Focal adhesion kinase-related non-kinase ameliorates liver fibrosis by inhibiting aerobic glycolysis via the FAK/Ras/c-myc/ENO1 pathway

BACKGROUND Hepatic stellate cell(HSC)hyperactivation is a central link in liver fibrosis development.HSCs perform aerobic glycolysis to provide energy for their activation.Focal adhesion kinase(FAK)promotes aerobic glycolysis in cancer cells or fibroblasts,while FAK-related non-kinase(FRNK)inhibits FAK phosphorylation and biological functions.AIM To elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.METHODS Mouse liver fibrosis models were established by administering CCl4,and the effect of FRNK on the degree of liver fibrosis in the model was evaluated.Transforming growth factor-β1 was used to activate LX-2 cells.Tyrosine phosphorylation at position 397(pY397-FAK)was detected to identify activated FAK,and the expression of the glycolysis-related proteins monocarboxylate transporter 1(MCT-1)and enolase1(ENO1)was assessed.Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region,which were validated with chromatin immunoprecipitation(ChIP)and dual luciferase reporter assays.RESULTS The pY397-FAK level was increased in human fibrotic liver tissue.FRNK knockout promoted liver fibrosis in mouse models.It also increased the activation,migration,proliferation and aerobic glycolysis of primary hepatic stellate cells(pHSCs)but inhibited pHSC apoptosis.Nevertheless,opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells.Mechanistically,the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis,which was inhibited by exogenous FRNK.CONCLUSION FRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway,thereby improving liver fibrosis.FRNK might be a potential target for liver fibrosis treatment.

Inhibition of focal adhesion kinase enhances antitumor response of radiation therapy in pancreatic cancer through CD8+ T cells

Objective:Pancreatic ductal adenocarcinoma(PDAC)is a deadly malignancy,due in large part to its resistance to conventional therapies,including radiotherapy(RT).Despite RT exerting a modest antitumor response,it has also been shown to promote an immunosuppressive tumor microenvironment.Previous studies demonstrated that focal adhesion kinase inhibitors(FAKi)in clinical development inhibit the infiltration of suppressive myeloid cells and T regulatory(T regs)cells,and subsequently enhance effector T cell infiltration.FAK inhibitors in clinical development have not been investigated in combination with RT in preclinical murine models or clinical studies.Thus,we investigated the impact of FAK inhibition on RT,its potential as an RT sensitizer and immunomodulator in a murine model of PDAC.Methods:We used a syngeneic orthotopic murine model to study the effect of FAKi on hypofractionated RT.Results:In this study we showed that IN10018,a small molecular FAKi,enhanced antitumor response to RT.Antitumor activity of the combination of FAKi and RT is T cell dependent.FAKi in combination with RT enhanced CD8+T cell infiltration significantly in comparison to the radiation or FAKi treatment alone(P<0.05).FAKi in combination with radiation inhibited the infiltration of granulocytes but enhanced the infiltration of macrophages and T regs in comparison with the radiation or FAKi treatment alone(P<0.01).Conclusions:These results support the clinical development of FAKi as a radiosensitizer for PDAC and combining FAKi with RT to prime the tumor microenvironment of PDAC for immunotherapy.

Focal adhesion kinase signaling is necessary for the hydrogen sulfide-enhanced proliferation,migration,and invasion of HTR8/SVneo human trophoblasts

Objective:Hydrogen sulfide(H_(2)S)has been elucidated that it promotes migration and invasion in human placenta trophoblasts.However,the signaling pathway underlying H_(2)S-based regulation of trophoblasts remains unknown.Hence,we investigated the potential effect of sodium hydrosulfide(NaHS),an exogenous H_(2)S donor,on extravillous trophoblasts.Methods:The Cell Counting Kit-8 was used to detect the proliferative activity of trophoblasts and to screen the optimal concentration of NaHS.The migration and invasion of HTR8/SVneo cells were measured by Transwell assays.Gene expression was determined by quantitative real-time PCR analysis.Protein expression was determined by western blot.Results:We found that NaHS could promote the proliferation,migration,and invasion of HTR8/SVneo cells.The phosphorylation of focal adhesion kinase(FAK),Src,and extracellular signal-regulated kinase(ERK)were activated by NaHS.Moreover,NaHS also upregulated the expression of matrix metalloproteinase-2(MMP-2)and MMP-9,downregulated the expression of E-cadherin in HTR8/SVneo cells.The application of NaHS could increase the expression of cystathionine-β-synthase.Conclusion:Both FAK-Src signaling and the upstream signaling cascade of ERK activation play a significant important role in NaHS-induced proliferation,migration,and invasion via upregulating activity of MMP-2,MMP-9,and downregulating E-cadherin in HTR8/SVneo cells.These novel findings may provide a strong foundation for the clinical application of H_(2)S donor drugs.

MicroRNA-7 regulates glioblastoma cell invasion via targeting focal adhesion kinase expression

Background Invasion growth is the most characteristic biological phenotype of glioblastoma, but the molecular mechanism in glioma cell invasion is poorly understood. Recent data have showed that microRNA plays an essential role in tumor invasion. Our study aimed to explore the mechanism of miR-7 involved in the control of glioblastoma cell invasion. Methods Glioma cell invasion was evaluated by transwell and scratch assays after up-regulation of miR-7 using miR-7 mimics in U87 and U251 cells. Luciferase reporter assay was used to determine focal adhesion kinase （FAK） as a target of miR-7. The levels of miR-7, matrix metalloproteinases （MMP）-2 and MMP-9 mRNA were detected by PCR assay, and the levels of FAK, MMP-2, MMP-9, total and phosphorylation serine/threonine kinase （AKT）, and extracellular signal-regulated kinase （ERK） 1/2 were measured by Western blotting analysis. Results Over-expression of miR-7 inhibited the invasion and migration activity of U87 and U251 cells. And up-regulation of miR-7 reduced FAK protein expression, Further, luciferase reporter assay showed that miR-7 modulated FAK expression directly by binding 3＇UTR of FAK mRNA. In addition, miR-7 repressed p-ERK1/2 and p-AKT level, MMP-2 and MMP-9 expression. Finally, the inverse relationship between FAK and miR-7 expression was certificated in human glioma tissues. Conclusion To our knowledge, these data indicate for the first time that miR-7 directly regulates cell invasion by targeting FAK in glioblastoma and that miR-7 could be a potential therapeutic target for glioblastoma intervention.

Expression, distribution and function of the focal adhesion kinase (pp125^(FAK)) during murine ectoplacental cone outgrowth in vitro

Mouse embryo implantation is a complex process that includes trophoblast cells derived from ectoplacental cone (EPC) adhesion to and migration through the extracellular matrix (ECM) of uterine endometrium and invasion into the decidua. At the time of implantation, fibronectin (FN) is abundant in the decidua and is distributed pericellularly around each individual stromal cell, and its receptor (integrin α-5β-1) expression on trophoblast populations is up-regulated. The focal adhesion kinase, a 125 ku protein tyrosine kinase (pp125 FAK), is tyrosine phosphorylated upon integrin engagement with its ECM ligand, and its tyrosine phosphorylation sites then serve as the binding sites which couple it with cellular proteins that contain Src SH2 or SH3 domains. Through these linkages, pp125 FAK may integrate multiple signals triggered by integrins. The model of EPC culture %in vitro% was used to study the expression, distribution and function of pp125 FAK during EPC outgrowth on FN. Results indicated that, pp125 FAK primarily expressed and distributed in cellular focal adhesions of the front edge of trophoblast outgrowth from EPC, and was localized in the peripheral region of the individual migrating trophblast cell; antibody or antisense oligodeoxynucleotide to pp125 FAK inhibited EPC attachment and outgrowth, as well as trophoblast cells spreading and migration. This experiment demonstrated that pp125 FAK as an integrin-mediated signaling molecule was involved in EPC outgrowth %in vitro%, and played an important role during trophoblast cells interaction with FN.

Focal adhesion kinase antisense oligodeoxynucleotides inhibit human pulmonary artery smooth muscle cells proliferation and promote human pulmonary artery smooth muscle cells apoptosis

Background Pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling At present, the mechanisms related to proliferation of PASMCs are not clear Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs) We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein Methods Cultured HPASMCs stimulated by fibronectin (40 μg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense FAK respectively Expression of FAK, Jun NH2 terminal kinase (JNK), cyclin dependent kinase 2 (CDK 2) and caspase 3 proteins were detected by immunoprecipitation and Western blots Cell cycle and cell apoptosis were analysed by flow cytometry In addition, cytoplasmic FAK expression was detected by immunocytochemical staining Results When compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense FAK ODNs group and increased in sense FAK ODNs group significantly Caspase 3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs When compared with mismatch sense ODNs group, the proportion of cells at G 1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly In contrast, compared with mismatch sense ODNs group, the proportion of cells at G 1 phase was increased significantly in antisense FAK ODNs group The level of cell apoptosis in antisense FAK group was higher than in the mismatch sense group and the latter was higher than sense FAK group In addition, the sense FAK ODNs group was strongly stained by immunocytochemistry, whereas the antisense FAK ODNs group was weakly stained Conclusions The results suggest that FAK relates to the proliferation of HPASMCs Antisense FAK ODNs inhibit HPASMCs proliferation and facilitate their apoptosis It is possible that FAK via JNK, CDK 2 signalling pathways enhances HPASMCs proliferation and via caspase 3 inhibits HPASMCs apoptosis

Regulation of mouse blastocyst adhesion,outgrowth and matrix metalloproteinase-2 by focal adhesion kinase

The interaction of extracellular matrix-integrin markedly influences the adhesion, outgrowth, differentiation and expression of serine proteinases by the blastocyst, so it is regarded as a vital factor in blastocyst implantation. Al- though the mechanism of extracellular interactions between extracellular matrix and integrins has been well elucidated, the roles of the signaling molecules in the extracellular ma- trix-integrin signal transduction pathway in blastocyst im- plantation are unknown. This limits the understanding of blastocyst implantation and ECM-integrin signal transduc- tion pathway. In the present study, in vitro blastocyst culture and indirect immunocytochemistry, matrix metallopro- teinases (MMPs) zymography and antisense oligodeoxynu- cleotide (ODN) were used to investigate the expression of a fundamental molecule of integrin-dependent signal trans- duction pathways, focal adhesion kinase (FAK), in mouse blastocysts and its influence on mouse blastocyst adhesion, outgrowth and MMP-2. The results showed that mouse blas- tocysts expressed FAK. FAK protein was clustered in the peripheral migrating trophoblast cells and dispersed in the central area of blastocyst outgrowth. Fibronectin triggered pro-MMP-2 and 64 kD MMP-2 activities. The antisense ODN to FAK attenuated pro-MMP-2 and 64 kD MMP-2 activities which decreased abruptly and tended to disappear with increasing concentrations of the antisense ODN. Both mouse blastocyst adhesion and outgrowth on fibronectin were also influenced by the antisense ODN. Up to 20 mg/mL of the antisense ODN concentration, the adhesion and out- growth rates were decreased in a dose-dependent manner. The results indicated that FAK influenced mouse blastocyst adhesion, outgrowth and MMP-2 activity by intracellular signal transduction. In other words, FAK regulates mouse implantation in terms of blastocyst adhesive and invasive abilities.

Integrins and focal adhesion kinase in the malignant behavior of gliomas

Glioblastoma multiforme(GBM)is the most common type of glioma and is associated with a very poor prognosis.The standard treatment includes radiotherapy concurrent with temozolomide,however recently the Food and Drug Administration approved bevacizumab for use in patients with progressive glioblastoma following prior therapy.The limited number of treatment options points to the need for novel effective therapeutic approaches.A promising approach is the use of tyrosine kinase inhibitors(TKIs)in GBM treatment.However,the results from the majority of clinical trials using TKIs are not very encouraging.One growing area is the development of tumor-homing peptides that resemble the integrin recognition sequence RGD.In this article,the role of integrins and focal adhesion kinase in malignant glioma is reviewed,and an experimental study is proposed that will apply a strategy for peptide-mediated delivery of compounds deep into tumor parenchyma using tumor-homing peptides.

GPER1 promotes estrogen receptor negative breast cancer cell migration and invasion via non-genomic activation of c-Src/NF-κB/focal adhesion kinase cascade

Breast cancer metastasis is the root cause of deaths from breast cancer.Currently,endocrine therapy resistance in estrogen receptor(ER)-positive(ER^(+))breast cancer remains a major clinical issue.Moreover,ER-negative(ER^(-))breast cancer is often associated with distant recurrence and death.G-protein-coupled ER(GPER1)participates in endocrine therapy resistance and is involved in the malignant progression of breast cancer.However,the underlying detailed mechanisms remain obscure.Here we investigated the role and mechanism of GPER1 in the activation of focal adhesion kinase(FAK)using ER^(+)or ER
breast cancer cell lines.In SK-Br-3 cells(ERa^(-)/β/GPER1^(+)),both 17b-estradiol(E2)and the GPER1 agonist G1 resulted in rapid FAK phosphorylation.This action is due to GPER1 interaction with the non-receptor tyrosine kinase c-Src and subsequent activation of nuclear factor kappa B(NF-kB)signaling.Silencing of GPER1,c-Src or the nuclear factor kappa B p65 subunit blocked E2-or G1-induced SK-Br-3 cell migration and invasion.In MCF-7 cells(ERa^(+)/β(+)/GPER1^(+)),silencing of GPER1,but not ERa or ERb,abolished FAK phosphorylation induced by E2 or G1.In MDA-MB-231 cells(ERa^(-)/β^(+)/GPER1^(-)),E2 or G1 was also unable to stimulate E2-induced FAK phosphorylation.However,E2 and G1 regained the ability to induce FAK phosphorylation under conditions of overexpression of GPER1.In conclusion,we demonstrated that GPER1,but not ERa or ERb,mediates FAK phosphorylation induced by E2 via the c-Src/p65 signaling pathway,which enhances cell migration and invasion.These findings may shed light on novel therapeutic strategies based on GPER1/FAK signaling pathways in suppression of breast cancer metastasis.

Degradation of FAK-targeting by proteolytic targeting chimera technology to inhibit the metastasis of hepatocellular carcinoma

Liver cancer is a prevalent malignant cancer,ranking third in terms of mortality rate.Metastasis and recurrence primarily contribute to the high mortality rate of liver cancer.Hepatocellular carcinoma(HCC)has low expression of focal adhesion kinase(FAK),which increases the risk of metastasis and recurrence.Nevertheless,the efficacy of FAK phosphorylation inhibitors is currently limited.Thus,investigating the mechanisms by which FAK affects HCC metastasis to develop targeted therapies for FAK may present a novel strategy to inhibit HCC metastasis.This study examined the correlation between FAK expression and the prognosis of HCC.Additionally,we explored the impact of FAK degradation on HCC metastasis through wound healing experiments,transwell invasion experiments,and a xenograft tumor model.The expression of proteins related to epithelial-mesenchymal transition(EMT)was measured to elucidate the underlying mechanisms.The results showed that FAK PROTAC can degrade FAK,inhibit the migration and invasion of HCC cells in vitro,and notably decrease the lung metastasis of HCC in vivo.Increased expression of E-cadherin and decreased expression of vimentin indicated that EMT was inhibited.Consequently,degradation of FAK through FAK PROTAC effectively suppressed liver cancer metastasis,holding significant clinical implications for treating liver cancer and developing innovative anti-neoplastic drugs.

Specific shRNA targeting of FAK influenced collagen metabolism in rat hepatic stellate cells

AIM:To investigate the effects and mechanism of disruption of focal adhesion kinase(FAK) expression on collagen metabolism in rat hepatic stellate cells(HSC).METHODS:The plasmids expressing FAK short hairpin RNA(shRNA) were transfected into HSC-T6 cells,and the level of FAK expression was determined by both real-time quantitative polymerase chain reaction(QPCR) and Western blotting analysis.The production of type collagen and type collagen in FAK-disrupted cells was analyzed by real-time Q-PCR.The level of collagen metabolism proteins,including matrix metalloproteinases-13(MMP-13) and tissue inhibitors of metalloproteinases-1(TIMP-1) was also determined by both real-time Q-PCR and Western blotting analysis.RESULTS:The transfection of FAK shRNA plasmids into HSC resulted in disrupted FAK expression.Compared with the HK group,the levels of type collagen and type collagen mRNA transcripts in FAK shRNA plas-mid group were signif icantly decreased(0.69 ± 0.03 vs 1.96 ± 0.15,P = 0.000;0.59 ± 0.07 vs 1.62 ± 0.12,P = 0.020).The production of TIMP-1 in this cell type was also signif icantly reduced at both mRNA and protein levels(0.49 ± 0.02 vs 1.72 ± 0.10,P = 0.005;0.76 ± 0.08 vs 2.31 ± 0.24,P = 0.000).However,the expression of MMP-13 mRNA could be significantly up-regulated by the transfection of FAK shRNA plasmids into HSC(1.74 ± 0.20 vs 1.09 ± 0.09,P = 0.000).CONCLUSION:These data support the hypothesis that shRNA-mediated disruption of FAK expression could attenuate extracellular matrix(ECM) synthesis and promote ECM degradation,making FAK a potential target for novel anti-f ibrosis therapies.

α1 and β1 integrins enhance the homing and differentiation of cultured prostate cancer stem cells

CD133＋ prostate cancer stem cells （PCSCs） have recently been identified in human prostate cancer tissues. The present study reports the integrin profile of prostate cancer progenitor cells and the role of α1 and β1 integrins in the homing and differentiation of PCSCs in vitro. PCSCs were isolated from the tissue specimens of patients with prostate cancer and the expression of surface integrins and adhesion patterns were determined. Our analysis of the expression of surface integrins and their adhesion patterns of prostate cancer stem cells derived from prostate cancer tissues revealed that the levels of β1 and α2β1 integrins were significantly higher （P 〈 0.05） than those of the other integrins. By contrast, peripheral blood-derived CD 133＋ cells from prostate cancer patients showed a high level of expression （P 〈 0.01） of α2β1,αvβ3, αvβ5, β1 and α1 integrins and a minimal expression of α4β1 integrins. Moreover, CD 133＋ cells derived from both prostate cancer tissues and peripheral blood exhibited an increased degree of attachment to extraceUular matrix proteins （P 〈 0.001） and a high expression level of α2β1 integrin. In vitro experiments using blocking antibodies indicated that α1 and β1 integrins have a role in the homing and differentiation of PCSCs. This is the first report to suggest the importance of integrins in mediating the homing and differentiation of PCSCs.

Key Role of CD151-integrin Complex in Lung Cancer Metastasis and Mechanisms Involved

Tetraspanin CD151 was found to be upregulated in malignant cell types and has been identified as a tumor metastasis promoter.In this study,we aimed to examine the role of the CD151-integrin complex in lung cancer metastasis and the underlying mechanisms.CD151 QRD194–196→AAA194–196 mutant was generated and used to transfect A549 human lung adenocarcinoma cells.We found that there was no significant difference in CD151 protein expression between CD151 and CD151-AAA mutant groups.In vitro,CD151-AAA mutant delivery abrogated the migration and invasion of A549 cells,which was promoted by CD151 gene transfer.Furthermore,CD151-AAA delivery failed to activate FAK and p130Cas signaling pathways.Western blot and immunohistochemical staining showed strong CD151 expression in lung cancerous tissues but not in adjacent normal tissues.Increased level of CD151 protein was observed in 20 of the patients and the positive rate of CD151 protein in specimens was 62.5%(20/32).In addition,CD151 was co-localized withα3 integrin at the cell-cell contact site in carcinoma tissues.These results suggested that the disruption of the CD151-α3 integrin complex may impair the metastasis-promoting effects and signaling events induced by CD151 in lung cancer.Our findings identified a key role for CD151-α3 integrin complex as a promoter in the lung cancer.

The Expression of Integrinβ1 and FAK in Pituitary Adenomas and Their Correlation with Invasiveness

The expression and possible role of integrin-focal adhesion kinase signal pathway in invasive pituitary adenomas were explored. Forty-nine human pituitary adenomas were detected for the expression of integrinβ1 （INTβ1） and focal adhesion kinase （FAK） by immunohistochemistry, and their correlation with the invasiveness of pituitary adenomas as well as between themselves was ana- lyzed. The results showed that INTβ1 was expressed in 46 cases （93.9%） and FAK in 36 cases （73.5%）, respectively, and their expression levels were highly correlated with tumor invasiveness, but not with the tumor types. It was suggested that the integrin-focal adhesion kinase signal pathway plays a role in the invasiveness of pituitary adenomas.

Safflower Yellow Inhibits Progression of Hepatocellular Carcinoma by Modulating Immunological Tolerance via FAK Pathway

Objective To explore the anti-tumor effect of safflower yellow(SY)against hepatocellular carcinoma(HCC)and the underlying potential mechanism.Methods An in vitro model was established by mixing Luc-Hepa1-6 cells and CD3^(+)CD8^(+)T cells,followed by adding programmed cell death protein 1(PD-1)antibody(Anti-mPD-1)with or without SY.The apoptosis was detected by flow cytometry and the level of inflammatory cytokines was determined by enzyme-linked immunosorbent assay.The protein levels of programmed cell death 1 ligand 1(PD-L1),chemokine ligand(CCL5),C-X-C motif chemokine ligand 10(CXCL10)were measured by Western blot.An in situ animal model was established in mice followed by treatment with anti-mPD-1 with or without SY.Bioluminescence imaging was monitored with an AniView 100 imaging system.To establish the FAK-overexpressed Luc-Hepa1-6 cells,cells were transfected with adenovirus containing pcDNA3.1-FAK for 48 h.Results The fluorescence intensity,apoptotic rate,release of inflammatory cytokines,and CCL5/CXCL10 secretion were dramatically facilitated by anti-mPD-1(P<0.01),accompanied by an inactivation of PD-1/PD-L1 axis,which were extremely further enhanced by SY(P<0.05 or P<0.01).Increased fluorescence intensity,elevated percentage of CD3+CD8+T cells,facilitated release of inflammatory cytokines,inactivated PD-1/PD-L1 axis,and increased CCL5/CXCL10 secretion were observed in Anti-mPD-1 treated mice(P<0.01),which were markedly enhanced by SY(P<0.05 or P<0.01).Furthermore,the enhanced effects of SY on inhibiting tumor cell growth,facilitating apoptosis and inflammatory cytokine releasing,suppressing the PD-1/PD-L1 axis,and inducing the CCL5/CXCL10 secretion in Anti-mPD-1 treated mixture of Luc-Hepa1-6 cells and CD3+CD8+T cells were abolished by FAK overexpression(P<0.01).Conclusion SY inhibited the progression of HCC by mediating immunological tolerance through inhibiting FAK.

LOX upregulates FAK phosphorylation to promote metastasis in osteosarcoma

Osteosarcoma is a malignant bone tumor that commonly occurs in the pediatric population.Despite the use of chemotherapy and surgery,metastasis remains to be the leading cause of death in patients with osteosarcoma.We have previously reported that cytoplasmic mislocalization of p27 is associated with a poor outcome in osteosarcoma.In this study,we further show that lysyl oxidase(LOx)expression was associated with p27 mislocalization.Lox is an enigmatic molecule that acts as a tumor suppressor or a metastatic promoter;however,its role in osteosarcoma is still unclear.Hence,we performed both in vitro and in vivo analyses to dissect the role of Lox in osteosarcoma.The result of our survival analysis indicated that Lox expression significantly correlated with a poor outcome in osteosarcoma with or without controlling for the initial metastasis status(P<0.05).Functionally,we found that higher LoX expression promoted osteosarcoma cell proliferation,migration,and invasiveness in vitro and produced a higher number of mice with pulmonary metastases in an orthotopic xenograft mouse model.Mechanistically,phospho-FAK was increased in osteosarcoma cells with high LOX expression.Our results further showed that FAK inhibition significantly reduced tumor cell proliferation and migration in vitro as well as LoX-mediated metastasis in mice.Together,our findings suggest that there is a novel link between p27 mislocalization and LOX expression.LOX plays a pivotal role in osteosarcoma metastasis by upregulating FAK phosphorylation.FAK inhibition may constitute a promising therapeutic strategy to reduce the development of metastasis in osteosarcoma with Lox overexpression.

Inhibition of epithelial-mesenchymal transition in A549 cell by transfected Napsin A

Background Epithelial-mesenchymal transition is a cellular process characterized by the loss of cell adhesion, inhibition of E-cadherin expression, and increased cell mobility. Cells without Napsin A are susceptible to transition. Further studies are required to investigate whether this transition can be reversed by restoration of Napsin A. Methods A Napsin A expression vector PLJM1-Napsin A plasmid was constructed and then transfected into the epithelial cell line A549 by lentivirus transfection to obtain A549-PLJM1-Napsin A cell line. Cell proliferation was assayed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide and cell cycle was measured by flow cytometry. The E-cadherin, type I collagen, and focal adhesion kinase mRNA level was detected by reverse transcription-polymerase chain reaction. The Napsin A, E-cadherin, type I collagen, and focal adhesion kinase protein level in A549 cells was detected by Western blotting. Results Transforming growth factor-β1 induced epithelial-mesenchymal transition in A549 cells, as demonstrated by significant reduction of E-cadherin mRNA and protein levels （P 〈0.01） as well as up-regulation of type I collagen （P 〈0.01）. Transfection of Napsin A in A549 cells can partially block the transforming growth factor-β1-regulated expression of E-cadherin and type I collagen （P 〈0.01）. In addition, transforming growth factor-β1-induced cell proliferation was inhibited by Napsin A （P 〈0.01）. Further study demonstrated that Napsin A caused G0/G1 arrest and inhibited the expression of focal adhesion kinase （P 〈0.01）, a key protein in the integrin signaling pathway, in the in vitro epithelial-mesenchymal transition model. Conclusions Sustained Napsin A expression in A549 cells can inhibit the transforming growth factor-β1-induced epithelial-mesenchymal transition. This may be due to the Napsin A-mediated inhibition of focal adhesion kinase expression and integrin signaling pathway.

Emerging targets in cancer drug resistance

Drug resistance is a complex phenomenon that frequently develops as a failure to chemotherapy during cancer treatment.Malignant cells increasingly generate resistance to various chemotherapeutic drugs through distinct mechanisms and pathways.Understanding the molecular mechanisms involved in drug resistance remains an important area of research for identification of precise targets and drug discovery to improve therapeutic outcomes.This review highlights the role of some recent emerging targets and pathways which play critical role in driving drug resistance.

A novel molecular mechanism for nitrated α-synuclein-induced cell death

Although previous studies have demonstrated the involvement of nitrated a-synuclein in neurodegenerative disorders(synucleinopathies),the effects of nitrated α-synuclein and the molecular mechanisms underlying its toxicity are still unclear.In the present study,nitrated α-synuclein with four 3-nitrotyrosines(Tyr^(39),Tyr^(125),Tyr^(133),and Tyr^(136))was obtained non-enzymatically by incubation with nitrite.The nitrated protein existed as a mixture of monomers,dimers,and polymers in solution.The nitrated a-synuclein could induce cell death in a time-and concentration-dependent manner when SH-SY5Y cells(a human neuroblastoma cell line)were incubated with the dimers and polymers.Treatment with anti-integrin α5β1 antibody partially rescued the SH-SY5Y cells from the cell death.Dot blotting and immunoprecipitation revealed that the nitrated protein bound to integrin on the cell membranes.Level of nitric oxide(NO)and calcium-independent inducible NO synthase(iNOS)activity increased during the initial stages of the treatment.The expression of phosphorylated focal adhesion kinase(FAK)decreased in the cells.Subsequently,an increase in caspase 3 activity was observed in SH-SY5Y cells.Our results demonstrate that activation of iNOS and inhibition of FAK may both be responsible for the cell death induced by nitrated α-synuclein.These data suggest that the cytotoxicity of nitrated a-synuclein is mediated via an integrin-iNOS/-FAK signaling pathway,and that the nitration of α-synuclein plays a role in neuronal degeneration.

Effect of Ligustrazine on Bone Marrow Microenvironment and Signal Transducti on Path in Irradiation Injured Mice

Objective:To evaluate the effect of ligust razine on bone marrow hematopoietic microenvironment and signal transduction in irradiation injured mice.Methods:After being irradiated by 6.0 Gy 60 Co γ -ray, the mice in the ligustrazine group were orally given ligustrazine 4 mg/mouse twice a day to the end of the experiment. For the control group, normal saline was given instead of ligustrazine and the mice in the normal group was untreated. The mice were sacrificed separately on the 3rd, 7th and 14th day after radiation, bone marrow of them was taken and m anaged as follows:(1) Make stromal cell culture to observe the growth state and the expression of focal adhesion kinase (FAK) of the cells; (2) Make the bo ne marrow section to examine the pathological and immunohistochemistry changes, including the expression of fetal liver kinase-1 (Flk-1) and F VIII :RAg. The data were recorded and compared among groups.Results:After radiation, the hematopoietic cells in bo ne marrow decreased and the micro vessels dilated and congested with bleeding; bone mar row became thinner, red colored with cells of irregular shape and few cells adhere to the bottom of culture flask. These changes began to recover at the 7th day and approached to normal within 2 weeks. The recovery was better, earlier and quick er in the ligustrazine group than that in the control group. The expression lev els of Flk-1 decreased significantly.Conclusion: Ligustrazine could promote the recovery of hematopoietic function of radiation damaged bone marrow, the mechanisms might be through imp roving the microenvironment and signal transduction path for recovery.