Mycophenolate mofetil modulates adhesion receptors of the beta1 integrin family on tumor cells: impact on tumor recurrence and malignancy

BACKGROUND: Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. METHODS: Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. RESULTS: Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. CONCLUSION: We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype

Mechanisms behind temsirolimus resistance causing reactivated growth and invasive behavior of bladder cancer cells in vitro

Background: Although mechanistic target of rapamycin (mTOR) inhibitors, such as temsirolimus, show promise in treating bladder cancer, acquired resistance often hampers efficacy. This study evaluates mechanisms leading to resistance. Methods: Cell growth, proliferation, cell cycle phases, and cell cycle regulating proteins were compared in temsirolimus resistant (res) and sensitive (parental—par) RT112 and UMUC3 bladder cancer cells. To evaluate invasive behavior, adhesion to vascular endothelium or to immobilized extracellular matrix proteins and chemotactic activity were examined. Integrin α and β subtypes were analyzed and blocking was done to evaluate physiologic integrin relevance. Results: Growth of RT112res could no longer be restrained by temsirolimus and was even enhanced in UMUC3res, accompanied by accumulation in the S- and G2/M-phase. Proteins of the cdk-cyclin and Akt-mTOR axis increased, whereas p19, p27, p53, and p73 decreased in resistant cells treated with low-dosed temsirolimus. Chemotactic activity of RT112res/UMUC3res was elevated following temsirolimus re-exposure, along with significant integrin α2, α3, and β1 alterations. Blocking revealed a functional switch of the integrins, driving the resistant cells from being adhesive to being highly motile. Conclusion: Temsirolimus resistance is associated with reactivation of bladder cancer growth and invasive behavior. The α2, α3, and β1 integrins could be attractive treatment targets to hinder temsirolimus resistance

Combining the receptor tyrosine kinase inhibitor AEE788 and the mammalian target of rapamycin (mTOR) inhibitor RAD001 strongly inhibits adhesion and growth of renal cell carcinoma cells

Background Treatment options for metastatic renal cell carcinoma (RCC) are limited due to resistance to chemo- and radiotherapy. The development of small-molecule multikinase inhibitors have now opened novel treatment options. The influence of the receptor tyrosine kinase inhibitor AEE788, applied alone or combined with the mammalian target of rapamycin (mTOR) inhibitor RAD001, on RCC cell adhesion and proliferation in vitro has been evaluated. Methods RCC cell lines Caki-1, KTC-26 or A498 were treated with various concentrations of RAD001 or AEE788 and tumor cell proliferation, tumor cell adhesion to vascular endothelial cells or to immobilized extracellular matrix proteins (laminin, collagen, fibronectin) evaluated. The anti-tumoral potential of RAD001 combined with AEE788 was also investigated. Both, asynchronous and synchronized cell cultures were used to subsequently analyze drug induced cell cycle manipulation. Analysis of cell cycle regulating proteins was done by western blotting. Results RAD001 or AEE788 reduced adhesion of RCC cell lines to vascular endothelium and diminished RCC cell binding to immobilized laminin or collagen. Both drugs blocked RCC cell growth, impaired cell cycle progression and altered the expression level of the cell cycle regulating proteins cdk2, cdk4, cyclin D1, cyclin E and p27. The combination of AEE788 and RAD001 resulted in more pronounced RCC growth inhibition, greater rates of G0/G1 cells and lower rates of S-phase cells than either agent alone. Cell cycle proteins were much more strongly altered when both drugs were used in combination than with single drug application. The synergistic effects were observed in an asynchronous cell culture model, but were more pronounced in synchronous RCC cell cultures. Conclusions Potent anti-tumoral activitites of the multikinase inhibitors AEE788 or RAD001 have been demonstrated. Most importantly, the simultaneous use of both AEE788 and RAD001 offered a distinct combinatorial benefit and thus may provide a therapeutic advantage over either agent employed as a monotherapy for RCC treatment

Molekulare Mechanismen im Blasen-Karzinom unter Temsirolimus-Resistenz

Primäre Tumore werden nach ihrem Entstehungsort benannt. Selbst Metastasen zeigen eine gewisse Ähnlichkeit mit ihrem ursprünglichen Gewebe. Somit gehören alle Geschwülste, die ursprünglich der Harnblase entstammen, zu den Harnblasenkarzinomen. Das Harnblasenkarzinom geht meist (90-95%) von der Schleimhaut der ableitenden Harnwege aus und wird als Urothel bezeichnet. Dementsprechend haben die meisten Patienten mit der Diagnose Blasenkarzinom ein Urothel-Blasenkarzinom. Die restlichen Blasenkarzinome entfallen auf Adenokarzinome, Plattenepithelkarzinome, kleinzellige Karzinome, Sarkome, Paragangliome, Melanome oder Lymphome (Humphrey A. et al. 2016, Moch H. et al 2016). Die Urothel-Blasenkarzinome können sowohl flach, als auch warzenförmig wachsen. Je nach Diagnostik „oberflächlich“ oder „muskelinvasiv“ lassen sich die Urothel-Blasenkarzinome in zwei Hauptgruppen unterteilen; Etwa 70% der Erkrankten haben dabei ein oberflächliches Urothel-Blasenkarzinom, das auf die Blasenschleimhaut begrenzt ist und durch eine Basistherapie, sog. Transurethrale Resektion (TUR-B) behandelt wird. Dabei werden die in der Schleimhaut gewachsenen Tumore getrennt reseziert. Therapieergänzend und/oder prophylaktisch wird die Blase danach mit einem Chemotherapeutikum gespült (intravesikale Instillation). Diese Zytostatika-Behandlung soll das Wiederauftreten eines Rezidivs verhindern bzw. eventuell verlangsamen (Iida K. et al. 2016, Celik O. et al. 2016)

Inhibition of <i>Acinetobacter baumannii</i> Biofilm Formation Using Different Treatments of Silica Nanoparticles

There exists a multitude of pathogens that pose a threat to human and public healthcare, collectively referred to as ESKAPE pathogens. These pathogens are capable of producing biofilm, which proves to be quite resistant to elimination. Strains of A. baumannii, identified by the “A” in the acronym ESKAPE, exhibit significant resistance to amoxicillin in vivo due to their ability to form biofilm. This study aims to inhibit bacterial biofilm formation, evaluate novel silica nanoparticles’ effectiveness in inhibiting biofilm, and compare their effectiveness. Amoxicillin was utilized as a positive control, with a concentration exceeding twice that when combined with silica NPs. Treatments included pure silica NPs, silica NPs modified with copper oxide (CuO.SiO2), sodium hydroxide (NaOH.SiO2), and phosphoric acid (H3PO4.SiO2). The characterization of NPs was conducted using scanning electron microscopy (SEM), while safety testing against normal fibroblast cells was employed by MTT assay. The microtiter plate biofilm formation assay was utilized to construct biofilm, with evaluations conducted using three broth media types: brain heart infusion (BHI) with 2% glucose and 2% sucrose, Loria broth (LB) with and without glucose and sucrose, and Dulbecco’s modified eagle medium/nutrient (DMEN/M). Concentrations ranging from 1.0 mg/mL to 0.06 µg/mL were tested using a microdilution assay. Results from SEM showed that pure silica NPs were mesoporous, but in the amorphous shape of the CuO and NaOH treatments, these pores were disrupted, while H3PO4 was composed of sheets. Silica NPs were able to target Acinetobacter biofilms without harming normal cells, with viability rates ranging from 61–73%. The best biofilm formation was achieved using a BHI medium with sugar supplementation, with an absorbance value of 0.35. Biofilms treated with 5.0 mg/mL of amoxicillin as a positive control alongside 1.0 mg/mL of each of the four silica treatments in isolation, resulting in the inhibition of absorbance values of 0.04, 0.13, 0.07, 0.09, and 0.08, for SiO2, CuO.SiO2, NaOH.SiO2 and H3PO4.SiO2, respectively. When amoxicillin was combined, inhibition increased from 0.3 to 0.04; NaOH with amoxicillin resulted in the lowest minimum biofilm inhibitory concentration (MBIC), 0.25 µg/mL, compared to all treatments and amoxicillin, whereas pure silica and composite had the highest MBIC, even when combined with amoxicillin, compared to all treatments, but performed better than that of the amoxicillin alone which gave the MBIC at 625 µg/mL. The absorbance values of MBIC of each treatment showed no significant differences in relation to amoxicillin absorbance value and relation to each other. Our study showed that smaller amoxicillin doses combined with the novel silica nanoparticles may reduce toxic side effects and inhibit biofilm formation, making them viable alternatives to high-concentration dosages. Further investigation is needed to evaluate in vivo activity

HDAC-inhibition counteracts everolimus resistance in renal cell carcinoma in vitro by diminishing cdk2 and cyclin A

Background: Targeted therapies have improved therapeutic options of treating renal cell carcinoma (RCC). However, drug response is temporary due to resistance development. Methods: Functional and molecular changes in RCC Caki-1 cells, after acquired resistance to the mammalian target of rapamycin (mTOR)-inhibitor everolimus (Cakires), were investigated with and without additional application of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA). Cell growth was evaluated by MTT assay, cell cycle progression and apoptosis by flow cytometry. Target molecules of everolimus and VPA, apoptotic and cell cycle regulating proteins were investigated by western blotting. siRNA blockade was performed to evaluate the functional relevance of the proteins. Results: Everolimus resistance was accompanied by significant increases in the percentage of G2/M-phase cells and in the IC50. Akt and p70S6K, targets of everolimus, were activated in Cakires compared to drug sensitive cells. The most prominent change in Cakires cells was an increase in the cell cycle activating proteins cdk2 and cyclin A. Knock-down of cdk2 and cyclin A caused significant growth inhibition in the Cakires cells. The HDAC-inhibitor, VPA, counteracted everolimus resistance in Cakires, evidenced by a significant decrease in tumor growth and cdk2/cyclin A. Conclusion: It is concluded that non-response to everolimus is characterized by increased cdk2/cyclin A, driving RCC cells into the G2/M-phase. VPA hinders everolimus non-response by diminishing cdk2/cyclin A. Therefore, treatment with HDAC-inhibitors might be an option for patients with advanced renal cell carcinoma and acquired everolimus resistance