Circ_0085495 knockdown reduces adriamycin resistance in breast cancer through miR-873-5p/integrin ß1 axis.

Circular RNAs (circRNAs) are reported to be related to cancer chemoresistance. However, the role of circ_0085495 in adriamycin (ADM) and its action mechanism has not been elucidated in breast cancer. Cell counting kit-8 was employed to detect cell viability. Quantitative real-time-PCR and western blot were performed to examine the gene and protein expression level. Flow cytometry and colony formation assay were conducted to measure cell apoptosis and proliferation. Cell migration and invasion were evaluated via transwell assay. The target association between molecules was confirmed by dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. Tumor xenograft assay was implemented to explore the role of circ_0085495 in vivo. Circ_0085495 and Integrin ß1 were upregulated, while miR-873-5p was downregulated in ADM-resistant cells. Circ_0085495 was a stable circRNA, mainly located in the cytoplasm. Depletion of circ_0085495 repressed ADM resistance, proliferation and metastasis of ADM-resistant breast cancer cells, which was weakened by miR-873-5p inhibition or integrin ß1 overexpression. Circ_0085495 sponged miR-873-5p to positively regulate integrin ß1 expression. Integrin ß1 knockdown also inhibited ADM resistance. Furthermore, circ_0085495 knockdown inhibited tumor growth in vivo. Circ_0085495 knockdown reduced ADM resistance in ADM-resistant cells through modulating miR-873-5p/integrin ß1 axis, indicating circ_0085495 as a promising target for overcoming ADM resistance in breast cancer patients.

DS-1 Inhibits Migration and Invasion of Non-small-cell Lung Cancer Cells Through Suppression of Epithelial to Mesenchymal Transition and Integrin ß1/FAK Signaling.

BACKGROUND/AIM: Epithelial to mesenchymal transition (EMT), and focal adhesion kinase (FAK) facilitate lung cancer cell motility and survival. We, therefore, investigated the antimigratory effect of 3,4-dihydroxy-5,4'-dimethoxybibenzyl (DS-1) on human lung cancer cells. MATERIALS AND METHODS: Cell viability and proliferation were examined by the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide assay. Filopodia formation, migration, and anchorage-independent growth assays were performed to assess metastatic behaviors while EMT-related proteins, integrins, and FAK-RhoA pathway were evaluated by western blot analysis. RESULTS: We found that DS-1 significantly inhibited the proliferation of lung cancer cells compared to the control. The aggressive behavior of cancer cells, including migration and invasion, was significantly reduced by DS-1. Anchorage-independent growth analysis provided evidence that DS-1 suppressed the growth and survival of cancer cells in detached conditions as indicated by the significant reduction in size and number of colonies. With regard to the mechanisms involved, we found that DS-1-suppressed EMT, as indicated by the reduction of EMT markers, namely N-cadherin, SNAIL and SLUG, and increased levels of the epithelial marker, E-cadherin. In addition, DS-1 was shown to reduce the level of integrin ß1 protein and FAK activation. CONCLUSION: DS-1 suppressed lung cancer metastasis via suppressing EMT, integrin ß1 expression and FAK-related signaling.

rAj-Tspin, a novel recombinant peptide from Apostichopus japonicus, suppresses the proliferation, migration, and invasion of BEL-7402 cells via a mechanism associated with the ITGB1-FAK-AKT pathway.

Purpose Our study aimed to investigate the antitumor effects of rAj-Tspin on BEL-7402 hepatocellular carcinoma cells and to explore the underlying mechanism. Method For the in vivo experiment, BEL-7402 cells were inoculated subcutaneously into the axilla of nude mice to generate a BEL-7402 cell-bearing model, and model mice were then treated with different doses of rAj-Tspin. A CCK-8 assay was used to evaluate the in vitro viability of BEL-7402 and LO2 cells after treatment with different concentrations of rAj-Tspin. The effects of rAj-Tspin on BEL-7402 cell apoptosis, migration and invasion were evaluated by AO/EB and Hoechst fluorescent staining and by scratch and Transwell assays, and the tumor-suppressive mechanism of rAj-Tspin was explored by Western blotting. Results rAj-Tspin suppressed the proliferation of BEL-7402 cells with an IC50 of 0.89 µM. The results of both microscopic analysis and Western blotting suggested that rAj-Tspin induced the apoptosis of BEL-7402 cells through a mitochondria-dependent pathway. Furthermore, rAj-Tspin disrupted EMT; this disruption ultimately caused BEL-7402 cells to lose their shape and decreased their migration and invasion. Moreover, rAj-Tspin might inhibit the proliferation and metastasis of BEL-7402 cells through the ITGB1-FAK-AKT pathway. Conclusion rAj-Tspin exerts an antitumor effect through the ITGB1-FAK-Akt signaling pathway and exhibits low toxicity at an effective dose.

Inhibition of ITGB1 enhance the anti-tumor effect of cetuximab in colorectal cancer cell.

BACKGROUND: Colorectal cancer is the second commonly seen cancer around the world and accounts for 13% of all human cancers. Among them, 25% of all case were diagnosed with metastasis and 50% occurs metastasis during the development of disease. Cetuximab is a chimeric monoclonal antibody against epidermal growth factor receptor, and is used for treatment of metastatic colorectal cancer alone or combined with chemotherapy or radiation therapy. Integrin-beta 1 (ITGB1), which is also known as CD29, and plays an important role in development of malignant cancers. However, the effect of ITGB1 in promoting the anti-tumor effect of cetuximab is not fully understand. METHODS: The model of ITGB1 inhibition and overexpression was firstly constructed in LS174T cells, and the viability of cells in each group was detected using CCK-8 assay. The expression of key factors in tumor formation process at transcription level was detected using real-time quantitative polymerase chain reaction method. The expression of key proteins in metastasis process, cell apoptosis and activation of Ras/Raf/MEK signaling pathway was detected using western blotting analysis. And the concentration of key factors of in tumor formation process in cultured medium of LS174T cells were detected using enzyme-linked immunosorbent assay method. RESULTS: We found that cetuximab could inhibit the proliferation of LS174T cells, and inhibition of ITGB1 enhanced this effect while overexpression of ITGB1 reduced this effect. We further found that cetuximab could inhibit the expression and secretion of extracellular matrix degradation related molecules in cultured medium and transcription level. Besides, we also found that the expression of key factors in angiogenesis and extracellular matrix degradation related proteins were also reduced after cetuximab treatment. These effects might be mediated by Ras/Raf/MAPK signaling pathway and enhanced after inhibition of ITGB1 expression. CONCLUSION: Inhibition of ITGB1 might be a new therapeutic method in colorectal cancer.

Beta 1 integrin signaling mediates pancreatic ductal adenocarcinoma resistance to MEK inhibition.

Pancreatic cancer, one of the deadliest human malignancies, has a dismal 5-year survival rate of 9%. KRAS is the most commonly mutated gene in pancreatic cancer, but clinical agents that directly target mutant KRAS are not available. Several effector pathways are activated downstream of oncogenic Kras, including MAPK signaling. MAPK signaling can be inhibited by targeting MEK1/2; unfortunately, this approach has been largely ineffective in pancreatic cancer. Here, we set out to identify mechanisms of MEK inhibitor resistance in pancreatic cancer. We optimized the culture of pancreatic tumor 3D clusters that utilized Matrigel as a basement membrane mimetic. Pancreatic tumor 3D clusters recapitulated mutant KRAS dependency and recalcitrance to MEK inhibition. Treatment of the clusters with trametinib, a MEK inhibitor, had only a modest effect on these cultures. We observed that cells adjacent to the basement membrane mimetic Matrigel survived MEK inhibition, while the cells in the interior layers underwent apoptosis. Our findings suggested that basement membrane attachment provided survival signals. We thus targeted integrin ß1, a mediator of extracellular matrix contact, and found that combined MEK and integrin ß1 inhibition bypassed trametinib resistance. Our data support exploring integrin signaling inhibition as a component of combination therapy in pancreatic cancer.

Simvastatin ameliorates altered mechanotransduction in uterine leiomyoma cells.

BACKGROUND: Uterine leiomyomas, the most common tumors of the female reproductive system, are characterized by excessive deposition of disordered stiff extracellular matrix and fundamental alteration in the mechanical signaling pathways. Specifically, these alterations affect the normal dynamic state of responsiveness to mechanical cues in the extracellular environment. These mechanical cues are converted through integrins, cell membrane receptors, to biochemical signals including cytoskeletal signaling pathways to maintain mechanical homeostasis. Leiomyoma cells overexpress ß1 integrin and other downstream mechanical signaling proteins. We previously reported that simvastatin, an antihyperlipidemic drug, has antileiomyoma effects through cellular, animal model, and epidemiologic studies. OBJECTIVE: This study aimed to examine the hypothesis that simvastatin might influence altered mechanotransduction in leiomyoma cells. STUDY DESIGN: This is a laboratory-based experimental study. Primary leiomyoma cells were isolated from 5 patients who underwent hysterectomy at the Department of Gynecology and Obstetrics of the Johns Hopkins University Hospital. Primary and immortalized human uterine leiomyoma cells were treated with simvastatin at increasing concentrations (0.001, 0.01, 0.1, and 1 µM, or control) for 48 hours. Protein and mRNA levels of ß1 integrin and extracellular matrix components involved in mechanical signaling were quantified by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence. In addition, we examined the effect of simvastatin on the activity of Ras homolog family member A using pull-down assay and gel contraction. RESULTS: We found that simvastatin significantly reduced the protein expression of ß1 integrin by 44% and type I collagen by 60% compared with untreated leiomyoma cells. Simvastatin-treated cells reduced phosphorylation of focal adhesion kinase down to 26%-60% of control, whereas it increased total focal adhesion kinase protein expression. Using a Ras homolog family member A pull-down activation assay, we observed reduced levels of active Ras homolog family member A in simvastatin-treated cells by 45%-85% compared with control. Consistent with impaired Ras homolog family member A activation, simvastatin treatment reduced tumor gel contraction where gel area was 122%-153% larger than control. Furthermore, simvastatin treatment led to reduced levels of mechanical signaling proteins involved in ß1 integrin downstream signaling, such as A-kinase anchor protein 13, Rho-associated protein kinase 1, myosin light-chain kinase, and cyclin D1. CONCLUSION: The results of this study suggest a possible therapeutic role of simvastatin in restoring the altered state of mechanotransduction signaling in leiomyoma. Collectively, these findings are aligned with previous epidemiologic studies and other reports and support the need for clinical trials.

Akermanite bioceramic enhances wound healing with accelerated reepithelialization by promoting proliferation, migration, and stemness of epidermal cells.

Reepithelialization is an important step of wound healing, which is mainly completed by proliferation and migration of epidermal cells. Akermanite is a Ca-, Mg-, and Si-containing bioceramic. This study evaluated the effects of Akermanite on wound healing and investigated the mechanisms. Using scald burn mice models, we demonstrated that local Akermanite treatment significantly accelerated wound healing by increasing reepithelialization and the stemness of epidermal cells. Epidermal cells were cultured in medium containing Akermanite extracts to explore the cellular mechanism of reepithelialization. Akermanite promoted the cell proliferation and migration, maintaining more cells in the S and G2 /M phases of the cell cycle. An additional study showed that Akermanite enhanced the expressions of integrinß1, Lgr4, Lgr5, and Lgr6, which are specific molecular markers of epidermal stem cells, accompanied by the activation of the Wnt/ß-catenin pathway. These results suggested that Akermanite accelerated reepithelialization by increasing the proliferation, migration, and stemness of epidermal cells in a manner related to the Wnt/ß-catenin pathway, which might contribute, at least partially, to accelerated wound healing by Akermanite therapy.

TNF-α Regulates ITGß1 and SYND4 Expression in Nucleus Pulposus Cells: Activation of FAK/PI3K Signaling.

Integrins can function synergistically with syndecan-4 (SYND4) and bind to the fibronectin (FN) matrix, resulting in the regulation of tissue regeneration. This study aimed to explore the effects of TNF-α on the formation of FN/ITGß1/SYND4 complex and the relative mechanism in NP cells. The expression of FN-ITG-SYND4 at the cellular level under TNF-α stimulation was detected by immunofluorescent staining, western blotting, and RT-PCR. ITGß1 is a crucial component of ITG FN-induced FAK signaling, which was detected using dual mode. And, the involved signaling down stream pathways were also detected. FN is a preferred adhesion substrate for NP cells and that integrin ß1 (ITGß1) and SYND4 work synergistically during ECM engagement in a focal adhesion kinase (FAK)-dependent fashion. The PI3k/Akt pathway is obviously down-regulated, resulting in decreased adherence capacity and increased anoikis. TNF-α induction could weaken FAK activity and downstream levels of phospho-PI3K and Akt, resulting in decreased adherence capacity and increased apoptosis. Thus, TNF-α is essential for the formation of FN/ITGß1/SYND4 complex in NP cells and further elucidates the inflammatory mechanism of NP cells degeneration.

Toxicological effects of bisphenol A exposure-induced cancer cells migration via activating directly integrin ß1.

Bisphenol A (BPA) is an important chemical that widely used in our life. Mounting evidences show that BPA can leak into environment, which associates with the health risks, such as initiation and metastasis of cancer, but the mechanisms still need to be interpreted. Integrin ß1 is the most known subunit in integrin family, its abnormal expression and activation are tightly linked to tumorigenesis and a number of hallmarks of cancer. Here we show that environmental concentration (10-8 M) of BPA exposure can quickly activate integrin ß1 to induce cancer cell migration, and this effect is proved as a direct interaction between BPA and integrin ß1, which is independent from classical or non-canonical estrogen receptors. The data further indicates that residues S134, D137 and E229 in integrin ß1 played important roles in the interaction as predicted by AutoDock Vina, and confirmed by spectroscopy and native-PAGE. The study is the first to show a tumorigenic mechanism of BPA on tumor metastasis by the direct activation of integrin ß1 molecule, and give rise to profound concerns about widespread use of BPA in the manufacture of plastics and human health.

The peptide mimicking small extracellular loop domain of CD82 inhibits tumor cell migration, adhesion and induces apoptosis by inhibiting integrin mediated signaling.

Within the extracellular domains of metastasis suppressor CD82, the large extracellular loop (EC2) has received much of the attention and its structure and function have been studied in detail. However, little attention has been given to the small extracellular loop (EC1 domain). To investigate the function role of EC1 in metastasis suppression of CD82, the peptide mimicking EC1 amino acid sequence (EC1-mP) was synthesized and its effect on cancer cells behavior was examined. Here, we reported that EC1-mP strongly inhibited cancer cell migration in vitro, attnuated the ability of cancer cells adhesion to fibronectin, and induced the apoptosis. Furthermore, the EC1-mP was showed to supprese the expressions of integrins α5 and ß1, as well as decreased the phosphorylation of FAK and expression of ILK in SW620 cells. Taken together, these results demonstrate that this small peptide has the functional role of CD82 intact molecule. This novel finding will improve our understanding of the mechanism by which CD82 inhibits metastasis, and suggested that EC1 mimic peptide may be a promising candidate for developing anti-metastasis drugs.

Altered O-glycosylation is associated with inherent radioresistance and malignancy of human laryngeal carcinoma.

Radioresistance (inherent or acquired) remains a major obstacle affecting the clinical outcome of radiotherapy for laryngeal carcinoma. Results from our laboratory and other groups suggest that aberrant glycosylation contributes to cancer acquired radioresistance. However, the role of glycosylation in inherent radioresistance of laryngeal carcinoma has not been fully uncovered. In this study, we investigated the glycan profiling of the inherent radioresistant (Hep-2max) and radiosensitive (Hep-2min) cell lines using lectin microarray analysis. The results revealed that the radioresistant cell line Hep-2max presented higher core 1-type O-glycans than the sensitive one. Further analysis of the O-glycan regulation by benzyl-α-GalNAc application in Hep-2max cells showed partial inhibition of the O-glycan biosynthesis and increased radiosensitivity. In addition, core 1 ß1, 3-galactosyltransferase (C1GALT1) overexpression in Hep-2min cells enhanced cell migration, invasion, and radioresistance. Conversely, knockdown of C1GALT1 in Hep-2max cells was able to suppress these malignant phenotypes. Moreover, mechanistic investigations showed that C1GALT1 modified the O-glycans on integrin ß1 and regulated its activity. The glycosylation-mediated radioresistance was further inhibited by anti-integrin ß1 blocking antibody. Importantly, we also observed that core 1-type O-glycans expression was correlated with advanced tumor stage, metastasis, and poor survival of laryngeal carcinoma patients. These findings suggest that altered O-glycosylation can lead to the inherent radioresistance and progression, and therefore may be important for enhancing the efficacy of radiotherapy in laryngeal carcinoma.

Differential effect of platelet-rich plasma fractions on ß1-integrin signaling, collagen biosynthesis, and prolidase activity in human skin fibroblasts.

The study was conducted to evaluate the effects of platelet-rich plasma (PRP), supernatant of PRP (SPRP) obtained by centrifugation, and supernatant of activated PRP (SActi-PRP) obtained by Ca2+ solution-treated PRP on collagen biosynthesis, prolidase activity, and ß1-integrin signaling in cultured human skin fibroblasts. Incubation of fibroblasts with 5% PRP for 24 h contributed to ~5-fold increase in collagen biosynthesis compared to the control. In the cells treated with 5% of SPRP or SActi-PRP, collagen biosynthesis showed a 3-fold increase of the control. PRP, SPRP, and SActi-PRP stimulated prolidase activity similar to collagen biosynthesis. Collagen biosynthesis and prolidase activity are regulated by ß1-integrin receptor signaling. Incubation of fibroblasts with PRP for 24 h contributed to a dose-dependent increase in the expression of ß1-integrin receptor, while SActi-PRP increased the process to a much lower extent. SPRP had no effect on the ß1-integrin receptor expression. All the studied fractions of blood increased the expression of FAK as well as the expression of phosphorylated MAP-kinases. However, PRP was found to be the most effective stimulator of expression of these particular kinases. These studies suggest that a complex of factors, including growth factors, adhesion molecules, and prolidase contained in PRP, all evoke growth and collagen-promoting activities in human dermal fibroblasts.

Effective treatment of HER2-amplified breast cancer by targeting HER3 and ß1 integrin.

The central role of HER2 as the disease driver and HER3 as its essential partner has made them rational targets for the treatment of HER2-amplifed breast cancers, and there is considerable interest in developing highly effective treatment regimens for this disease that consist of targeted therapies alone. Much of these efforts are focused on dual targeting approaches, particularly dual targeting of the HER2-HER3 tumor driver complex itself, or vertical combinations that target downstream PI3K or Akt in addition to HER2. There is also potential in lateral combinations based on evidence implicating cross-talk with other membrane receptor systems, particularly integrins, and such lateral combinations can potentially involve either HER2 or HER3. We established a preclinical model of targeting HER3 using doxycycline-inducible shRNA and determined the efficacy of a ß1 integrin inhibitor in combination with targeting HER3. We report that targeting HER3 and ß1 integrin provides a particularly effective combination therapy approach for HER2-amplified cancers, surpassing the combination of HER2 and ß1 integrin targeting, and evading some of the safety concerns associated with direct HER2-targeting. This further validates HER3 as a major hub mediating the tumorigenic functions of HER2 and identifies it as a high value target for lateral combination therapy strategies.

MicroRNA-124 inhibits the progression of adjuvant-induced arthritis in rats.

OBJECTIVE: MicroRNAs (miRNAs) are small endogenous, non-coding RNAs that act as post-transcriptional regulators. We analysed the in vivo effect of miRNA-124 (miR-124, the rat analogue of human miR-124a) on adjuvant-induced arthritis (AIA) in rats. METHODS: AIA was induced in Lewis rats by injecting incomplete Freund's adjuvant with heat-killed Mycobacterium tuberculosis. Precursor (pre)-miR-124 was injected into the right hind ankle on day 9. Morphological changes in the ankle joint were assessed by micro-CT and histopathology. Cytokine expression was examined by western blotting and real-time RT-PCR. The effect of miR-124 on predicted target messenger RNAs (mRNAs) was examined by luciferase reporter assays. The effect of pre-miR-124 or pre-miR-124a on the differentiation of human osteoclasts was examined by tartrate-resistant acid phosphatase staining. RESULTS: We found that miR-124 suppressed AIA in rats, as demonstrated by decreased synoviocyte proliferation, leucocyte infiltration and cartilage or bone destruction. Osteoclast counts and expression level of receptor activator of the nuclear factor κB ligand (RANKL), integrin ß1 (ITGB1) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were reduced in AIA rats treated with pre-miR-124. Luciferase analysis showed that miR-124 directly targeted the 3'UTR of the rat NFATc1, ITGB1, specificity protein 1 and CCAAT/enhancer-binding protein α mRNAs. Pre-miR-124 also suppressed NFATc1 expression in RAW264.7 cells. Both miR-124 and miR-124a directly targeted the 3'-UTR of human NFATc1 mRNA, and both pre-miR-124 and pre-miR-124a suppressed the differentiation of human osteoclasts. CONCLUSIONS: We found that miR-124 ameliorated AIA by suppressing critical prerequisites for arthritis development, such as RANKL and NFATc1. Thus, miR-124a is a candidate for therapeutic use for human rheumatoid arthritis.

Bisphenol a exposure promotes the migration of NCM460 cells via estrogen receptor-mediated integrin ß1/MMP-9 pathway.

Bisphenol A (BPA) is a widely used industrial chemical and also an environmental endocrine disruptor (EED), which serves as a monomer in the manufacture of polycarbonate plastics. BPA enters human body mainly through oral intake, and has been reported as being linked to oncogenesis in many tissues. However, the association of BPA intake with gastrointestinal cancer, such as colon cancer, has received less attention. The present study was conducted to investigate the effects of BPA on the migration of normal colon epithelial cells (NCM460 cells) and further elucidate the underlying mechanisms. Our data showed that 1 × 10(-8) M (equivalent to environmental concentration) of BPA potently promoted the migration of NCM460 cells. Interestingly, BPA treatment induced an increase of integrin ß1 expression, and the functional blocking of integrin ß1 abolished the migration-promoting effects of BPA. Moreover, the results showed that it was estrogen receptor ß but not estrogen receptor α that was involved in this migration promotion. In addition, cellular exposure of BPA stimulated the expression and activity of MMP-9, a well-known factor of cell migration. Taken together, these results indicate that environmental concentration of BPA exposure promotes cell migration through activating ERß-mediated integrin ß1/MMP-9 pathway, suggesting exposure to BPA in the colon may present a potential cancer risk. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 799-807, 2016.

MAP4K4 regulates integrin-FERM binding to control endothelial cell motility.

Cell migration is a stepwise process that coordinates multiple molecular machineries. Using in vitro angiogenesis screens with short interfering RNA and chemical inhibitors, we define here a MAP4K4-moesin-talin-ß1-integrin molecular pathway that promotes efficient plasma membrane retraction during endothelial cell migration. Loss of MAP4K4 decreased membrane dynamics, slowed endothelial cell migration, and impaired angiogenesis in vitro and in vivo. In migrating endothelial cells, MAP4K4 phosphorylates moesin in retracting membranes at sites of focal adhesion disassembly. Epistasis analyses indicated that moesin functions downstream of MAP4K4 to inactivate integrin by competing with talin for binding to ß1-integrin intracellular domain. Consequently, loss of moesin (encoded by the MSN gene) or MAP4K4 reduced adhesion disassembly rate in endothelial cells. Additionally, α5ß1-integrin blockade reversed the membrane retraction defects associated with loss of Map4k4 in vitro and in vivo. Our study uncovers a novel aspect of endothelial cell migration. Finally, loss of MAP4K4 function suppressed pathological angiogenesis in disease models, identifying MAP4K4 as a potential therapeutic target.

Simultaneous ß1 integrin-EGFR targeting and radiosensitization of human head and neck cancer.

BACKGROUND: Signaling from integrins and receptor tyrosine kinases (RTKs) contributes substantially to therapy resistance of malignant tumors. We investigated simultaneous ß1 integrin-epidermal growth factor receptor (EGFR) targeting plus radiotherapy in human head and neck squamous cell carcinomas (HNSCCs). METHODS: Ten HNSCC cell lines were grown in three-dimensional laminin-rich extracellular matrix cell cultures and two of them as tumor xenografts in nude mice (n = 12-16 per group). Targeting of ß1 integrin and EGFR with monoclonal inhibitory antibodies (AIIB2 and cetuximab, respectively) was combined with x-ray irradiation. Clonogenic survival, tumor growth, and tumor control (evaluated by Kaplan-Meier analysis), apoptosis, phosphoproteome (interactome, network betweeness centrality analysis), receptor expression (immunohistochemistry), and downstream signaling (western blotting) were assessed. Various mutants of the integrin signaling mediator focal adhesion kinase (FAK) were employed for mechanistic studies. All statistical tests were two-sided. RESULTS: Compared with ß1 integrin or EGFR single inhibition, combined ß1 integrin-EGFR targeting resulted in enhanced cytotoxicity and radiosensitization in eight out of 10 tested HNSCC cell lines, which responded with an FAK dephosphorylation after ß1 integrin inhibition. In vivo, simultaneous anti-ß1 integrin/anti-EGFR treatment and radiotherapy of UTSCC15 responder xenografts enabled better tumor control compared with anti-EGFR monotherapy and irradiation (hazard ratio [HR] = 6.9, 95% confidence interval [CI] = 1.6 to 30.9, P = .01), in contrast to the SAS nonresponder tumor model (HR = 0.9, 95% CI = 0.4 to 2.3, P = .83). Mechanistically, a protein complex consisting of FAK- and Erk1-mediated prosurvival signals for radiation resistance, which was effectively compromised by ß1 integrin and EGFR blocking. CONCLUSIONS: Concomitant targeting of ß1 integrin and EGFR seems a powerful and promising approach to overcome radioresistance of HNSCCs.

Interleukin-8 induces DNA synthesis, migration and down-regulation of cleaved caspase-3 in cultured human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Migration of the junctional epithelium occurs in association with the formation of a periodontal pocket. Although the migration of junctional epithelium is known to be related to the proliferation and migration of gingival junctional epithelial cells, the mechanism has not been clarified. In patients with periodontitis, the levels of interleukin-8 (IL-8) in both gingival tissue and gingival crevicular fluid are dramatically increased. IL-8 has broad bioactive functions. In this study, we examined the role of IL-8 in DNA synthesis, migration and protection against apoptosis in cultured human gingival epithelial cells (HGEC). MATERIAL AND METHODS: DNA synthesis was estimated by measuring the incorporation of bromodeoxyuridine. The migration of gingival epithelial cells was assessed in a wound-healing assay. The expression of integrin beta-1 was analyzed using immunofluorescence confocal microscopy and western blotting. Cleaved caspase-3 was detected using western blotting and a Caspase-Glo assay kit. RESULTS: IL-8 increased the synthesis of DNA in HGEC, and the maximal effect was seen at 25 or 50 ng/mL of IL-8. In addition, 50 ng/mL of IL-8 induced cell migration, and a neutralizing antibody of integrin beta-1 inhibited the migration. IL-8 also activated expression of integrin beta-1. Furthermore, IL-8 reduced the Aggregatibacter actinomycetemcomitans-induced increase in caspase-3 expression in HGEC. CONCLUSION: IL-8 may facilitate the migration of gingival junctional epithelium by enhancing DNA synthesis, migration and preventing apoptosis of gingival epithelial cells.

Effects of low-intensity pulsed ultrasound on the expression of cyclooxygenase-2 in mandibular condylar chondrocytes.

AIMS: To determine the effect of low-intensity pulsed ultrasound (LIPUS) on cyclooxygenase-2 (COX-2) expression and related mechanisms by using cultured articular chondrocytes derived from porcine mandibular condyles after treatment with interleukin-1 beta (IL-1ß). METHODS: Chondrocytes were derived from porcine mandibular condylar cartilage and cultured. The cells were treated with or without 10 ng/mL IL-1ß. At the same time, the cells were exposed to LIPUS for 20 minutes. After LIPUS exposure, the conditioned medium was changed to a fresh one without IL-1ß, and the cells were incubated for 0 to 24 hours. The effects of LIPUS on IL-1ß-treated chondrocytes were examined in terms of the expression of p-integrin ß1, COX-2, and phosphorylated extracellular signal-related kinase (p-ERK) 1/2 by real-time polymerase chain reaction (PCR) and Western blot analyses. Differences in the means among multiple groups were examined by one-way analysis of variance (ANOVA) for all groups at each time point, followed by a Scheffé multiple comparison test as a post-hoc test; Student t test was also used. RESULTS: COX-2 mRNA level was upregulated by the treatment with IL-1ß and was suppressed significantly (P < .01) by LIPUS exposure. Furthermore, LIPUS enhanced gene expression and phosphorylation of integrin ß, and it inhibited the expression of p-ERK 1/2. CONCLUSION: LIPUS exposure inhibited IL-1ß-induced COX-2 expression through the integrin ß1 receptor followed by the phosphorylation of ERK 1/2. Despite the restricted duration of its effect, LIPUS is suggested to be a potential candidate as a preventive and auxiliary treatment to suppress the degradation of articular chondrocytes in temporomandibular joint osteoarthritis.