PTL and Ro efficiently improved tumor growth inhibition compared to treatment with either agent alone in an in vivo subcutaneous tumor model. In conclusion, the modulation of HO-1 expression substantially improved the anticancer effect of PTL. The combination of PTL and Ro could prove to be a valuable chemotherapeutic strategy for CC.
Subject(s)
Humans , Active Transport, Cell Nucleus/drug effects , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Line, Tumor , Cell Nucleus/metabolism , Cholangiocarcinoma/drug therapy , Drug Resistance, Neoplasm/drug effects , Enzyme Activation , Enzyme Inhibitors/pharmacology , Heme Oxygenase-1/genetics , Lactones/chemistry , NF-E2-Related Factor 2/genetics , Protein Kinase C-alpha/antagonists & inhibitors , RNA, Small Interfering/genetics , Sesquiterpenes/chemistry , Signal Transduction/drug effectsABSTRACT
Objective To investigate the effect of syndecan-4 on the proliferation and extracellular matrix (ECM) secretion of human mesangial cells(HMC) stimulated by basic fibroblast growth factor (bFGF) and to evaluate the role of syndecan-4-PKCα pathway. Methods The expression of syndecan-4 in HMC was observed by immunofluorescence. After the down-regulation of syndecan-4 in HMC by RNA interference, the cell proliferation was detected by MTT. The secretion of fibronectin (FIN), type IV collagen, type Ⅰ collagen was assessed by ELISA. The copy number of syndecan-4 and PKCα was measured by fluorescent quantitation PCR at different time points. Results Syndecan-4 was expressed in HMC. bFGF could promote the cell proliferation and ECM secretion together with the PKCα copy number per million house-keeping genes of HMC, which could be reversed by the syndecan-4 siRNA transfection (MtT: 48-60 h, P<0.01; FiN: 24 h, P<0.01, 48-96 h, P<0.05; type Ⅳ collagen: 72-96 h, P<0.05; PKCa: 0 h, P<0.05, 12-48 h, P< 0.01). Conclusion Syndecan-4 may regulate the proliferation and ECM secretion of HMC stimulated by bFGF through syndecan-4-PKCα pathway.
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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.
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Expression of matrix metalloproteinase-9 (MMP-9) is associated with airway remodeling and tissue injury in asthma. However, little is known about how MMP-9 is up-regulated in airway epithelial cells. In this study, we show that phorbol myristate acetate (PMA) induces MMP-9 expression via a protein kinase Calpha(PKCalpha)-dependent signaling cascade in BEAS-2B human lung epithelial cells. Pretreatment with either GF109203X, a general PKC inhibitor, or Go6976, a PKCalpha/beta isozyme inhibitor, inhibited PMA-induced activation of the MMP-9 promoter, as did transient transfection with PKCalpha antisense oligonuclotides. PMA activated NF-kappaB by phosphorylating IkappaB in these cells and this was also inhibited by GF109203X and Go6976, suggesting that PKCalpha acts as an upstream regulator of NF-kappaB in PMA-induced MMP-9 induction. Our results indicate that a "PKCalpha-NF-kappaB"-dependent cascade is involved in the signaling leading to PMA-induced MMP-9 expression in the lung epithelium.
Subject(s)
Humans , Up-Regulation/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Protein Kinase C-alpha/metabolism , NF-kappa B/metabolism , Matrix Metalloproteinase 9/metabolism , Lung/drug effects , Epithelial Cells/drug effects , Cell LineABSTRACT
Objective: To study the clinical significance of expression of the protein kinase C-α PKC-α) in non-small cell lung cancer (NSCLC) tissues and four types of NSCLC cell lines. Methods: PKC-α protein expression was examined by immunohistochemical SP method using tissue microarray technology in 160 cases with tumor tissue including 91 cases at stage I, and 69 cases at stage II-III and 40 cases of adjacent tissues. The mRNA and protein expression of PKC-α in NSCLC cell lines (A 549, A 549-DDP, H460 and H1299) were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Results: Expression of PKC-α protein was positive in 53.1% (85/ 160) of cancerous tissues and 7.5% (3/40) of adjacent tissues (P < 0.01). But the expression of PKC-α had no significant difference between different types of NSCLC (adenocarcinoma, squamous carcinoma, and bronchioalveolar carcinoma). The positive rate of PKC-α protein was not related with the gender and age of NSCLC patients, but related with TNM staging and lymph node metastasis of NSCLC especially adenocarcinoma (P < 0.05). The expression of PKC-α protein was related with the differentiation degree of adenocarcinoma (P <0. 05), but not with the differentiation degree of squamous carcinoma. PKC-α were expressed in all NSCLC cell lines (A 549, A 549-DDP, H 460, and H 1299) at mRNA and protein level. A 549-DDP cells had the highest expression level of PKC-α. Conclusions: There exists high expression of PKC-α protein in NSCLC tissues and cell lines. The expression of PKC-α protein may play a crucial role in invasion, metastasis and development of NSCLC especially in adenocarcinoma. There is significant difference in the mRNA and protein expression levels of PKC-α in different NSCLC cell lines.
ABSTRACT
Background and purpose:Protein kinase C (PKC) is a potentially important target for cancer therapy due to its potential role in carcinogenesis. Abnormal expression and increasing activity of PKC-?are present in non-small cell lung cancer (NSCLC).The aim of this study is to investigate PKC-? protein expressions in lung cancer tissues by tissue microarray and their significance. Methods:Using tissue microarray, PKC-? protein expression in lung cancer patients was examined by immunohistochemistry. 182 samples of umor tissues and 18 samples of adjacent tissues were taken;of the 182 samples, 160 were NSCLC tissues and 22 were SCLC;102 samples from stage I cases and 58 from stageⅡ-Ⅲ cases.Results:Expression of PKC-? protein was observed in 48.3% (88/182) of cancerous tissues and 16.7% (3/18) of adjacent tissues (P
ABSTRACT
Ceramides are the main lipids in the stratum corneum and are generated during cellular stress and apoptosis by de novo synthesis or by the action of sphingomyelinase. In addition, they are lipid second messengers produced by sphingolipid metabolism and trigger important cell responses, including protein kinase C-alpha (PKC-alpha) activation and the stimulation of signal transduction pathways with apoptosis and stress-activated protein kinases (SAPK), such as c-jun N-terminal kinase (JNK). Thus, ceramides have anti-proliferative and apoptotic effects. This study measured the changes in the levels of epidermal ceramides and ceramide-related apoptotic signaling molecules in psoriasis patients. Samples from lesional and non-lesional epidermis were obtained from psoriasis patients. Total ceramides were fractionated using thin-layer chromatography, and the levels of PKC-alpha and JNK expression were measured using Western blot analysis with specific antibodies. The ceramide level was reduced significantly, and this was associated with the downregulation of apoptotic signaling molecules, such as PKC-alpha and JNK, in the lesional epidermis of psoriasis patients. These results suggest that the decreased level of ceramides downregulates the apoptotic pathway, leading to epidermal proliferation in psoriasis.