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Immunotherapy has revolutionized the landscape of cancer treatment. However, single immunotherapy only works well in a small subset of patients. Combined immunotherapy with antitumor synergism holds considerable potential to boost the therapeutic outcome. Nevertheless, the synergistic, additive or antagonistic antitumor effects of combined immunotherapies have been rarely explored. Herein, we established a novel combined cancer treatment modality by synergizing p21-activated kinase 4 (PAK4) silencing with immunogenic phototherapy in engineered extracellular vesicles (EVs) that were fabricated by coating M1 macrophage-derived EVs on the surface of the nano-complex cores assembled with siRNA against PAK4 and a photoactivatable polyethyleneimine. The engineered EVs induced potent PAK4 silencing and robust immunogenic phototherapy, thus contributing to effective antitumor effects in vitro and in vivo. Moreover, the antitumor synergism of the combined treatment was quantitatively determined by the CompuSyn method. The combination index (CI) and isobologram results confirmed that there was an antitumor synergism for the combined treatment. Furthermore, the dose reduction index (DRI) showed favorable dose reduction, revealing lower toxicity and higher biocompatibility of the engineered EVs. Collectively, the study presents a synergistically potentiated cancer treatment modality by combining PAK4 silencing with immunogenic phototherapy in engineered EVs, which is promising for boosting the therapeutic outcome of cancer immunotherapy.
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Objective:To observe the effects of p21 activated kinase 4 (PAK4) on the mitochondrial function and biological behavior in retinal vascular endothelial cells.Methods:The experimental study was divided into two parts: in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 12 healthy C57BL/6J male mice were randomly divided into normal control group and diabetes group, with 6 mice in each group. Diabetes mice were induced by streptozotocin to establish diabetes model. Eight weeks after modeling, quantitative real-time polymerase chain reaction and Western blots were performed to detect the expression of PAK4 in diabetic retinas. In vitro cell experiments: the human retinal microvascular endothelial cells (hRMEC) were divided into three groups: conventional cultured cells group (N group), empty vector transfected (Vector group); pcDNA-PAK4 eukaryotic expression plasmid transfected group (PAK4 group). WB and qPCR were used to detect transfection efficiency, while scratching assay, cell scratch test was used to detect cell migration in hRMEC of each group. In vitro white blood cell adhesion experiment combined with 4 ', 6-diamino-2-phenylindole staining was used to detect the number of white blood cells adhering to hRMEC in each group. The Seahorse XFe96 cell energy metabolism analyzer measures intracellular mitochondrial basal respiration, adenosine triphosphate (ATP) production, maximum respiration, and reserve respiration capacity. The t-test was used for comparison between the two groups. Single factor analysis of variance was used for comparison among the three groups. Results:In vivo animal experiments: compared with normal control group, the relative expression levels of PAK4 mRNA and protein in retina of diabetic mice were significantly increased, with statistical significance ( t=25.372, 22.419, 25.372; P<0.05). In vitro cell experiment: compared with the N group and Vector group, the PAK4 protein, mRNA relative expression and cell mobility in the hRMEC of PAK4 group were significantly increased, with statistical significance ( F=36.821, 38.692, 29.421; P<0.05). Flow cytometry showed that the adhesion number of leukocytes on hRMEC in PAK4 group was significantly increased, and the difference was statistically significant ( F=39.649, P<0.01). Mitochondrial pressure measurement results showed that the capacity of mitochondrial basic respiration, ATP production, maximum respiration and reserve respiration in hRMEC in PAK4 group was significantly decreased, with statistical significance ( F=27.472, 22.315, 31.147, 27.472; P<0.05). Conclusion:Over-expression of PAK4 impairs mitochondrial function and significantly promotes leukocyte adhesion and migration in retinal vascular endothelial cells.
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p21-activated kinase 5 (PAK5), a kind of the PAK family of serine / threonine kinases, modulates various cellular processes, including cytoskeletal remodeling, cell proliferation, migration and metastasis. Although studies have suggested that PAK5 is a key regulator of breast cancer progression, the link of PAK5 to senescence has not been reported yet. In this study, the CRISPR/ Cas9 lentivirus infection method was used to construct two PAK5 stably knockdown cell lines of human breast cancer (MDA-MB-231 and BT474). The effect of PAK5 knockdown on the proliferation of breast cancer cells was detected by CCK-8 and clone formation assays. The effect of PAK5 knockdown on cell cycle was detected by flow cytometry. The effect of PAK5 knockdown on cell senescence was observed by β-galactosidase staining. Western blotting was performed to detect the expression of senescence-related proteins including p53, p21 and p16 in breast cancer cells. Cells were treated by CHX and MG132 to explore the possible mechanism of PAK5 regulating p53 protein expression. The results showed that knockdown of PAK5 inhibited cell proliferation and blocked cells in the G
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The p21-activated kinase 1 (PAK1) is a member of the P21-activated protein kinase family that plays an important role in the proliferation and on cogenesis of pancreatic cancer. PAK1 is an important target for the treatment of pancreatic cancer. At present, akinase inhibitor targeting PAK1 is still in the preclinical research stage. Therefore, screening for new PAK1 kinase inhibitors is of great significance. In this study the natural compound celastrol was found to have a significant inhibitory effect on PAK1, with an IC50 value of 3.614 μmol·L-1. Molecular docking results showed that celastrol had good binding to PAK1. An MTT assay indicated that celastrol inhibited the proliferation of pancreatic cancer cells BxPC-3 and PANC-1. Mechanistic studies revealed that the inhibition of pancreatic cancer cells by celastrol was reversed by PAK1 siRNA. Celastrol inhibited PAK1 and the subsequent activation of downstream signaling pathways, thereby activating apoptosis signaling pathways and triggering apoptosis in pancreatic cancer cells. These findings suggested that celastrol induced apoptosis in pancreatic cancer cells by suppressing the PAK1 kinase signaling pathway and has potential value for the treatment of pancreatic cancer.
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Objective@#To investigate the expressions of migration and invasion inhibitory protein (MIIP) and p21-activated kinase 1 (PAK1) in endometrial carcinoma (EC) and their correlation with clinicopathological features.@*Methods@#The protein levels of MIIP and PAK1 in 135 paraffin-embedded EC tissues, 55 atypical hyperplasia of endometrium (AHE) and 88 normal endometrium (NE) tissues were quantified by immunohistochemistry, the clincial significance and the relationship of these two proteins were also analyzed.@*Results@#The positive rates of MIIP expression in NE, AHE and EC tissues were 52.3%(46/88), 41.8% (23/55) and 34.8% (47/135), respectively. The expression of MIIP in EC was significantly lower than that of MIIP in NE (P<0.05). The positive rates of PAK1 expression in NE, AHE and EC tissues were 45.5% (40/88), 50.9% (28/55) and 62.2% (84/135), respectively. The expression of PAK1 in EC tissues was significantly higher than that of PAK1 in NE tissues (P<0.05). The expression of MIIP in EC tissues was significantly associated with myometrial invasion, International Federation of Gynaecology and Obstetrics (FIGO) stage and lymph node metastasis (P<0.05). The expression of PAK1 in EC tissues was significantly related with differentiation, myometrial invasion, FIGO stage and lymph node metastasis (P<0.05). The expressions of MIIP and PAK1 in EC tissues were marginally related with the overall survival of patients (P=0.092, P=0.052). The expression of MIIP in EC was negatively correlated with PAK1 (r=-0.329, P<0.001).@*Conclusions@#The down-regulation of MIIP and up-regualtion of PAK1 paticipate in the initiation and development of EC, which are correlated with the poor prognosis of EC. The protein expression of MIIP is inversely related with PAK1 in EC.
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AIM:To investigate the expression and clinical significance of PAK4 in the cell lines and tissues of non-small cell lung cancer (NSCLC).METHODS:PAK4 expression in human bronchial epithelial (HBE) cells, NSCLC cell lines, NSCLC tissues and adjacent non-tumor tissues were assessed by immunohistochemistry, real-time PCR and Western blot.Prognostic value of PAK4 expression was evaluated by Kaplan-Meier analysis and Cox regression.RE-SULTS:PAK4 was over-expressed in the NSCLC cell lines at both mRNA and protein levels compared with HBE cells ( P<0.05).PAK4 was over-expressed in the NSCLC tissues at both mRNA and protein levels compared with adjacent non-tumor tissues (P<0.05).PAK4 was over-expressed in the metastatic NSCLC tissues compared with the primary NSCLC tissues (P<0.05).Higher PAK4 staining scores were positively correlated with differentiation, lymph node metastasis, distant metastasis, and clinical stage.Kaplan-Meier analysis and log-rank test showed that overall survival was significantly different between the patients with up-regulated PAK4 and the patients with down-regulated PAK4 ( P<0.05) .PAK4 over-expression was associated with NSCLC progression.CONCLUSION:Increased PAK4 expression was associated with tumor invasion, metastasis and prognosis in the patients with NSCLC.PAK4 is an important prognostic marker and potential ther-apeutic target in NSCLC.
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AIM:To explore the mechanism of p21-activated kinase 4 (PAK4) on non-small-cell lung cancer (NSCLC) migration and invasion.METHODS:After A549 and NCI-H520 cell lines were transfected with PAK4-siRNA or negative control , the expression of PAK4 at mRNA and protein levels was detected by real-time PCR and Western blot , re-spectively .The invasion and migration of A 549 cells and NCI-H520 cells were measured by Matrigel invasion assay and Transwell migration assay.LIMK1, cofilin, and their respective phosphorylation were examined by Western blot .The inter-action of PAK4 and LIMK1 was investigated by co-immunoprecipitation assay .The relationship between PAK4 and LIMK1 phosphorylation was examined by a protein kinase assay in the A 549 cells and NCI-H520 cells.The expression of PAK4 and p-LIMK1 in 10 human NSCLC tissues was examined by Western blot .A549 cells and NCI-H520 cells were individually or commonly transfected with PAK4-siRNA or LIMK1 plasmid in order to observe the cell migration and invasion .RE-SULTS:After A549 cells and NCI-H520 cells were transfected with PAK4-siRNA for 48 h, the expression of PAK4 at mR-NA and protein levels , and the numbers of invasion and migration cells in PAK 4-siRNA group were lower than those in con-trol group.Compared with control group , the phosphorylation of LIMK1 and cofilin was lower in PAK4-siRNA group, whereas the total expression levels of LIMK 1 and cofilin did not change .The results of co-immunoprecipitation assays showed that PAK4 specifically interacted with LIMK1 in A549 and NCI-H520 cells.LIMK1 phosphorylation in the presence of PAK4 (K350M) was significantly lower than that in the presence of PAK 4 (WT) or PAK4 (S445N) in the protein ki-nase assay.The PAK4 upregulation was positively correlated with the level of p-LIMK1 (P<0.05).After A549 cells and NCI-H520 cells were co-transfected with PAK4-siRNA and LIMK1 plasmid, the migration and invasion cell numbers in co-transfection group were higher than those in PAK 4-siRNA transfection group .CONCLUSION: PAK4 promotes the inva-sive and migratory abilities of NSCLC , which is mediated by LIMK 1 phosphorylation .
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Objective To investigate the effect of p21-activated kinase 1 on chemotherapy sensitivity of 5-fluorouracil. Methods Cell proliferation was measured by CCK8 and apoptosis rate by flow cytometry or Hoechst staining; the expression of Bcl-xl, Bcl-2, XIAP were determined by Western Blot. Results 5-FU combined shRNA-Pak1 group (combination group) could be significantly inhibited in terms of proliferation (P <0.05). The percentage of apoptosis rate in combined group was the highest and the difference among groups indicated statistical significance (P < 0.05). The expression of Bcl-xl, Bcl-2, XIAP in combination group was significantly inhibited compared with 5-FU group or shRNA-Pak1 group. Conclusion PAK1 inhibited by RNA interference can enhance chemotherapy sensitivity of 5-Fu on growth inhibition and apoptosis induction in colon cancer significantly.
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CardiovascuIar disease is the number one cause for morbidity and mortaIity worIdwide. Possi-biIities for new therapies in the emerging fieId of cardiac signaIIing prompted extensive research on myocardiaI re-modeIIing over the past decades. In this review,we as-sembIe an overview of the recent findings on the muIti-functionaI enzyme,p21-activated kinase 1( Pak1),a member of a serine/ threonine protein kinase famiIy in the heart,particuIarIy its cardiac protective effects. We pres-ent a modeI for Pak1 signaIing that provides a mecha-nism for specificaIIy affecting cardiac ceIIuIar processes. We discuss its cardiac protective effects such as anti-hy-pertrophy,anti-ischaemic injury and roIe in maintaining ventricuIar Ca2+ homeostasis and eIectrophysioIogicaI stabiIity under physioIogicaI, β-adrenergic and hyper-trophic stress conditions.We aIso discuss the potentiaIs of Pak1 activation by naturaIIy occurring sphingosine and its anaIogues FTY720,and bioactive peptides designed to diminish Pak1 auto-inhibition as noveI thera-peutics for major cardiovascuIar diseases.
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Objective To investigate the effect of ginsenosides-Rb1(Gs-Rb1) on doxorubicin (Dox)-induced heart failure (HF), and the related mechanisms of connexin 43 (CX43) thereof. Methods Rats with Dox-induced HF were ran-domly divided into Dox group (n=15) and Gs-Rb1 group (n=17), and the health age-matched rats were as control (n=15). In addition, cardiomyocytes were randomly divided into Dox group, Gs-Rb1 group and control group. After the intervention, echocardiography or apoptotic ratio (AR) was analyzed, respectively. The expression levels of p21-activated kinase 1 (PAK1), protein phosphatase type 2A (PP2A) and CX43 were detected by Western bolt or RT-PCR analysis, respectively. Re-sults Gs-Rb1 significantly improved heart function in rats with HF, decreased left ventricular mass index and inhibited the cell apoptosis induced by Dox. Both mRNA and protein expressions of CX43 were significantly decreased in Dox group than those of control group. The expression of CX43 was significantly increased in Gs-Rb1 group, which was significantly lower than that of control group. There was no significant difference in PAK1 between Dox group and control group (P>0.05). The expression of PAK1 was significantly up-regulated by Gs-Rb1. The PP2A protein was significantly up-regulated in Dox group than that of control group, which was significantly higher in Gs-Rb1 group than that of Dox group. Conclusion Gs-Rb1 improved HF by adjusting CX43, which may be mediated by PAK1-PP2A.
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p21 activated kinase 4 (PAK4) is a member of a family of serine/threonine kinase.PAK4plays an important role in a variety of cellular functions including cell cycle,cell proliferation,apoptosis,and cytoskeletal reorganization.Recently,PAK4 has been shown to overexpress in a variety of malignancies,and contribute to cancer cell migration and invasion through multiple signalling pathways.
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Objective To investigate the role of PAK1 in the development of pancreatic cancer.Methods 33 cases of pancreatic cancer in Renmin Hospital of XianTao were selected in this study. 33cases of pericancerous normal tissues were collected as control group.Immunohistochemical staining were performed to detect the expression of PAK1 in 33 cases of pancreatic cancer and pericancerous normal specimens.Results By Immunohistochemical staining PAK1 were mainly expressed in the cytoplasm. The positive rate of PAK1 expression in pancreatic cancer were 69.7 ％(23 / 33),which was significantly higher than that of control group[27.3 ％(9 / 33),x2=11.89,P =0.001].In addition,overexpression of PAK1 was more frequently in Ⅲ+Ⅳ stage pancreatic cancer tissue[93.3 ％(14/15)],compared with Ⅰ + Ⅱ stage [50.0 ％( 9 / 18 ),x2=5.367,P=0.021]. Conclusion The PAK1 protein may be associated with the occurrence and development of pancreatic cancer.
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It has been known that the Alzheimer's disease(AD)is related closely with a synaptic failure,and the p21-activated kinase(PAK)is well documented to play an important role in the regulation of the synaptie functions.However,the relationship between thePAK and the pathology of AD is unclear.In the present study,we examined the expressions of the PAK3(one subtype ofPAK),phospho-rylated-PAK(pPAK) and β-amyloid42(Aβ42,β-amyloid with 42 peptides)in an APP/PS1 double transgenie mouse model of AD andthe morphologies of geurOtlS in the hippocampus at different ages.The Western Blot results showed that the expression of PAK remainedunchanged,while,the expression of pPAK decreased largely at the age of 32 weeks and further decreased significantly with aging in thehippocampus of the APP/PS1 transgenic mouse.A1342 levels in the hippocampus were detected to increase as early as the age of 22 weeks,and kept the increase to continue with aging.The morphological results showed no obvious neuron loss in the sections of Nissl staining,while serious distonion and disorder of the dendrites of the hippocampal neurons were observed on the sections of Gelgi staining in theAPP/PS1 transgenic mouse.The present results suggested that it seemed something wrong in the processes of phospholization of PAK,butnot in the expression of the PAK itself;the toxic Aβ42 might affect the PAK in its phospholization,which in turn directly influence thedendritic development in the hippocampal neurons and cause the dendrites distorting and disordering.
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Objective To investigate the role of p21-activated kinase 1 (PAK1) in colorectal mucosal carcinogenesis and the relationship between PAK1 expression and biological behavior of colorectal carcinoma. Method PAK1 was detected with immunohistochemical method in 10 normal colorectal mucosas,40 colorectal villous or tubular adenomas and 60 colorectal carcinomas. Results The positive rate for PAK1 was 10.0% in normal colorectal mucesas,and 25.0%, 33.3% and 33.3% in slight, moderate and severe dys-plastic adenomas, respectively, 65.0% was found in colorectul carcinomas. The positive rate for PAK1 in col-orectal carcinomas was higher than that in normal colorectal mucosas(P<0.01), colorectal villous or tubular adenomas(P<0.01), slight dysplastic adenomas(P<0.01) and moderate dysplastic adenomas (P<0.05).The positive rate for PAK1 of poor differentiated colorectal carcinomas was higher than that of high differentiated ones (P<0.05), and the positive rate for PAK1 of patients with lymph node metastasis was higher than that of patients without lymph node metastasis (P<0.05). In clinical stages, the positive rate for PAK1 in Dukes C and D stages patients was higher than that in Dukes A stage patients, respectively (P<0.05). Conclusion PAK1 maybe play some role in the process of carcinogenesis of colorectal mucesa, and be used as an useful marker for assessment of the biological behavior and prognosis of colorectal carcinoma.
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p21-Activated kinases including p21-activated kinase 2 contributed to the regulation of actin cytoskeleton and cell dynamics. In order to investigate the function of PAK2 on the maturation of Xenopus oocyte, PAK2-NT(PAK2-N-terminal,PAK2-NT) and PAK2-NTm (PAK2-N-terminal mutation) mRNA were microinjected into Xenopus oocyte respectively. Under fluorescent microscopy germinal vesicle breakdown was observed during cytokinesis. To further observe the relationship of oocyte cytokinesis, polar body formation and Cdc42 activity, confocal microscopy with time-lapse was employed . As a result, occurrences of germinal vesicle breakdown in oocytes were similar to those oocytes injected with PAK2-NT mRNA or injected with PAK2-NTm mRNA,but no cytokinesis and polar body formation were observed in oocytes injected with PAK2-NT mRNA or PAK2-NTm mRNA. These results indicated that PAK2 involved in Xenopus oocytes cytokinesis and polar body formation independent of Cdc42 activity.
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Objective Study the function of p21-activated kinase 2 in Xenopus oocyte cytokinesis. Methods Xenopus oocyte was used as a model,and MPF activity and PAK2 phosphorylation status were observed during oocyte maturation.The specific inhibitor of PAK2 activity,PAK2-NT(PAK2-N-terminal) was used to microinject into Xenopus oocyte.Under fluorescent microscope germinal vesicle breakdown was observed during cytokinesis.To further observe the changes of F-actin and spindle,laser scanning confocal microscopy with time-lapse method was employed. Results Occurrences of germinal vesicle breakdown in control oocytes was similar to those oocytes injected with PAK2-NT mRNA,but no cytokinesis was observed in oocytes injected with PAK2-NT mRNA.Conclusion These results indicated that PAK2 might involved in Xenopus oocytes cytokinesis.