Tyrosine-kinase inhibition results in EGFR clustering at focal adhesions and consequent exocytosis in uPAR down-regulated cells of Head and Neck cancers

<p>Abstract</p> <p>Background</p> <p>Antisense (AS) induced down-regulation of uPAR in ACCS adenoid-cyctic carcinoma cells decreased the cellular adhesion and invasion on various extracellular matrices. Additionally, ACCS-AS cells showed an increased EGFR expression and other behavioral similarities to NA-SCC, a typical highly proliferative but less invasive squamous cell carcinoma (SCC) cell line of the head and neck. ACCS, ACCS-AS and NA-SCC cells were used to elucidate the relationships between uPAR down-regulation and EGFR inhibition.</p> <p>Results</p> <p>Tyrosine kinase inhibitor Gefitinib (IRESSA, ZD 1839) significantly reduced the chemotactic cell migration and adhesion. This was associated with reduced EGFR and ERK activation. In addition, anti-proliferative effect of gefitinib in uPAR down-regulated ACCS-AS was significantly higher than parental ACCS, to levels comparable to gefitinib-sensitive NA-SCC cells. This was evidenced by both reduced dosage and duration of treatment. Furthermore, time-lapse videography showed that treatment with gefitinib was also associated with cell rounding and loss of pseudopodia, mostly in ACCS-AS rather than parental ACCS cells. There were also evidences of formation and exocytosis of vacuole-like structures in ACCS-AS, as well as NA-SCC, but not in parental ACCS cells. Interestingly, immunocytochemistry showed that the exocytotic vacuoles actually contained de-activated EGFR.</p> <p>Conclusion</p> <p>Our results suggested that down-regulation of uPAR affected the fate of EGFR in high EGFR expressing cells. Furthermore, combining the uPAR down-regulation with EGFR inhibition showed a synergistic anti-tumor effect and might provide an alternative method to increase anti-proliferative effect of tyrosine kinase inhibitors with lower doses and duration to reduce their side effects during cancer control.</p

Impaired germ cell development due to compromised cell cycle progression in Skp2-deficient mice

BACKGROUND: The gonads are responsible for the production of germ cells through both mitosis and meiosis. Skp2 is the receptor subunit of an SCF-type ubiquitin ligase and is a major regulator of the progression of cells into S phase of the cell cycle, which it promotes by mediating the ubiquitin-dependent degradation of p27, an inhibitor of cell proliferation. However, the role of the Skp2-p27 pathway in germ cell development remains elusive. RESULTS: We now show that disruption of Skp2 in mice results in a marked impairment in the fertility of males, with the phenotypes resembling Sertoli cell-only syndrome in men. Testes of Skp2(-/- )mice manifested pronounced germ cell hypoplasia accompanied by massive apoptosis in spermatogenic cells. Flow cytometry revealed an increased prevalence of polyploidy in spermatozoa, suggesting that the aneuploidy of these cells is responsible for the induction of apoptosis. Disruption of the p27 gene of Skp2(-/- )mice restored germ cell development, indicating that the testicular hypoplasia of Skp2(-/- )animals is attributable to the antiproliferative effect of p27 accumulation. CONCLUSION: Our results thus suggest that compromised cell cycle progression caused by the accumulation of p27 results in aneuploidy and the induction of apoptosis in gonadal cells of Skp2(-/- )mice. The consequent reduction in the number of mature gametes accounts for the decreased fertility of these animals. These findings reinforce the importance of the Skp2-p27 pathway in cell cycle regulation and in germ cell development

Effect of combined treatment with alendronate and calcitriol on femoral neck strength in osteopenic rats

<p>Abstract</p> <p>Background</p> <p>Hip fracture is associated with pronounced morbidity and excess mortality in elderly women with postmenopausal osteoporosis. Many drugs have been developed to treat osteoporosis and to reduce the risk of osteoporotic fractures. We investigated the effects of combined alendronate and vitamin D₃treatment on bone mass and fracture load at the femoral neck in ovariectomized (OVX) rats, and evaluated the relationship between bone mass parameters and femoral neck strength.</p> <p>Methods</p> <p>Thirty 12-week-old female rats underwent either a sham-operation (n = 6) or OVX (n = 24). Twenty weeks later, OVX rats were further divided into four groups and received daily doses of either saline alone, 0.1 mg/kg alendronate, 0.1 μg/kg calcitriol, or a combination of both two drugs by continuous infusion via Alzet mini-osmotic pumps. The sham-control group received saline alone. After 12 weeks of treatment, femoral necks were examined using peripheral quantitative computed tomography (pQCT) densitometry and mechanical testing.</p> <p>Results</p> <p>Saline-treated OVX rats showed significant decreases in total bone mineral content (BMC) (by 28.1%), total bone mineral density (BMD) (by 9.5%), cortical BMC (by 26.3%), cancellous BMC (by 66.3%), cancellous BMD (by 29.0%) and total cross-sectional bone area (by 30.4%) compared with the sham-control group. The combined alendronate and calcitriol treatments improved bone loss owing to estrogen deficiency. On mechanical testing, although OVX significantly reduced bone strength of the femoral neck (by 29.3%) compared with the sham-control group, only the combined treatment significantly improved the fracture load at the femoral neck in OVX rats to the level of the sham-controls. The correlation of total BMC to fracture load was significant, but that of total BMD was not.</p> <p>Conclusion</p> <p>Our results showed that the combined treatment with alendronate and calcitriol significantly improved bone fragility of the femoral neck in OVX osteopenic rats.</p

Small interfering RNA library screen identified polo-like kinase-1 (PLK1) as a potential therapeutic target for breast cancer that uniquely eliminates tumor-initiating cells

Introduction: Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs), molecularly defined as being CD44high/CD24-/low. TICs are resilient to chemotherapy and radiation. However, no currently accepted molecular target exists against TNBC and, moreover, TICs. Therefore, we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs. Methods: A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44high subpopulation and sorted CD44high/CD24-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1), including its expression in breast cancer cell lines, effect on the CD44high/CD24-/low TIC subpopulation, growth inhibition, mammosphere formation, and apoptosis, as well as the activity of the PLK1 inhibitor, BI 2536. Results: Of the 85 kinases identified in the screen, 28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC), BT474-M1 (ER+/HER2+, a metastatic variant), and HR5 (ER+/HER2+, a trastuzumab-resistant model) cells and showed a broad spectrum of growth inhibition. Importantly, 12 of 28 kinases also reduced the CD44high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44high/CD24-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines, representing all of the breast cancer subtypes, and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth, mammosphere formation, and apoptosis as did PLK1 siRNAs. Finally, whereas paclitaxel, doxorubicin, and 5-fluorouracil enriched the CD44high/CD24-/low population compared with control in SUM149, subsequent treatment with BI 2536 killed the emergent population, suggesting that it could potentially be used to prevent relapse. Conclusion: Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44high/CD24-/low TIC subpopulation and mammosphere formation. Thus, PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer.Experimental Medicine, Division ofMedical Genetics, Department ofMedicine, Department ofMedicine, Faculty ofPediatrics, Department ofOther UBCReviewedFacult

Gefitinib led to reduced spreading and cell rounding which was more obvious in ACCS-AS cells

There was also vacuolar formation observed as early as 10 minutes in ACCS-AS cell lines which continued and affected most of cell population with time. Representative vacuole formations on ACCS-AS cells (red arrowheads) are shown. Time-lapse videography also showed that formed vacuoles were exocytosed (see supplemented data). An example of masses speculated to be already exocytosed vacuole sacs are shown (green arrowheads).<p><b>Copyright information:</b></p><p>Taken from "Tyrosine-kinase inhibition results in EGFR clustering at focal adhesions and consequent exocytosis in uPAR down-regulated cells of Head and Neck cancers"</p><p>http://www.molecular-cancer.com/content/7/1/47</p><p>Molecular Cancer 2008;7():47-47.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2464604.</p><p></p

Gefitinib inhibited the phosphorylation of EGFR and ERK and reduced Cyclin D1 expression in all cells

Total EGFR and total ERK are also shown for comparison. β-actin western blots and Coomassie blue staining of gels were used for confirmation of equal loading.<p><b>Copyright information:</b></p><p>Taken from "Tyrosine-kinase inhibition results in EGFR clustering at focal adhesions and consequent exocytosis in uPAR down-regulated cells of Head and Neck cancers"</p><p>http://www.molecular-cancer.com/content/7/1/47</p><p>Molecular Cancer 2008;7():47-47.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2464604.</p><p></p

The effects of increasing concentrations of gefitinib on growth of ACCS, ACCS-AS and NA-SCC cells are shown

Cells were incubated with vehicle, ., non-stimulated, or increasing concentration of gefitinib for 24, 48, and 72 hours. Comparison of cellular growth in response to gefitinib (3.75 μM), EGF (100 ng/ml) and vehicle ., non-stimulated. The represent SD of results of separate wells. * p < 0.05, versus similar dosage in parental ACCS cells at 24 and 48 hrs. ** p < 0.05, versus similar dosage in ACCS cells at 24 and 48 hrs.<p><b>Copyright information:</b></p><p>Taken from "Tyrosine-kinase inhibition results in EGFR clustering at focal adhesions and consequent exocytosis in uPAR down-regulated cells of Head and Neck cancers"</p><p>http://www.molecular-cancer.com/content/7/1/47</p><p>Molecular Cancer 2008;7():47-47.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2464604.</p><p></p

RT- PCR confirmed the uPAR down-regulation in ACCS-AS cells and constitutively low uPAR expression in NA-SCC cells

ACCS-AS cells showed marked increase in mRNA expression of EGFR, compared to the parental ACCS cells. Constitutive high expression of EGFR in NA-SCC cells is also shown.<p><b>Copyright information:</b></p><p>Taken from "Tyrosine-kinase inhibition results in EGFR clustering at focal adhesions and consequent exocytosis in uPAR down-regulated cells of Head and Neck cancers"</p><p>http://www.molecular-cancer.com/content/7/1/47</p><p>Molecular Cancer 2008;7():47-47.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2464604.</p><p></p