Jasmina Makarević

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

BackgroundTumor 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.MethodsTumor 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.ResultsAdhesion 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 MMFs effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes.ConclusionWe 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.

Acetylation of histone H3 prevents resistance development caused by chronic mTOR inhibition in renal cell carcinoma cells

Chronic mTOR inhibition may induce resistance development in renal cell carcinoma (RCC). We analyzed whether long-term exposure of RCC cells to the mTOR-inhibitor RAD001 evokes resistance and whether additional targeting histone deacetylases (HDAC) by valproic acid (VPA) overcomes RAD001 resistance. It is demonstrated that responsiveness to either drug alone is lost over time, evidenced by increased cell growth, proliferation and de-differentiation. However, drug sensitivity was conserved when RAD001 and VPA were applied in concert. Molecular analysis particularly revealed strong re-activation of Akt under chronic RAD001 or diminished histone H3 acetylation under chronic VPA single drug exposure. Combined drug application did not inactivate Akt but rather resulted in H3 acetylation remaining high while RCC cell growth was still reduced. siRNA experiments revealed that histone H3 acetylation is responsible for preserving drug sensitivity in RCCs.

Resistance after Chronic Application of the HDAC-Inhibitor Valproic Acid Is Associated with Elevated Akt Activation in Renal Cell Carcinoma In Vivo

Targeted drugs have significantly improved the therapeutic options for advanced renal cell carcinoma (RCC). However, resistance often develops, negating the benefit of these agents. In the present study, the molecular mechanisms of acquired resistance towards the histone deacetylase (HDAC) inhibitor valproic acid (VPA) in a RCC in vivo model were investigated. NMRI:nu/nu mice were transplanted with Caki-1 RCC cells and then treated with VPA (200 mg/kg/day). Controls remained untreated. Based on tumor growth dynamics, the mice were divided into “responders” and “non-responders” to VPA. Histone H3 and H4 acetylation and expression of cell signaling and cell cycle regulating proteins in the RCC mouse tumors were evaluated by Western blotting. Tumor growth of VPA responders was significantly diminished, whereas that of VPA non-responders even exceeded control values. Cdk1, 2 and 4 proteins were strongly enhanced in the non-responders. Importantly, Akt expression and activity were massively up-regulated in the tumors of the VPA non-responders. Chronic application (12 weeks) of VPA to Caki-1 cells in vitro evoked a distinct elevation of Akt activity and cancer cells no longer responded with cell growth reduction, compared to the short 2 week treatment. We assume that chronic use of an HDAC-inhibitor is associated with (re)-activation of Akt, which may be involved in resistance development. Consequently, combined blockade of both HDAC and Akt may delay or prevent drug resistance in RCC.

The cdk1-cyclin B complex is involved in everolimus triggered resistance in the PC3 prostate cancer cell line

The growth potential of PC3 prostate cancer cells, sensible (PC3(par)) or resistant (PC3(res)) to the mTOR inhibitor everolimus (RAD001) was investigated. Cell growth and proliferation of PC3(res) was similar to that of PC3(par), and late apoptosis increased in PC3(par) but decreased in PC3(res) following treatment with low dosed everolimus. PC3(res) accumulated in the G2/M-phase, accompanied by cdk1, cdk2 and cyclin B elevation. Knocking down cdk1 or cyclin B distinctly blocked the growth activity of PC3(res). One reason for everolimus resistance may be up-regulation of the cdk1-cyclin B complex in prostate cancer cells, leading to enhanced progression towards G2/M.

CCL2 Chemokine as a Potential Biomarker for Prostate Cancer: A Pilot Study

Purpose Prostate specific antigen is not reliable in diagnosing prostate cancer (PCa), making the identification of novel, precise diagnostic biomarkers important. Since chemokines are associated with more aggressive disease and poor prognosis in diverse malignancies, we aimed to investigate the diagnostic relevance of chemokines in PCa. Materials and Methods Preoperative and early postoperative serum samples were obtained from 39 consecutive PCa patients undergoing radical prostatectomy. Serum from 15 healthy volunteers served as controls. Concentrations of CXCL12, CXCL13, CX3CL1, CCL2, CCL5, and CCL20 were measured in serum by Luminex. The expression activity of CXCR3, CXCR4, CXCR5, CXCR7, CXCL12, CXCL13, CX3CR1, CXCL1, CCR2, CCR5, CCR6, CCR7, CCL2, and CCL5 mRNA was assessed in tumor and adjacent normal tissue of prostatectomy specimens by quantitative real-time polymerase chain reaction. The associations of these chemokines with clinical and histological parameters were tested. Results The gene expression activity of CCL2 and CCR6 was significantly higher in tumor tissue compared to adjacent normal tissue. CCL2 was also significantly higher in the blood samples of PCa patients, compared to controls. CCL5, CCL20, and CX3CL1 were lower in patient serum, compared to controls. CCR2 tissue mRNA was negatively correlated with the Gleason score and grading. Conclusion Chemokines are significantly modified during tumorigenesis of PCa, and CCL2 is a promising diagnostic biomarker.

Molecular targeting of prostate cancer cells by a triple drug combination down-regulates integrin driven adhesion processes, delays cell cycle progression and interferes with the cdk-cyclin axis

BackgroundSingle drug use has not achieved satisfactory results in the treatment of prostate cancer, despite application of increasingly widespread targeted therapeutics. In the present study, the combined impact of the mammalian target of rapamycin (mTOR)-inhibitor RAD001, the dual EGFr and VGEFr tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on prostate cancer growth and adhesion in vitro was investigated.MethodsPC-3, DU-145 and LNCaP cells were treated with RAD001, AEE788 or VPA or with a RAD-AEE-VPA combination. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT-assay, flow cytometry and western blotting, respectively. Furthermore, tumor cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins as well as migratory properties of the cells was evaluated, and integrin α and β subtypes were analyzed. Finally, effects of drug treatment on cell signaling pathways were determined.ResultsAll drugs, separately applied, reduced tumor cell adhesion, migration and growth. A much stronger anti-cancer effect was evoked by the triple drug combination. Particularly, cdk1, 2 and 4 and cyclin B were reduced, whereas p27 was elevated. In addition, simultaneous application of RAD001, AEE788 and VPA altered the membranous, cytoplasmic and gene expression pattern of various integrin α and β subtypes, reduced integrin-linked kinase (ILK) and deactivated focal adhesion kinase (FAK). Signaling analysis revealed that EGFr and the downstream target Akt, as well as p70S6k was distinctly modified in the presence of the drug combination.ConclusionsSimultaneous targeting of several key proteins in prostate cancer cells provides an advantage over targeting a single pathway. Since strong anti-tumor properties became evident with respect to cell growth and adhesion dynamics, the triple drug combination might provide progress in the treatment of advanced prostate cancer.

Mycophenolate mofetil increases adhesion capacity of tumor cells in vitro.

Background. The immunosuppressive drug mycophenolate mofetil (MMF) reduces expression of the heterophilic binding elements intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and thereby prevents attachment of alloactivated leukocytes to donor endothelium. The authors speculated that MMF might further diminish receptors of the immunoglobulin superfamily which, however, act as homophilic binding elements. Because decrease of homophilic adhesion receptors correlates with tumor dissemination and metastasis, MMF could trigger development or recurrence of neoplastic tumors. Methods. The authors analyzed the influence of MMF on homotypic adhesion receptors and its consequence for tumor cell attachment to an endothelial cell monolayer. Neuroblastoma (NB) cells, which self-aggregate by means of the homophilic-binding element neural cell adhesion molecule (NCAM), were used. Effects of MMF on the 140- and 180-kDa NCAM isoforms were investigated quantitatively by flow cytometry, Western blot, and reverse-transcriptase (RT) polymerase chain reaction (PCR). The relevance of NCAM for tumor cell binding was proven by treating NB with NCAM antisense oligonucleotides. Results. MMF profoundly increased the number of adherent NB cells, with a maximum effect at 0.1 &mgr;M, compared with controls. Decrease of NCAM on the cell surface was detected by flow cytometry. Western blot and RT-PCR demonstrated reduced protein and RNA levels of the 140- and 180-kDa isoforms. Treatment of NB cells with NCAM antisense oligonucleotides showed that reduced NCAM expression leads to enhanced tumor cell adhesion. Conclusions. MMF decreases NCAM receptors, which is associated with enhanced tumor cell invasiveness. The authors conclude that an MMF-based immunosuppressive regimen might increase the risk of tumor metastasis if this process is predominantly conveyed by means of homophilic adhesion proteins.

Low dosed interferon alpha augments the anti-tumor potential of histone deacetylase inhibition on prostate cancer cell growth and invasion.

We evaluated whether low‐dosed interferon alpha (IFNa) may augment the anti‐tumor potential of the histone deacetylase (HDAC)‐inhibitor valproic acid (VPA) on prostate cancer cells in vitro and in vivo. PC‐3, DU‐145, or LNCaP prostate cancer cells were treated with VPA (1 mM), IFNa (200 U/ml), or with the VPA‐IFNa combination. Tumor cell growth, cell cycle progression, and cell cycle regulating proteins were then investigated by the MTT assay, flow cytometry, and western blotting. Tumor cell adhesion to endothelium or to immobilized extracellular matrix proteins, as well as migratory properties of the cells, were evaluated. Integrin α and β adhesion molecules and alterations of cell signaling pathways were analyzed. Finally, effects of the drug treatment on prostate cancer growth in vivo were determined in the NOD/SCID mouse model. VPA reduced tumor cell adhesion, migration, and growth in vitro. A much stronger anti‐cancer potential was evoked by the VPA‐IFNa combination, although IFNa in itself did not block growth or adhesion. The same effect was seen when tumor growth was evaluated in vivo. Molecular analysis revealed distinct elevation of histone H3 acetylation caused by VPA which was further up‐regulated by VPA‐IFNa, whereas IFNa alone did not alter H3 acetylation. The combinatorial benefit became obvious in Akt phosphorylation, p21 and p27 and integrin α1, α3, and β1 expression. Application of low‐dosed IFNa to a VPA based regimen profoundly boosts the anti‐tumor properties of VPA. The combined use of VPA and low‐dosed IFNa may therefore be an innovative option in treating advanced prostate cancer. Prostate 72:1719–1735, 2012.

Amygdalin blocks bladder cancer cell growth in vitro by diminishing cyclin A and cdk2.

Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25–10 mg/ml) on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP). Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR) related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalins practical value as an anti-tumor drug.

Molecular analysis of sunitinib resistant renal cell carcinoma cells after sequential treatment with RAD001 (everolimus) or sorafenib

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 μM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 μM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short‐term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross‐resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR‐inhibitor for second‐line therapy could be the strategy of choice after first‐line sunitinib failure.