Integrin-linked Kinase Protein Expression Research Articles

Plantamajoside alleviates hypoxia-reoxygenation injury through integrin-linked kinase/c-Src/Akt and the mitochondrial apoptosis signaling pathways in H9c2 myocardial cells

Myocardial ischemia–reperfusion injury(MIRI) is one of the common complications after myocardial infarction surgery, Oxidative stress is among the main mechanisms of myocardial ischemia–reperfusion injury. Plantamajoside (PMS), the main effective ingredient in the genus Plantain, has been reported to possess an antioxidation, anti-inflammatory and anti-apoptosis role. However, whether PMS can attenuate myocardial ischemia–reperfusion injury is not yet known. Herein, we explored the effects of PMS on hypoxia-reoxygenation (H/R) injury in H9c2 cardiomyocytes and the underling molecular mechanisms of the treatment. Network pharmacological analysis screened the top 31 key genes in the treatment of MIRI disease treated with PMS, and the result of molecular docking further illustrated the roles that the PMS play in the treatment of MIRI through its interference with integrin-linked kinase (ILK) target protein. PMS was not cytotoxic in the concentration range of 5–40 μM and increased cell survival after H/R injury in a concentration-dependent manner without affecting proliferation or growth. PMS significantly reduced the levels of lactate dehydrogenase, malonic dialdehyde, reactive oxygen species and cell apoptosis, and increased soperoxide dismutase activity compared with those of the H/R injury group. PMS promoted the protein and mRNA expression of ILK and Bcl-2, the protein expression of p-Akt, and reduced the protein and mRNA expression of Bax, Caspase-3, and Cytochrome c, the protein expression of p–c-Src. PMS has protective effects against H/R injury in H9c2 cells, and its protective mechanism may be related to reactive oxygen species clearance, activation of the ILK/c-Src/Akt pathway and inhibition of the mitochondrial apoptosis.

Chronic Venous Disease in Pregnant Women Causes an Increase in ILK in the Placental Villi Associated with a Decrease in E-Cadherin.

Chronic venous disease (CVD) is a multifactorial vascular disorder frequently manifested in lower limbs in the form of varicose veins (VVs). Women are a vulnerable population for suffering from CVD, especially during pregnancy, when a plethora of changes occur in their cardiovascular system. Previous studies have indicated a worrisome association between CVD in pregnancy with the placental structure and function. Findings include an altered cellular behavior and extracellular matrix (ECM) composition. Integrin-linked kinase (ILK) is a critical molecule involved in multiple physiological and pathological conditions, and together with cadherins, is essential to mediate cell to ECM and cell to cell interplay, respectively. Thus, the aim of this study was to evaluate the implication of ILK and a set of cadherins (e-cadherin, cadherin-6 and cadherin-17) in placentas of women with CVD in order to unravel the possible pathophysiological role of these components. Gene expression (RT-qPCR) and protein expression (immunohistochemistry) studies were performed. Our results show a significant increase in the gene and protein expression of ILK, cadherin-6 and cadherin-17 and a decrease of e-cadherin in the placenta of women with CVD. Overall, this work shows that an abnormal expression of ILK, e-cadherin, cadherin-6 and cadherin-17 may be implicated in the pathological changes occurring in the placental tissue. Further studies should be conducted to determine the possible associations of these changes with maternal and fetal well-being.

Effect of herbal cake-partitioned moxibustion on renal function and expression of renal connective tissue growth factor, integrin-linked kinase and bone morphogenetic protein-7 in chronic renal failure rabbits

To observe the effect of herbal cake-partitioned moxibustion on renal function and expression of connective tissue growth factor (CTGF), integrin-linked kinase (ILK) and bone morphogenetic protein-7 (BMP-7) in chronic renal failure (CRF) rabbits, so as to reveal its mechanisms underlying improvement of CRF. Twenty-four male New Zealand rabbits were randomly divided into control, model, medication and herbal cake-partitioned moxibustion (moxibustion) groups (n=6 rabbits in each group). The CRF model was established by gavage of suspension of Adenine (150 mg·kg-1·d-1) for 21 days. Herbal cake-partitioned moxibustion was applied to "Mingmen"(GV4) and bilateral "Shenshu"(BL23), "Pishu"(BL20) and for 5 moxa-cones every time. Rabbits of the medication group was treated by gavage of Losartan Potassium (2.33 mg·kg-1·d-1). All the treatments were conducted once daily,12 times a course for consecutive 3 courses with a two-day rest after each course of treatment. Serum creatinine (Scr), blood urea nitrogen (BUN) and 24 h urine protein contents were detected by using an automatic biochemical analyzer. The expression levels of CTGF, ILK and BMP-7 proteins and mRNA in the kidney tissue were determined by Western blot and quantitative real time-PCR, separately. Following modeling, Scr and BUN and 24 h urine protein contents were significantly increased in the model group in comparison with the control group (P<0.01). After the intervention, Scr and BUN contents were all significantly decreased in both the moxibustion and medication groups relevant to the model group (P<0.01), suggesting an improvement of the renal function. Compared with the control group, the expression levels of ILK and CTGF mRNAs and proteins were obviously up-regulated (P<0.01), and those of BMP-7 mRNA and protein significantly decreased in the model group (P<0.01). In comparison with the model group, the expression levels of ILK and CTGF mRNAs and proteins were significantly down-regulated in the two treatment groups (P<0.01, P<0.05), and those of BMP-7 mRNA and protein markedly increased in the two treatment groups (P<0.01). In comparison with the medication group, the expression level of ILK protein was significantly up-regulated (P<0.01) and BMP-7 protein was significantly down-regulated (P<0.01) in the moxibustion group. No significant differences were found between the medication and moxibustion groups in down-regulating the levels of Scr, BUN and 24 h urine protein and expression of ILK mRNA, CTGF mRNA and protein and BMP-7 mRNA(P>0.05). Herbal cake-partitioned moxibustion can improve renal function in CRF rabbits, which may be related to its effects in suppressing the expression of ILK and CTGF, and in up-regulating the expression of BMP-7 in the kidney tissue.

Role of integrin-linked kinase in the hypoxia-induced phenotypic transition of pulmonary artery smooth muscle cells: Implications for hypoxic pulmonary hypertension

The phenotypic transition of pulmonary artery smooth muscle cells (PASMCs) from a contractile/differentiated to synthetic/de-differentiated phenotype is an important mechanism for the occurrence and development of hypoxic pulmonary hypertension (HPH). Integrin-linked kinase (ILK) is an early hypoxic response factor whose kinase activity is significantly affected during early hypoxia. Myocardin and ETS-like protein 1 (Elk-1) are co-activators of serum response factor (SRF) and can bind to SRF to mediate the phenotypic transition of PASMCs. However, little is known about the role of ILK on the phenotypic transition of these PASMCs. Thus, in our study, we explored the role of ILK in this process. We found that the expression of ILK and myocardin decreased gradually with the increase in hypoxia exposure time in the pulmonary arteries of rats. We observed that hypoxia exposure for 1 h caused an increase in the phosphorylation of Elk-1 but did not affect the expression of ILK, myocardin, or SRF. Exposure to hypoxic treatment for 1 h decreased ILK kinase activity and caused Elk-1 to suppress myocardin binding to SRF and the smooth muscle (SM) α-actin gene promoters. In addition, hypoxia exposure for 24 h decreased the expression of ILK, myocardin, SM α-actin, and calponin but increased the expression of osteopontin. Silencing of the myocardin gene significantly decreased the expression of SM α-actin and calponin but increased the expression of osteopontin. Silencing of the ILK gene significantly decreased the expression of myocardin, SM α-actin, and calponin but increased the expression of osteopontin. ILK overexpression reversed the effects of 24 h of hypoxia on the expression of myocardin, SM α-actin, calponin, and osteopontin and reversed the decrease in binding of myocardin to the SM α-actin promoter caused by 24 h of hypoxia exposure. Thus, our results suggest that ILK initiates the phenotypic transition of PASMCs. The underlying mechanism may involve hypoxia downregulating ILK kinase activity and protein expression, causing Elk-1 to compete with myocardin for binding to the SM α-actin promoter, which downregulates the expression of the downstream target myocardin and results in the phenotypic transition of PASMCs from a contractile to a synthetic phenotype. This may be an important mechanism in the development of HPH.

PPARβ/δ Agonist GW501516 Inhibits Tumorigenicity of Undifferentiated Nasopharyngeal Carcinoma in C666-1 Cells by Promoting Apoptosis.

Activation of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) had been linked to inhibition on the proliferation and apoptosis in a few cancer cell lines. However, limited data exists regarding the role of PPARβ/δ in nasopharyngeal carcinoma (NPC). This study was undertaken to determine the effect of PPARβ/δ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the human NPC cell lines. Gene and protein expression of PPARβ/δ were reduced specifically in the poor- and un-differentiated NPC cell lines as compared with the control NP-69 cells. Ligand activation of PPARβ/δ by GW501516, a specific PPARβ/δ selective agonist, inhibited cell proliferation and colony formation strikingly, and induced a G2/M phase arrest in the EBV positive undifferentiated NPC C666-1 cells relative to the control cells. Moreover, GW501516 induced C666-1 cell apoptosis in a caspase and BAX dependent manner. In accordance with the in vitro result, GW501516 significantly suppressed the ectopic NPC xenograft tumorigenicity that derived from the C666-1 NPC cells in BALB/c nu/nu mice. This effect is greatly associated with its inhibition on the gene and protein expression of integrin-linked kinase (ILK) through activation of the AMPKα-dependent signaling pathways. Collectively, we showed that PPARβ/δ expression is in reverse correlation with the degree of differentiation in the NPC cell lines, and revealed the anti-tumorigenic effects of GW501516 in NPC cells by activation of AMPKα. This study suggested that PPARβ/δ targeting molecules may be useful for the poor-, and particularly un-differentiated NPC chemoprevention.

Clinical significance of integrin-linked kinase in laryngeal squamous cell carcinoma

ObjectiveTo investigate the expression of integrin-linked kinase (ILK) and its relationship with clinicopathological parameters in laryngeal squamous cell carcinoma (LSCC). Methods116 patients who had previously undergone complete resection of tumor for LSCC were studied retrospectively. The level of ILK expression in tumor tissues and adjacent nontumor tissues were determined by immunohistochemistry. ResultsIncreased expression of ILK was found in 65.5% of cases. The expression of ILK protein was significantly associated with tumor grade (p=0.046), lymph node metastasis (p=0.020), and pTNM stage (p=0.019). Kaplan–Meier survival estimates showed a significant correlation between ILK expression and patient survival rate (log-rank p<0.05). The multivariate survival analysis revealed that N status was statistically significant prognostic factor (p<0.001). Other parameters, such as ILK expression, cannot predict disease prognosis separately. ConclusionIncreased expression of integrin-linked kinase is associated with lymph node metastases and patient survival rate in laryngeal squamous cell carcinoma. However, it does not appear to be an independent prognostic predictor in LSCC.

The Focal Adhesion Complex Ilk-Pinch-Parvin Is Key to Niche Interactions and Survival of Quiescent Stem Cells in Chronic Myeloid Leukemia In Vitro and In Vivo

Growing evidence indicates that interactions of cancer cells with their microenvironment in vivo can influence disease progression and therapy resistance, including chronic myeloid leukemia (CML). Focal adhesions that modulate cell attachments, migration, proliferation and intracellular signaling pathways are considered critical mediators of some of these interactions. However, the potential role of focal adhesion components in mediating survival and therapeutic responses of leukemic stem cells is largely unknown.Transcriptional profiling of CD34+ cells from 6 CML patients and 3 healthy donors revealed that the expression of Integrin-linked kinase (ILK), PINCH1 and β-Parvin, major constituents of focal adhesions, is significantly increased in CD34+ CML cells, in particular in cells from drug-nonresponders (p<0.05). Quantitative real-time PCR confirmed these observations in CD34+ cells obtained from additional 30 CML patients and 14 normal healthy adults (p<0.05). Furthermore, we found that the primitive leukemic stem-cell enriched Lin-CD34+CD38- portion from CML patients expressed the highest levels of ILK, PINCH1, and β-Parvin transcripts compared to the more prevalent Lin-CD34+CD38+ progenitor population or mature CD34-cells in the same samples (n=6, p<0.05). In addition, ILK protein expression was increased in primitive CML cells compared to normal donors, in particular when CML cells were co-cultured with BM niche cells.Stable knockdown (KD) of 3 different targeting sequences of ILK in CD34+ CML cells resulted in decreased cell viability (30-80%, p<0.05) and proliferation (2-12-fold) associated with a significantly enhanced frequency of apoptotic cells compared to control-transduced cells (60-80% vs. 30%, p<0.05). Interestingly, these effects of ILK KD were not rescued by co-cultures with BM niche cells in vitro. Cell cycle analysis indicated a reduction in the proportion of surviving cells in S-phase upon ILK suppression. In addition, Western blotting showed that effective suppression of ILK led also to a decrease in β-Parvin and PINCH1 protein expression but not their transcript levels, suggesting that the ILK-PINCH-PARVIN complex is not stable under these conditions and may not be able to mediate critical interactions between primitive CML cells and BM niche components. In agreement, short- and long-term assays of stem/progenitor activity in the presence of BM niche cells demonstrated a significant reduction of colonies upon ILK suppression that was almost entirely abolished with simultaneous ABL1 tyrosine kinase inhibitor (TKI) treatment (p<0.05). Moreover, in vivo studies with 2 different mouse strains (NRG and the humanized NRG-3GS model) emphasized that primitive ILK KD CML cells showed greatly reduced in vivo regenerative activity as compared to control-transduced cells (<2% vs. 13% human cells in the BM of NRG mice, and 3% vs. 18% in NRG-3GS mice 25 weeks post-transplantation).To investigate whether ILK can be targeted pharmacologically, we utilized QLT0267, a validated and selective ILK kinase inhibitor. Similarly to ILK suppression, inhibition of the ILK kinase resulted in a modest decrease of cell viability, reduced short-and long-term stem/progenitor activity, and increased apoptosis of bulk CD34+ as well as more primitive Lin-CD34+CD38- CML cells from drug-nonresponder patients with strong synergistic effects upon simultaneous ABL1 kinase inhibition in vitro. In addition, oral gavage of QLT0267 combined with dasatinib significantly enhanced survival of leukemic mice and eradicated infiltrated leukemic cells in multiple hematopoietic tissues in an aggressive NSG mouse model of BCR-ABL+human leukemia. Most interestingly, dual inhibition of ILK and BCR-ABL1 decreased the proportion of quiescent leukemic stem cells compared to single agent treatments. RNA sequencing of these cells indicated a deregulation of MYC and novel signaling targets, with differences between dividing and non-dividing cell subpopulations.In summary, genetic and pharmacological inhibition of ILK significantly impaired survival, proliferation and quiescence of drug-nonresponder CML stem cells and sensitized them to TKIs both in vitro and in vivo. These findings suggest that ILK plays a critical role in regulating CML stem cell activity and that targeting ILK and BCR-ABL1 simultaneously may offer an improved novel therapeutic strategy. DisclosuresNo relevant conflicts of interest to declare.

Effects of lentiviral RNA interference-mediated downregulation of integrin-linked kinase on biological behaviors of human lens epithelial cells.

To investigate the effects of lentivirus (LV) mediated integrin-linked kinase (ILK) RNA interference (RNAi) on biological behaviors of human lens epithelial cells (LECs). Human cataract LECs and immortalized human LEC line, human lens epithelial (HLE) B-3 cells were transfected by lentiviral vector expressing ILK-specific short hairpin RNA (shRNA) and then stimulated by transforming growth factor-β (TGF-β), the silencing of ILK gene and protein was identified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot methods; biological behaviors including cell cycle and apoptosis, cell morphology, α-smooth muscle actin (SMA) stress fiber formation and cell migration were examined. Remarkable decreases of ILK protein expression were detected in LECs carrying lentiviral ILK-shRNA vector; flow cytometry revealed arresting of cell cycle progression through the G1/S transition and higher apoptosis rate in ILK-RNAi-LV transfected cells. Less α-SMA stress fiber formation and migration was observed in ILK-RNAi-LV transfected LECs. The present study demonstrated that ILK was an important regulator for LECs proliferation and migration. LV mediated ILK RNAi is an effective way to decrease ILK-regulated cell growth by arresting cell cycle progression and increasing cell apoptosis, as well as, to prevent cell migration by inhibiting TGF-β induced α-SMA stress fiber formation. Thus, LV mediated ILK RNAi might be useful to prevent posterior capsular opacification.

Integrin-linked kinase affects signaling pathways and migration in thyroid cancer cells and is a potential therapeutic target

Integrin-linked kinase (ILK) is a serine-threonine kinase that regulates interactions between the cell and the extracellular matrix. In many cancers, overexpression of ILK leads to increased cell proliferation, motility, and invasion. We hypothesized that ILK functions as a regulator of viability and migration in thyroid cancer cells. Eleven human thyroid cancer cell lines were screened for ILK protein expression. The cell lines with the greatest expression were treated with either ILK small interfering RNA (siRNA) or a novel ILK inhibitor, T315, and the effects were evaluated via Western blot and migration assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assays were performed to assess cell viability. siRNA against ILK decreased phosphorylation of downstream effectors Akt and MLC, as well as decreased migration. Treatment with T315 showed a dose-related decrease in both Akt and MLC phosphorylation, as well as decreased migration. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assays showed T315 to have an half maximal inhibitory concentration of less than 1μM in cell lines with high ILK expression. ILK is expressed differentially in thyroid cancer cell lines. Both ILK siRNA and T315 inhibit motility of thyroid cancer cell lines, and T315 is shown to be cytotoxic at low concentrations. Altogether, our study suggests that ILK may represent an important kinase in aggressive thyroid cancers.

Knockdown of ILK inhibits glioma development via upregulation of E-cadherin and downregulation of cyclin D1.

Integrin-linked kinase (ILK) is a highly conserved serine-threonine protein kinase that interacts with cytoplasmic domains of integrin subunits in tumor tissues. However, the relationship between gliomas and ILK is elusive. The present study aimed to investigate the role of ILK in a human glioma cell line (U251). ILK stable expressing vector, U251ILK-PGFP-V-RS-shRNA, was established and named as U251-si. The empty-PGFP-V-RS-shRNA (U251-N) was employed as the control. Quantitative real-time PCR and western blot analysis were used to detect ILK and E-cadherin mRNA and protein expression, respectively. Cell cycle analysis was employed to examine the cell cycle distribution. Cell migration was detected using a wound healing assay, and cell invasion was detected using a Transwell invasion assay. Tumor size and weight were also examined. The results indicated that ILK was expressed at a lower level at both the mRNA and protein levels in the U251-si group compared with the U251-N group (p<0.01). ILK knockdown suppressed cell proliferation of the glioma cells. Knockdown of ILK reduced the migratory and invasive potentials of the glioma cells. Inhibition of ILK expression upregulated E-cadherin and downregulated cyclin D1 in the glioma cells compared to the U251-N group (p<0.05). Knockdown of ILK in the U251 cells attenuated the ability of U251 cells to form tumors in nude mice and impaired glioma cell in vivo tumorigenicity. In conclusion, knockdown of ILK inhibits glioma cell migration, invasion and proliferation through upregulation of E-cadherin and downregulation of cyclin D1. Our results suggest that ILK may serve as a promising therapeutic target for glioma.

RNA silencing of integrin-linked kinase increases the sensitivity of the A549 lung cancer cell line to cisplatin and promotes its apoptosis.

The expression of integrin-linked kinase (ILK) has been reported to be involved in the regulation of integrin-mediated processes, including cancer cell proliferation, migration and invasion. Previous studies have demonstrated that inhibition of ILK may be an underlying approach for treating cancer. However, whether the knock down of ILK affects growth and apoptosis of lung cancer cells remains to be elucidated. Importantly, whether downregulation of ILK increases the sensitivity of lung cancer cells to cisplatin and amplifies cell apoptosis also remains to be elucidated. In the present study, ILK downregulation was mediated by lentivirus-mediated RNA interference. The expression levels of associated genes were determined by reverse-transcription quantitative polymerase chain reaction and western blotting. Cell proliferation was evaluated using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and clone formation assay. The cell cycle and apoptosis were analyzed using flow cytometry. The current data revealed that lentivirus-mediated ILK gene silencing alone inhibited A549 cell proliferation and promotes cell cycle arrest, however, had no detectable effect on cell apoptosis. However, combined treatment with lentivirus-mediated ILK interference and cisplatin chemotherapy induced significantly more cell apoptosis than mono-chemotherapy or knockdown. The increased cell apoptosis and proliferation inhibition were attributed to abnormal downstream protein expression of ILK, including phospho-glycogen synthase kinase 3β, p-AKT, activator protein-1, β-catenin, cyclin D1 and matrix metalloproteinase-9. ILK inhibition may suppress the proliferation of A549 and increase A549 sensitivity to cisplatin. The combined treatment of ILK gene knockdown and chemotherapy has the potential to improve anticancer efficacy.

Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression.

Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression.

Integrin-Linked Kinase As a Key Mediator of Stromal Cell-Enhanced Resistance of Primitive CML Cells to Tyrosine Kinase Inhibitors

The human bone marrow (BM) compartment consists of a heterogeneous, multi-functional network of cells and extracellular matrix that interact with hematopoietic stem cells. Growing evidence suggests that this microenvironment also likely adds to the resistance of chronic myeloid leukemia (CML) stem cells to tyrosine kinase inhibitor (TKI) therapy in vivo. Integrin-linked kinase (ILK) is a serine/threonine kinase that is an important constituent of focal adhesions, a regulator of spindle formation during mitosis, and a key mediator of multiple signaling pathways. However, its potential role in the regulation of primitive CML cells and their response to TKIs is unknown.Our RNA-seq analysis revealed that ILK expression and its downstream targets, such as AKT, are highly upregulated in pre-treatment CD34+ CML stem/progenitor cells (n=6) compared to normal BM controls (n=3, p<0.05). We confirmed this finding by qRT-PCR analysis of CD34+ cells obtained from 28 CML patients (including some who subsequently became clinically resistant to imatinib mesylate (IM) treatment) compared to CD34+ BM cells from 9 normal adults (p<0.05). Elevated expression of ILK protein in CD34+ CML cells was also demonstrated by Western blot analysis (threefold, n=4, p<0.05). Interestingly, we also found ILK transcript levels to be much higher in the more primitive and rarer CD38- CML stem cell-enriched subset of the lin-CD34+ population as compared to the more mature and prevalent CD38+ subset or the terminally differentiated lin+CD34- cells (p<0.01). Both ILK transcripts and intracellular protein levels were elevated in CD34+ CML cells, but not in CD34- differentiated cells, in response to IM treatment in vitro, especially in the presence of expanded BM stromal cells, suggesting differential regulation of ILK expression in primitive CML cells and their more mature counterparts.We next asked whether ILK influences primitive CML cell responses to TKIs by using a clinically validated and selective ILK inhibitor (QLT0267) to suppress ILK activity in colony assays of CD34+ CML cells. QLT0267 plus TKIs significantly reduced the yield of colonies obtained compared to any single agent or combination of TKIs and this enhanced cell killing was most pronounced on cells from IM non-responders (n=6; p<0.01). Analysis of the lineages affected showed that the combination of IM or dasatinib (DA) with QLT0267 had a more pronounced effect on myeloid colony formation (CFU-GM), at concentrations where either or both TKIs had little effect (i.e. 85-90% inhibition vs. 30-45%). Moreover, the simultaneous suppression of ILK and BCR-ABL activities also effectively inhibited the growth of more primitive CML cells (from IM non-responder patients) when these were co-cultured with stromal cells in 6-week long-term cultures (LTC), in contrast to the lack of these effects in the presence of single TKIs or TKI combinations.Mechanistically, we found that the combination of QLT0267 with a TKI enhanced the induction of apoptosis of CD34+ CML cells in suspension cultures within the first three days (from 10-15% to 30-45% apoptotic cells relative to untreated controls) and CFSE (carboxy-fluorescein diacetate succinimidyl diester) tracking of cell division showed that QLT0267 specifically targets quiescent CML stem cells from IM-resistant patients (n=4; p<0.05). In addition, treatment with the ILK inhibitor alone or in combination with a TKI enhanced apoptosis of primitive CML cells in vitro by abolishing the protective effect of BM stromal cells observed under TKI treatment alone (25-30% apoptotic cells vs. 3-8%) and combination treatments confirmed strong synergy between DA and QLT0267 (CI value <0.3). Importantly, QLT0267 (up to 10 μM) was not toxic to normal CD34+BM cells in either short- or long-term culture systems (with and without stromal cells).Together, these findings indicate that ILK is a critical player in the regulation of TKI response/resistance of primitive CML cells. The enhanced and selective effect obtained by dual inhibition of both targets, particularly in the presence of protective stromal cells to mimic their response within the BM microenvironment, may offer an important new therapeutic possibility. DisclosuresNo relevant conflicts of interest to declare.

Astragaloside IV ameliorates diabetic nephropathy involving protection of podocytes in streptozotocin induced diabetic rats

Podocyte loss and dysfunction play key role during the development of diabetic nephropathy (DN). The aim of this study was to observe the protective effects of astragaloside IV on podocyte in diabetic rats and explore its mechanisms preliminary. Healthy male Sprague-Dawley (SD) rats were randomized into normal control group, diabetic nephropathy group and diabetic nephropathy with AS-IV treatment group. DN was induced by intraperitoneal injection of streptozotocin (STZ). AS-IV treatment started 2 weeks before STZ injection and lasted 14 weeks. 24h Urinary proteins were measured 4, 8 and 12 weeks after STZ injection. Body weight, blood glucose, blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured 12 weeks after STZ injection. Renal pathology, podocyte morphological changes, podocyte density, protein and mRNA expression of integrin α3, integrin β1 and integrin-linked kinase (ILK) were detected by histopathology, electron microscopy, immunohistochemistry, western blot and real-time PCR, respectively. Hyperglycemia, proteinuria, mesangial expansion and podocyte loss, increased protein expression of ILK and decreased protein expression of integrin α3 and integrin β1 were detected in diabetic rats. AS-IV treatment ameliorated podocyte loss, renal histopathology and podocyte foot process effacement, decreased proteinuria, partially restored protein expression of integrin α3, integrin β1 and ILK. These findings suggested that AS-IV may protect podocyte and ameliorate diabetic nephropathy by inhibiting the expression of ILK and restoring the expression of integrin α3β1 in diabetic rats.

Repression of integrin-linked kinase by antidiabetes drugs through cross-talk of PPARγ- and AMPKα-dependent signaling: Role of AP-2α and Sp1

Nasopharyngeal carcinoma (NPC) is one of the most common cancers of the head and neck, particularly in Southern China and Southeast Asia with high treatment failure due to the development of local recurrence and distant metastasis. The molecular mechanisms related to the progression of NPC have not been fully understood. In this study, we showed that antidiabetes drugs rosiglitazone and metformin inhibit NPC cell growth through reducing the expression of integrin-linked kinase (ILK). Blockade of PPARγ and AMPKα overcame the effects of rosiglitazone and metformin on ILK protein. Importantly, overexpression of ILK abrogated the effect of rosiglitazone and metformin on NPC cell growth. Furthermore, these agents reduced ILK promoter activity, which was not observed in AP-2α, but not Sp1 site mutation in ILK gene promoter. In addition, silencing of AP-2α or overexpression of Sp1 reversed the effect of these agents on ILK protein expression and cell growth. Chromatin immunoprecipitation (ChIP) assay showed that rosiglitazone induced AP-2α, while metformin reduced Sp1 protein binding to the DNA sequences in the ILK gene promoter. Intriguingly, overexpression of Sp1 abolished the effect of rosiglitazone on AP-2α protein expression. Collectively, we show that rosiglitazone and metformin inhibit ILK gene expression through PPARγ- and AMPKα-dependent signaling pathways that are involved in the regulation of AP-2α and Sp1 protein expressions. The effect of combination of rosiglitazone and metformin demonstrates greater extent than single agent alone. The cross-talk of PPARγ and AMPKα signaling enhances the synergistic effects of rosiglitazone and metformin. This study unveils novel mechanisms by which oral antidiabetes drugs inhibit the growth of human NPC cells.

Expression of integrin-linked kinase in adenoid cystic carcinoma of salivary glands correlates with epithelial–mesenchymal transition markers and tumor progression

Integrin-linked kinase (ILK) plays a key role in cell-excellular matrix interactions mediated by integrins and several growth factors, regulating cell proliferation, apoptosis, differentiation, and migration. It has also been implicated in the development and progression in several malignancies involving epithelial to mesenchymal transition (EMT). However, the correlations between ILK and EMT markers and the progression of salivary adenoid cystic carcinoma (SACC) have not been well elucidated. Here, by immunohistochemistry, we studied the expression of ILK, Snail, E-cadherin, and N-cadherin in 94 SACC specimens and analyzed their correlations with clinicopathologic characteristics. Positive expression of ILK protein was detected in 76.6 % of the tumors. Increased expression of ILK and Snail and decreased E-cadherin expression correlated strongly with tumor solid type (P = 0.017, P = 0.008, and P = 0.038, respectively), advanced TNM stage (P = 0.021, P = 0.034, and P = 0.009, respectively), and increased risk of recurrence (P = 0.023, P = 0.011, and P = 0.039, respectively) and distant metastasis (P < 0.001, P < 0.001, and P = 0.001, respectively). Moreover, up-regulation of Snail and N-cadherin and down-regulation of E-cadherin correlated significantly with ILK over-expression (P < 0.001, P = 0.001, and P < 0.001, respectively) and a neural-invasive phenotype (P = 0.017, P = 0.002, and P < 0.001, respectively). Taken together, our results suggest that ILK may have an important role in progression and metastasis of SACC, possibly through EMT involving up-regulation of Snail and consequent aberrant expression of E-cadherin and N-cadherin. ILK should be considered as a potential therapeutic molecular target for patients with SACC.

Silencing of integrin-linked kinase suppresses in vivo tumorigenesis of human ovarian carcinoma cells

Integrin-linked kinase (ILK) plays a role in the regulation of multiple cellular functions (e.g., promoting cell migration and proliferation, but inhibiting cell adhesion). This study investigated the inhibitory effects of ILK gene knockdown on the regulation of invivo tumorigenesis of human ovarian carcinomacells in nude mouse xenografts. HO-8910 cells were transfected with an ILK antisense oligonucleotide (ILK-ASO) to silence the ILK gene. Expression of ILK mRNA and protein was evaluated by RT-PCR and western blotting, respectively. The cell cycle was assessed by flow cytometric analysis. Cells with or without ILK-ASO transfection were subcutaneously injected into nude mice. The mouse body weight, tumor formation, tumor size and tumor weight were determined up to 30 days after inoculation. Tumor cells transfected with ILK-ASO had significantly decreased ILK mRNA and protein expression (P<0.01) when compared to the control cells. ILK gene silencing significantly increased the number of cells in the G0/G1 phase (67.61 vs. 43.29%, χ2=1197.15, P<0.01). After tumor cell inoculation, tumor cells transfected with ILK-ASO showed significantly delayed tumor formation when compared to control (9.10±0.74 vs. 5.30±0.67 days, respectively; P<0.01). In addition, tumor growth was suppressed in the 30 days following inoculation (P<0.01 compared with the controls). The average tumor weight in the ILK-ASO group was statistically lower than that of the control group (1.29±0.11 vs. 1.57±0.13 g, respectively; P<0.01). This study demonstrated that ILK-ASO transfection efficiently downregulated ILK expression in human ovarian carcinoma HO-8910 cells and that ILK gene silencing suppressed tumor growth in nude mice xenografts.

Overexpression of integrin-linked kinase (ILK) is associated with tumor progression and an unfavorable prognosis in patients with colorectal cancer

Integrin-linked kinase (ILK), an intracellular serine-threonine kinase, has been reported to be overexpressed in multiple types of human malignancies, including colorectal cancer (CRC). The prognostic value of ILK in CRC, however, remains unknown. In the present study, expression of ILK in 25 paired primary CRC samples and adjacent noncancerous tissues were quantified using real-time PCR and Western blotting. ILK protein expression was analyzed in 102 archived, paraffin-embedded CRC samples using immunohistochemistry. The correlation between ILK expression and clinicopathological factors was evaluated by the χ(2) test. Patients' overall survival was analyzed by Kaplan-Meier method. We found that both ILK mRNA and protein expression levels were significantly up-regulated in primary CRC samples compared with their corresponding normal tissues. Immunohistochemical analysis revealed relative high expression of ILK in 43 of 102 (42.2 %) primary CRC samples. Statistical analysis showed a significant correlation of ILK expression with tumor differentiation, lymph node metastasis, tumor invasion, and tumor-node-metastasis stage. Patients with tumors displaying high-level ILK expression showed significantly shorter overall survival (P = 0.028, log-rank test). More importantly, multivariate analysis indicated that high ILK protein expression was an independent prognostic factor for CRC patients (P = 0.026). Taken together, our data suggest that ILK overexpression is associated with tumor progression and a poor prognosis in CRC patients and may represent a novel potential prognostic marker for patients with CRC.

Abstract 643: Gender -based Differences In Left Ventricular Remodeling After Myocardial Infarction: Insight Into The Protective Role Of Integrin-linked Kinase

There is conflicting data about gender differences with regard to left ventricular (LV) remodeling after myocardial infarction (MI). Moreover, the molecular basis for gender-based differences in LV remodeling remains unknown. This study evaluated whether gender differences exist in LV remodeling after MI. Given that integrin-linked kinase (ILK) influences LV remodeling, we also examined whether gender affects the modulation of ILK post-MI. Female and male Wistar rats were assigned to one of three groups: a sham group, a moderate MI group (size: 20-39% of LV area) and a large MI group (size: ≥40% of LV area). The MI was produced by coronary occlusion and echocardiographic analysis as well as ILK expression was performed six weeks post-MI. The MI resulted in systolic dysfunction and enlargement of end-diastolic as well as end-systolic dimension of the left ventricle as a function of necrotic area size for both genders. However, female rats with large MI showed a lower diastolic (5.4±1 mm 2 ) and systolic dilatation (3.9±0.9 mm 2 ) than respective male rats (8.2±0.7 mm 2 and 5.7±1.1 mm 2 , respectively). Both gene (moderate MI: 1.85±0.15; large MI: 2.35±0.16, p&lt;0.001) and protein (moderate MI: 3.21±0.09; large MI: 4.75±0.24, p&lt;0.001) levels of ILK were increased as a function of MI size in female rats. However, only male rats in the large MI group showed an altered ILK mRNA level (moderate MI: 1.51±0.10; large MI: 1.69±0.08, p&lt;0.001). A negative linear correlation was evident between LV dimensions and ILK protein expression in female rats with large MI (left ventricle diastolic area:ILK protein content: r 2 =0.52, p=0.003; left ventricle systolic area:ILK protein content: r 2 =0.68, p&lt;0.001). In conclusion, post-MI ILK expression is altered in a gender-specific manner and higher ILK levels found in females may be sufficient to improve geometry of LV; however, may not be sufficient to improve LV function.

Integrin-linked Kinase Functions as a Tumor Promoter in Bladder Transitional Cell Carcinoma

The aim of this study was to elucidate the role of the integrin-linked kinase (ILK) gene in development of human bladder transitional cell carcinoma (BTCC). Expression of ILK protein and ILK mRNA in 56 cases of human BTCC tissue and in 30 cases of adjacent normal bladder tissue was detected by immunohistochemistry S-P and reverse transcription polymerase chain reaction (RT-PCR), respectively. Four specific miRNA RNAi vectors targeting human ILK were synthesized and transfected into BIU-87 cells by liposome to obtain stable expression cell strains. The influence of ILK on proliferation of BTCC was detected by MTT, FCM on athymic mouse tumorigenesis. The positive rate of ILK protein in BTCC tissue (53.6%) was much higher than adjacent normal bladder tissue (10.0%) (p<0.05). Similarly, expression of ILK mRNA in BTCC tissue (0.540 ± 0.083) was significantly higher than in adjacent normal bladder tissue (0.492 ± 0.070) (p<0.05). MTT showed that the proliferation ability of miRNA-ILK transfected group was clearly decreased (p<0.05), the cell cycle being arrested in G0/G1-S, an tumorigenesis in vivo was also significantly reduced (p<0.05). ILK gene transcription and protein expression may be involved in the development of BTCC, so that ILK might be the new marker for early diagnosis and the new target for gene treatment.