Novel compounds with potent CDK9 inhibitory activity for the treatment of myeloma.

Cyclin-dependent kinases (CDKs) and Polo-like kinases (PLKs) play key role in the regulation of the cell cycle. The aim of our study was originally the further development of our recently discovered polo-like kinase 1 (PLK1) inhibitors. A series of new 2,4-disubstituted pyrimidine derivatives were synthesized around the original hit, but their PLK1 inhibitory activity was very poor. However the novel compounds showed nanomolar CDK9 inhibitory activity and very good antiproliferative effect on multiple myeloma cell lines (RPMI-8226).

Synthesis and characterization of amino acid substituted sunitinib analogues for the treatment of AML.

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted delivery may reduce such undesired side effects, our goal was to develop novel amino acid substituted derivatives of sunitinib which are potent candidates to be used conjugated with antibodies and peptides. In the current paper we present the synthesis, physicochemical and in vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with peptide-based delivery systems via their carboxyl group. We determined the solubility, pKa, CHI and LogP values of the compounds along with their inhibition potential against FLT3-ITD mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the growth of the MV4-11 leukemia cell line at submicromolar concentration.

Prognostic impact of B-cell density in cutaneous melanoma.

Studies on the prognostic importance of tumor-infiltrating lymphocytes have mainly focused on T cells, while little is known about the role of tumor-infiltrating B lymphocytes. We investigated the prevalence of CD20(+) B cells by immunohistochemistry in primary melanoma samples of 106 patients and analyzed in relation to clinicopathological parameters and patients' survival. The majority of samples contained a significant amount of B lymphocytes, predominantly dispersed in the stroma surrounding tumor deposits (mean peritumoral and intratumoral densities: 178.7 ± 156.1 vs. 4.9 ± 6.9 cells/mm², respectively). B cells organized in follicle-like aggregates were also observed in 26% of the samples. B-cell density correlated with that of activated (CD25(+) or OX40(+)) T lymphocytes. Infiltration by CD20(+) lymphocytes did not correlate with tumor thickness, while the presence of B-cell aggregates was observed more frequently in thick melanomas. On the other hand, B-cell infiltration was more pronounced in nonmetastatic or lymph node metastatic tumors, compared to visceral metastatic ones. Accordingly, high number of these cells provided significant survival advantage (P = 0.0391 and P = 0.0136 for intra- and peritumoral infiltration, respectively). Furthermore, combination of peritumoral B-cell density with the number of activated T lymphocytes identified patient subgroups with different disease outcome, which was most favorable in the case of high density, while very poor in the case of low density of both cell types. Multivariate survival analysis identified tumor thickness and CD20(+)/OX40(+) cell density combination as significant independent prognostic factors. Taken together, our results show correlation between low number of CD20(+) B lymphocytes and melanoma progression, indicating a possible role of tumor-infiltrating B cells in antitumoral immune response. It was also reflected in better outcome of the disease since the density of B lymphocytes alone as well as in combination with that of activated T cells proved of prognostic importance in patients with malignant melanoma.

EGFR Alterations Influence the Cetuximab Treatment Response and c-MET Tyrosine-Kinase Inhibitor Sensitivity in Experimental Head and Neck Squamous Cell Carcinomas.

Background: Anti-EGFR antibody therapy is still one of the clinical choices in head and neck squamous cell carcinoma (HNSCC) patients, but the emergence of cetuximab resistance questioned its effectiveness and reduced its applicability. Although several possible reasons of resistance against the antibody treatment and alternative therapeutic proposals have been described (EGFR alterations, activation of other signaling pathways), there is no method to predict the effectiveness of anti-EGFR antibody treatments and to suggest novel therapeutics. Our study investigated the effect of EGFR R521K alteration on efficiency of cetuximab therapy of HNSCC cell lines and tried to find alternative therapeutic approaches against the resistant cells. Methods: After genetic characterization of HNSCC cells, we chose one wild type and one R521K+ cell line for in vitro proliferation and apoptosis tests, and in vivo animal models using different therapeutic agents. Results: Although the cetuximab treatment affected EGFR signalization in both cells, it did not alter in vitro cell proliferation or apoptosis. In vivo cetuximab therapy was also ineffective on R521K harboring tumor xenografts, while blocked the tumor growth of EGFR-wild type xenografts. Interestingly, the cetuximab-resistant R521K tumors were successfully treated with c-MET tyrosine kinase inhibitor SU11274. Conclusion: Our results suggest that HNSCC cell line expressing the R521K mutant form of EGFR does not respond well to cetuximab treatment in vitro or in vivo, but hopefully might be targeted by c-MET tyrosine kinase inhibitor treatment.

Inclusion complexation of the anticancer drug pomalidomide with cyclodextrins: fast dissolution and improved solubility.

Pomalidomide (POM), a potent anticancer thalidomide analogue was characterized in terms of cyclodextrin complexation to improve its aqueous solubility and maintain its anti-angiogenic activity. The most promising cyclodextrin derivatives were selected by phase-solubility studies. From the investigated nine cyclodextrins - differing in cavity size, nature of substituents, degree of substitution and charge - the highest solubility increase was observed with sulfobutylether-ß-cyclodextrin (SBE-ß-CD). The inclusion complexation between POM and SBE-ß-CD was further characterized with a wide variety of state-of-the-art analytical techniques, such as nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), circular dichroism spectroscopy, fluorescence spectroscopy as well as X-ray powder diffraction method (XRD). Job plot titration by NMR and the AL-type phase-solubility diagram indicated 1:1 stoichiometry in a liquid state. Complementary analytical methods were employed for the determination of the stability constant of the complex; the advantages and disadvantages of the different approaches are also discussed. Inclusion complex formation was also assessed by molecular modelling study. Solid state complexation in a 1:1 M ratio was carried out by lyophilization and investigated by IR and XRD. The complex exhibited fast-dissolution with immediate release of POM, when compared to the pure drug at acidic and neutral pH. Kinetic analysis of POM release from lyophilized complex shows that Korsmeyer-Peppas and Weibull model described the best the dissolution kinetics. The cytotoxicity of the complex was tested against the LP-1 human myeloma cell line which revealed that supramolecular interactions did not significantly affect the anti-cancer activity of the drug. Overall, our results suggest that the inclusion complexation of POM with SBE-ß-CD could be a promising approach for developing more effective POM formulations with increased solubility.

High VEGFR-3-positive circulating lymphatic/vascular endothelial progenitor cell level is associated with poor prognosis in human small cell lung cancer.

PURPOSE: The newly identified bone marrow-derived cell population, called lymphatic/vascular endothelial progenitor cells (LVEPC), has been shown to contribute to lymph capillary growth in experimental tumor systems. The clinical significance of these cells has not yet been investigated in a human malignancy. Our aim was to study whether peripheral blood circulating LVEPCs participate in the progression of human small cell lung cancer (SCLC). EXPERIMENTAL DESIGN: A total of 88 patients with limited-stage SCLC and 32 tumor-free control subjects were included. Peripheral blood circulating LVEPC labeled with CD34 and vascular endothelial growth factor receptor-3 (VEGFR3) antibodies and the serum levels of the key lymphangiogenic molecule VEGF-C were measured by flow cytometry and ELISA, respectively. RESULTS: CD34-positive/VEGFR3-positive LVEPC levels were significantly increased in patients (versus controls; P<0.01), and there was also a significant relationship between LVEPC counts and lymph node metastasis (P<0.01). High pretreatment circulating LVEPC numbers correlated with poor overall survival (P<0.01). Although we observed significantly elevated VEGF-C concentrations in patients (versus controls; P<0.01), there was no significant correlation between VEGF-C and LVEPC levels. Moreover, no significant differences in peripheral blood VEGF-C levels were seen between patients subgrouped by clinicopathologic variables including tumor and lymph node stages and survival. CONCLUSIONS: Peripheral blood levels of bone marrow-derived LVEPCs are significantly increased in patients with SCLC and correlate with lymphatic involvement and prognosis. This is the first study that shows evidence of increased numbers of circulating LVEPC in patients with a malignant tumor.

[Endocrine factors influencing melanoma progression]. / A melanóma progresszióját befolyásoló endokrin tényezok.

According to recent findings that beside cancers traditionally considered as hormone-dependent, several other tumor types show different behavior in the two sexes, indicating the possible role of endocrine factors in the course of these diseases. The possibility that endocrine factors may influence the clinical course of human malignant melanoma is suggested by the higher survival rate in premenopausal vs. postmenopausal women or men of any ages. However, investigations on the sex hormone receptor status of human cutaneous melanomas and experiments attempting to support the epidemiological results yielded conflicting results. In our human melanoma cell lines we failed to detect steroid receptors at protein level, while quantitative PCR demonstrated that their mRNA expression level was orders of magnitude lower compared to the positive control cell lines. Sex hormones did not influence the in vitro features of the human melanoma cells considerably. On the other hand, glucocorticoid receptor was present both at mRNA and protein level, although dexamethasone was effective in vitro only at high doses. Our previous experiments showed that intrasplenic injection of human melanoma cells resulted in a significantly higher number of liver colonies in male than in female SCID mice. We now show that this difference evolves during the first day. After injection into the tail vein we did not observe gender-dependent difference in the efficiency of pulmonary colonization. Examining the pattern of metastasis formation after intracardiac injection, we have found differences between the two sexes in the incidence or number of colonies only in the case of the liver but not in other organs. We concluded that the observed phenomenon is specific to the liver; therefore we investigated the effects of 2-methoxyestradiol, an endogenous metabolite of estradiol produced mainly in the liver, with an estrogen receptor-independent antitumor activity. 2ME2 effectively inhibited melanoma cell proliferation by inducing apoptosis and an arrest in the G2/M phase. The mechanism of action involved microtubules, mitochondrial damage and caspase activation as well. In SCID mice, 2ME2 was effective in reducing primary tumor weight and the number of liver colonies after intrasplenic injection of human melanoma cells, and causing significantly higher rate of apoptotic cells in the colonies.

Targeted drug combination therapy design based on driver genes.

Targeted therapies against cancer types with more than one driver gene hold bright but elusive promise, since approved drugs are not available for all driver mutations and monotherapies often result in resistance. Targeting multiple driver genes in different pathways at the same time may provide an impact extensive enough to fight resistance. Our goal was to find synergistic drug combinations based on the availability of targeted drugs and their biological activity profiles and created an associated compound library based on driver gene-related protein targets. In this study, we would like to show that driver gene pattern based customized combination therapies are more effective than monotherapies on six cell lines and patient-derived primary cell cultures. We tested 55-102 drug combinations targeting driver genes and driver pathways for each cell line and found 25-85% of these combinations highly synergistic. Blocking 2-5 cancer pathways using only 2-3 targeted drugs was sufficient to reach high rates of tumor cell eradication at remarkably low concentrations. Our results demonstrate that the efficiency of cancer treatment may be significantly improved by combining drugs against multiple tumor specific drivers.

Discovery and development of extreme selective inhibitors of the ITD and D835Y mutant FLT3 kinases.

Aberrant activation of FMS-like tyrosine receptor kinase 3 (FLT3) is implicated in the pathogenesis of acute myeloid leukemia (AML) in 20-30% of patients. In this study we identified a highly selective (phenylethenyl)quinazoline compound family as novel potent inhibitors of the FLT3-ITD and FLT3-D835Y kinases. Their prominent effects were confirmed by biochemical and cellular proliferation assays followed by mice xenograft studies. Our modelling experiments and the chemical structures of the compounds predict the possibility of covalent inhibition. The most effective compounds triggered apoptosis in FLT3-ITD AML cells but had either weak or no effect in FLT3-independent leukemic and non-leukemic cell lines. Our results strongly suggest that our compounds may become therapeutics in relapsing and refractory AML disease harboring various ITD and tyrosine kinase domain mutations, by their ability to overcome drug resistance.

Circulating bone marrow-derived endothelial progenitor cells: characterization, mobilization, and therapeutic considerations in malignant disease.

Until recently, tumor vascularization was thought to occur exclusively through angiogenesis. However, recent studies using different animal models of cancer suggested the importance of bone marrow-derived endothelial progenitor cells (EPCs) (i.e. postnatal vasculogenesis) in tumor vascularization and growth. EPCs are present in the peripheral blood, their levels are increased in response to certain signals/cytokines, and they home into the neovascular bed of malignant tissues. Furthermore, at the clinical level, evidence is emerging that changes in EPC levels might predict the efficacy of anticancer drug combinations that include antiangiogenic agents. On the basis of these observations, EPCs have attractive potential diagnostic and therapeutic applications for malignant diseases. In this paper, we review biological features of EPCs and speculate on the utility of these progenitor cells for medical oncology.

Identification and clinical significance of circulating endothelial progenitor cells in human non-small cell lung cancer.

Until recently, it was generally accepted that vascularization of tumors arises exclusively from endothelial sprouting. Whether circulating bone marrow-derived endothelial progenitor cells (EPC) participate in the progression of non-small cell lung cancer (NSCLC) has not yet been evaluated. EPCs labeled with CD34, CD133, and vascular endothelial growth factor receptor-2 (VEGFR2) antibodies were counted by flow cytometry in the peripheral blood of 53 NSCLC patients. Furthermore, by means of a quantitative reverse transcription-PCR approach, we measured VEGFR2, CD133, CD34, and VE-cadherin mRNA in the peripheral blood samples of the same patient population. EPCs in tumor samples were identified by confocal microscopy using CD31, CD34, CD133, and VEGFR2 antibodies. Although immunofluorescent labeling of microvessels made clear that incorporation of EPCs is a rare phenomenon in NSCLC tissue (9 of 22 cases), circulating EPC levels before therapeutic intervention were increased in NSCLC patients (P < 0.002, versus healthy controls), and high pretreatment circulating EPC numbers correlated with poor overall survival (P < 0.001). Furthermore, in the subgroup of responders to treatment, the posttreatment EPC numbers in the peripheral blood were significantly lower compared with nonresponding patients. Interestingly, pretreatment mRNA levels of CD133, VE-cadherin, and CD34 were not significantly increased in NSCLC patients, whereas VEGFR2 expression was increased by 80-fold. Moreover, posttreatment VEGFR2 mRNA level in the peripheral blood was significantly higher in the subgroup of nonresponding patients when compared with posttreatment level of patients responding to antitumor therapy. Circulating levels of bone marrow-derived EPCs are significantly increased in NSCLC patients and correlate with clinical behavior.

Inhibition of epidermal growth factor receptor improves antitumor efficacy of vemurafenib in BRAF-mutant human melanoma in preclinical model.

Oncogenic activation of the epidermal growth factor receptor (EGFR) signaling pathway occurs in a variety of tumor types, albeit in human melanoma, the contribution of EGFR is still unclear. The potential role of EGFR was analyzed in four BRAF-mutant, one NRAS-mutant and one wild-type NRAS-BRAF-carrying human melanoma cell lines. We have tested clinically available reversible tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, irreversible EGFR-TKI pelitinib and a reversible experimental compound PD153035 on in-vitro proliferation, apoptosis, migration as well as in-vivo metastatic colonization in a spleen-liver model. The presence of the intracellular domain of EGFR protein and its constitutive activity were demonstrated in all cell lines. Efficacies of EGFR-TKIs showed significant differences, and irreversible inhibition had the strongest antitumor potential. Compared with BRAF-mutant cells, wild-type BRAF was associated with relative resistance against gefitinib. In combination with gefitinib, selective mutant BRAF-inhibitor vemurafenib showed additive effect in all BRAF-mutant cell lines. Treatment of BRAF-mutant cells with gefitinib or pelitinib attenuated in-vitro cell migration and in-vivo colonization. Our preclinical data suggest that EGFR is a potential target in the therapy of BRAF-mutant malignant melanoma; however, more benefits could be expected from irreversible EGFR-TKIs and combined treatment settings.

[Human recombinant erythropoietin-alpha increases the efficacy of irradiation in preclinical model]. / A rekombináns humán erythropoietin-alfa fokozza a humán laphámrák sugárérzékenységét preklinikai modellben.

According to recent data erythropoietin receptor (EPOR) is expressed not only by bone marrow erythroid progenitors but by endothelial- and cancer cells and it was suggested that erythropoietin (EPO) may affect their functions as well. We have analyzed the effects of recombinant human erythropoietin-alpha (rHuEPOalpha) on radiation sensitivity of EPOR+ human epidermoid carcinoma (A431) xenograft model. In vivo rHuEPOalpha treatment was started after tumor cell inoculation into SCID mice. 5 Gy irradiation was performed on day 14, the effect of which was measured on day 22. Hemoglobin level, tumor-associated microvessels and HIF-1alpha expression of the xenograft were monitored during the experiment. rHuEPOalpha administration prevented the development of tumor-induced anemia of SCID mice and reduced the level of HIF-1alpha expression of the xenograft tumor without affecting tumor growth. We have found that rHuEPOalpha treatment significantly increased the efficacy of antitumor effect of irradiation which was partly mediated by complex effects on tumor-associated microvessels. In vitro rHuEPOalpha did not affect proliferation of A431 cells but enhanced the antiproliferative and proapoptotic effects of irradiation. We concluded that rHuEPOalpha administration positively modulated the antitumoral effects of irradiation at least by two pathways, decreasing systemic hypoxia resulting in decreased tumoral HIF-1alpha expression and enhancing direct effects on tumor-associated microvessels.

Optimized conversion of antiproliferative lignans pinoresinol and epipinoresinol: Their simultaneous isolation and identification by centrifugal partition chromatography and high performance liquid chromatography.

High amount of the valuable lignan pinoresinol (PR) was determined in Carduus nutans fruit (7.8mg/g) for the first time. A preparative separation method using two consecutive, identical steps of centrifugal partition chromatography (CPC) was developed in order (i) to isolate PR and (ii) to subsequently isolate PR and its 7' epimer epipinoresinol (EPR) simultaneously after an optimized acid treatment which resulted in PR epimerization forming equal amounts of PR and EPR, from C. nutans fruit. As optimal conditions, a two-phase solvent system consisting of methyl tert-butyl ether:acetone:water (4:3:3, v/v/v) for CPC separation, and an acid treatment performed at 50°C for 30min for the epimerization were applied. Thus, 33.7mg and 32.8mg PR and EPR, in as high as 93.7% and 92.3% purity, were isolated from 10.0gC. nutans fruit, representing 86.4% and 84.1% efficiency, respectively. Conversion characteristic of PR and EPR in acidic medium, determined as a function of time and temperature of acid treatment provides their unambiguous identification by on-line high performance liquid chromatography (HPLC). Antiproliferative assay of isolated PR and EPR in two different types of colon cancer cell lines (HCT116 and SW480) confirmed that both epimers caused a more significant decrease of viability in HCT116 cells than in SW480 cells, suggesting their similar mechanism of antiproliferative action.

KRAS-mutation status dependent effect of zoledronic acid in human non-small cell cancer preclinical models.

BACKGROUND: In non-small cell lung cancer (NSCLC) KRAS-mutant status is a negative prognostic and predictive factor. Nitrogen-containing bisphosphonates inhibit prenylation of small G-proteins (e.g. Ras, Rac, Rho) and thus may affect proliferation and migration. In our preclinical work, we investigated the effect of an aminobisphosphonate compound (zoledronic acid) on mutant and wild type KRAS-expressing human NSCLC cell lines. RESULTS: We confirmed that zoledronic acid was unable to inhibit the prenylation of mutant K-Ras unlike in the case of wild type K-Ras. In case of in vitro proliferation, the KRAS-mutant human NSCLC cell lines showed resistance to zoledronic acid wild-type KRAS-cells proved to be sensitive. Combinatory application of zoledronic acid enhanced the cytostatic effect of cisplatin. Zoledronic acid did not induce significant apoptosis. In xenograft model, zoledronic acid significantly reduced the weight of wild type KRAS-EGFR-expressing xenograft tumor by decreasing the proliferative capacity. Futhermore, zoledronic acid induced VEGF expression and improved in vivo tumor vascularization. MATERIALS AND METHODS: Membrane association of K-Ras was examined by Western-blot. In vitro cell viability, apoptotic cell death and migration were measured in NSCLC lines with different molecular background. The in vivo effect of zoledronic acid was investigated in a SCID mouse subcutaneous xenograft model. CONCLUSIONS: The in vitro and in vivo inhibitory effect of zoledronic acid was based on the blockade of cell cycle in wild type KRAS-expressing human NSCLC cells. The zoledronic acid induced vascularization supported in vivo cytostatic effect. Our preclinical investigation suggests that patients with wild type KRAS-expressing NSCLC could potentially benefit from aminobisphosphonate therapy.

Design and synthesis of new imidazo[1,2-a]pyridine and imidazo[1,2-a]pyrazine derivatives with antiproliferative activity against melanoma cells.

Melanoma is an aggressive form of skin cancer and it is generally associated with poor prognosis in patients with late-stage disease. Due to the increasing occurrence of melanoma, there is a need for the development of novel therapies. A new series of diarylamide and diarylurea derivatives containing imidazo[1,2-a]pyridine or imidazo[1,2-a]pyrazine scaffold was designed and synthesized to investigate their in vitro efficacy against the A375P human melanoma cell line. We found several compounds expressing submicromolar IC50 values against the A375P cells, from which 15d, 17e, 18c, 18h, 18i demonstrated the highest potencies with IC50 below 0.06 µM.

Sensitivity of Melanoma Cells to EGFR and FGFR Activation but Not Inhibition is Influenced by Oncogenic BRAF and NRAS Mutations.

BRAF and NRAS are the two most frequent oncogenic driver mutations in melanoma and are pivotal components of both the EGF and FGF signaling network. Accordingly, we investigated the effect of BRAF and NRAS oncogenic mutation on the response to the stimulation and inhibition of epidermal and fibroblast growth factor receptors in melanoma cells. In the three BRAF mutant, two NRAS mutant and two double wild-type cell lines growth factor receptor expression had been verified by qRT-PCR. Cell proliferation and migration were determined by the analysis of 3-days-long time-lapse videomicroscopic recordings. Of note, a more profound response was found in motility as compared to proliferation and double wild-type cells displayed a higher sensitivity to EGF and FGF2 treatment when compared to mutant cells. Both baseline and induced activation of the growth factor signaling was assessed by immunoblot analysis of the phosphorylation of the downstream effectors Erk1/2. Low baseline and higher inducibility of the signaling pathway was characteristic in double wild-type cells. In contrast, oncogenic BRAF or NRAS mutation did not influence the response to EGF or FGF receptor inhibitors in vitro. Our findings demonstrate that the oncogenic mutations in melanoma have a profound impact on the motogenic effect of the activation of growth factor receptor signaling. Since emerging molecularly targeted therapies aim at the growth factor receptor signaling, the appropriate mutational analysis of individual melanoma cases is essential in both preclinical studies and in the clinical trials and practice.

Prenylation inhibition-induced cell death in melanoma: reduced sensitivity in BRAF mutant/PTEN wild-type melanoma cells.

While targeted therapy brought a new era in the treatment of BRAF mutant melanoma, therapeutic options for non-BRAF mutant cases are still limited. In order to explore the antitumor activity of prenylation inhibition we investigated the response to zoledronic acid treatment in thirteen human melanoma cell lines with known BRAF, NRAS and PTEN mutational status. Effect of zoledronic acid on proliferation, clonogenic potential, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were investigated in vitro with SRB, TUNEL and PARP cleavage assays and videomicroscopy and immunoblot measurements, respectively. Subcutaneous and spleen-to-liver colonization xenograft mouse models were used to evaluate the influence of zoledronic acid treatment on primary and disseminated tumor growth of melanoma cells in vivo. Zoledronic acid more efficiently decreased short-term in vitro viability in NRAS mutant cells when compared to BRAF mutant and BRAF/NRAS wild-type cells. In line with this finding, following treatment decreased activation of ribosomal protein S6 was found in NRAS mutant cells. Zoledronic acid demonstrated no significant synergism in cell viability inhibition or apoptosis induction with cisplatin or DTIC treatment in vitro. Importantly, zoledronic acid could inhibit clonogenic growth in the majority of melanoma cell lines except in the three BRAF mutant but PTEN wild-type melanoma lines. A similar pattern was observed in apoptosis induction experiments. In vivo zoledronic acid did not inhibit the subcutaneous growth or spleen-to-liver colonization of melanoma cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. Nevertheless, we also demonstrated that therapeutic sensitivity might be influenced by the PTEN status of BRAF mutant melanoma cells. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target prenylation in melanoma cells.

Sex-dependent liver colonization of human melanoma in SCID mice--role of host defense mechanisms.

The possibility that endocrine factors may influence the clinical course of malignant melanoma is suggested by the superior survival data of women. In preclinical models we observed a higher rate of colony formation by human melanoma cells in male compared to female SCID mice, but only in the case of the liver and not in other organs. The gender difference could be seen at an early phase of colony formation. On the other hand, in our human melanoma cell lines we failed to detect steroid receptor protein expression, and treatment with sex hormones did not considerably influence their in vitro behavior. Investigating the possible contribution of host cells to the observed gender difference, we performed in vivo blocking experiments applying pretreatment of the animals with Kupffer cell inhibitor gadolinium chloride and the NK cell inhibitor anti-asialo GM1 antibody. While Kupffer cell blockade enhanced melanoma liver colonization equally in the two sexes, a more prominent increase was observed in female than in male mice in the case of NK cell inhibition. Further supporting the importance of NK cells in the lower liver colonization efficiency of melanoma cells in females, gender difference in colony formation was lost in NSG mice lacking NK activity. Although in humans no organ selectivity of gender difference in melanoma progression has been observed according to data in the literature, our results possibly indicate a contribution of natural host defense mechanisms to gender difference in survival of patients with melanoma or other tumor types as well.

Peripheral vascular endothelial growth factor level is associated with antidepressant treatment response: results of a preliminary study.

BACKGROUND: Recent investigations have revealed multiple actions of vascular endothelial growth factor (VEGF) in the nervous system. The role of VEGF in the molecular background of mood disorders has also been proposed. In this study we were interested in investigating a possible association between VEGF levels and treatment response in patients with a current episode of major depression (MDE). METHODS: 34 patients with MDE were enrolled in our study. Depressive symptoms were monitored by the Montgomery-Åsberg Depression Rating Scale at baseline (V(1)) and after a 4-week treatment period (V(2)). Patients with less than a 50% improvement in MADRS total scores during this period were regarded as non-responders. RESULTS: Plasma VEGF levels did not change during the treatment period in either the total sample or in the responder and non-responder subsamples. There was a strong trend for higher baseline VEGF levels in the non-responder group than in the responder group (p=0.055) and this difference-as a weak trend-was still detectable at the end of the treatment period (p=0.097). Regression analysis revealed that the baseline VEGF level was a significant predictor for the endpoint MADRS score (p=0.02). LIMITATIONS: Sample size was relatively small; sample consists of both patients with MDD and bipolar disorder. CONCLUSIONS: Our preliminary results raise the possibility that baseline levels of peripheral VEGF may predict treatment response in patients with mood disorders. Considering the limitations of our study, further investigations should resolve whether VEGF is a useful biomarker for treatment response in depression in clinical practice.