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The mode of the antitumoral activity of multimutated oncolytic herpes simplex virus type 1 G207 has not been fully elucidated yet. Because the antitumoral activity of many drugs involves the inhibition of tumor blood vessel formation, we determined if G207 had an influence on angiogenesis. Monolayers of human umbilical vein endothelial cells and human dermal microvascular endothelial cells, but not human dermal fibroblasts, bronchial epithelial cells, and retinal glial cells, were highly sensitive to the replicative and cytotoxic effects of G207. Moreover, G207 infection caused the destruction of endothelial cell tubes in vitro. In the in vivo Matrigel plug assay in mice, G207 suppressed the formation of perfused vessels. Intratumoral treatment of established human rhabdomyosarcoma xenografts with G207 led to the destruction of tumor vessels and tumor regression. Ultrastructural investigations revealed the presence of viral particles in both tumor and endothelial cells of G207-treated xenografts, but not in adjacent normal tissues. These findings show that G207 may suppress tumor growth, in part, due to inhibition of angiogenesis.
Adhesion molecules of the integrin beta1 family are thought to be involved in the malignant progression renal cell carcinoma (RCC). Still, it is not clear how they contribute to this process. Since the hematogenous phase of tumour dissemination is the rate-limiting step in the metastatic process, we explored beta1 integrin alterations on several RCC cell lines (A498, Caki1, KTC26) before and after contacting vascular endothelium in a tumour-endothelium (HUVEC) co-culture assay. Notably, alpha2, alpha3 and alpha5 integrins became down-regulated immediately after the tumour cells attached to HUVEC, followed by re-expression shortly thereafter. Integrin down-regulation on RCC cells was caused by direct contact with endothelial cells, since the isolated endothelial membrane fragments but not the cell culture supernatant contributed to the observed effects. Integrin loss was accompanied by a reduced focal adhesion kinase (FAK) expression, FAK activity and diminished binding of tumour cells to matrix proteins. Furthermore, intracellular signalling proteins RCC cells were altered in the presence of HUVEC membrane fragments, in particular 14-3-3 epsilon, ERK2, PKCdelta, PKCepsilon and RACK1, which are involved in regulating tumour cell motility. We, therefore, speculate that contact of RCC cells with the vascular endothelium converts integrin-dependent adhesion to integrin-independent cell movement. The process of dynamic integrin regulation may be an important part in tumour cell migration strategy, switching the cells from being adhesive to becoming motile and invasive.
Histone deacetylase (HDAC) inhibitors represent a promising class of antineoplastic agents which affect tumour growth, differentiation and invasion. The effects of the HDAC inhibitor valproic acid (VPA) were tested in vitro and in vivo on pre-clinical renal cell carcinoma (RCC) models. Caki-1, KTC-26 or A498 cells were treated with various concentrations of VPA during in vitro cell proliferation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and to evaluate cell cycle manipulation. In vivo tumour growth was conducted in subcutaneous xenograft mouse models. The anti-tumoural potential of VPA combined with low-dosed interferon-α (IFN-α) was also investigated. VPA significantly and dose-dependently up-regulated histones H3 and H4 acetylation and caused growth arrest in RCC cells. VPA altered cell cycle regulating proteins, in particular CDK2, cyclin B, cyclin D3, p21 and Rb. In vivo, VPA significantly inhibited the growth of Caki-1 in subcutaneous xenografts, accompanied by a strong accumulation of p21 and bax in tissue specimens of VPA-treated animals. VPA–IFN-α combination markedly enhanced the effects of VPA monotherapy on RCC proliferation in vitro, but did not further enhance the anti-tumoural potential of VPA in vivo. VPA was found to have profound effects on RCC cell growth, lending support to the initiation of clinical testing of VPA for treating advanced RCC.
The antitumor effect of curcumin in urothelial cancer cells is enhanced by light exposure in vitro
(2019)
The natural compound curcumin exerts antitumor properties in vitro, but its clinical application is limited due to low bioavailability. Light exposure in skin and skin cancer cells has been shown to improve curcumin bioavailability; thus, the object of this investigation was to determine whether light exposure might also enhance curcumin efficacy in bladder cancer cell lines. RT112, UMUC3, and TCCSUP cells were preincubated with low curcumin concentrations (0.1-0.4 μg/ml) and then exposed to 1.65 J/cm2 visible light for 5 min. Cell growth, cell proliferation, apoptosis, cell cycle progression, and cell cycle regulating proteins along with acetylation of histone H3 and H4 were investigated. Though curcumin alone did not alter cell proliferation or apoptosis, tumor cell growth and proliferation were strongly blocked when curcumin was combined with visible light. Curcumin-light caused the bladder cancer cells to become arrested in different cell phases: G0/G1 for RT112, G2/M for TCCSUP, and G2/M- and S-phase for UMUC3. Proteins of the Cdk-cyclin axis were diminished in RT112 after application of 0.1 and 0.4 μg/ml curcumin. Cell cycling proteins were upregulated in TCCSUP and UMUC3 in the presence of 0.1 μg/ml curcumin-light but were partially downregulated with 0.4 μg/ml curcumin. 0.4 μg/ml (but not 0.1 μg/ml) curcumin-light also evoked late apoptosis in TCCSUP and UMUC3 cells. H3 and H4 acetylation was found in UMUC3 cells treated with 0.4 μg/ml curcumin alone or with 0.1 μg/ml curcumin-light, pointing to an epigenetic mechanism. Light exposure enhanced the antitumor potential of curcumin on bladder cancer cells but by different molecular action modes in the different cell lines. Further studies are necessary to evaluate whether intravesical curcumin application, combined with visible light, might become an innovative tool in combating bladder cancer.
Although the mechanistic target of rapamycin (mTOR) inhibitor, everolimus, has improved the outcome of patients with renal cell carcinoma (RCC), improvement is temporary due to the development of drug resistance. Since many patients encountering resistance turn to alternative/complementary treatment options, an investigation was initiated to evaluate whether the natural compound, sulforaphane (SFN), influences growth and invasive activity of everolimus-resistant (RCCres) compared to everolimus-sensitive (RCCpar) RCC cell lines in vitro. RCC cells were exposed to different concentrations of SFN and cell growth, cell proliferation, apoptosis, cell cycle, cell cycle regulating proteins, the mTOR-akt signaling axis, adhesion to human vascular endothelium and immobilized collagen, chemotactic activity, and influence on surface integrin receptor expression were investigated. SFN caused a significant reduction in both RCCres and RCCpar cell growth and proliferation, which correlated with an elevation in G2/M- and S-phase cells. SFN induced a marked decrease in the cell cycle activating proteins cdk1 and cyclin B and siRNA knock-down of cdk1 and cyclin B resulted in significantly diminished RCC cell growth. SFN also modulated adhesion and chemotaxis, which was associated with reduced expression of the integrin subtypes α5, α6, and β4. Distinct differences were seen in RCCres adhesion and chemotaxis (diminished by SFN) and RCCpar adhesion (enhanced by SFN) and chemotaxis (not influenced by SFN). Functional blocking of integrin subtypes demonstrated divergent action on RCC binding and invasion, depending on RCC cell sensitivity to everolimus. Therefore, SFN administration could hold potential for treating RCC patients with established resistance towards everolimus.
Background: Measurement of prostate-specific antigen (PSA) advanced the diagnostic and prognostic potential for prostate cancer (PCa). However, due to PSA’s lack of specificity, novel biomarkers are needed to improve risk assessment and ensure optimal personalized therapy. A set of protein molecules as potential biomarkers was therefore evaluated in serum of PCa patients.
Methods: Serum samples from patients undergoing radical prostatectomy (RPE) for biopsy-proven PCa without neoadjuvant treatment were compared to serum samples from healthy subjects. Preliminary screening of 119 proteins in 10 PCa patients and 10 controls was carried out by the Proteome Profiler Antibody Array. Those markers showing distinct differences between patients and controls were then further evaluated by ELISA in the serum of 165 PCa patients and 19 controls. Uni- and multivariate as well as correlation analysis were performed to test the capability of these molecules to detect disease and predict pathological outcome.
Results: Screening showed that soluble (s)E-cadherin, E-selectin, MMP2, MMP9, TIMP1, TIMP2, Galectin and Clusterin warranted further evaluation. sE-Cadherin, TIMP1, Galectin and Clusterin were significantly over- and MMP9 under-expressed in PCa compared to controls. The concentration of sE-cadherin, MMP2 and Clusterin correlated negatively and that of MMP9 and TIMP1 positively with the Gleason Sum at prostatectomy. Only sE-cadherin significantly correlated with the highest Gleason pattern. Compared to serum PSA, sE-cadherin provided an independent and better matching predictive ability for discriminating PCas with an upgrade at RPE and aggressive tumors with a Gleason Sum ≥7.
Conclusions: sE-cadherin performed most favorably from a large panel of serum proteins in terms of diagnostic and predictive potential in curatively treatable PCa. sE-cadherin merits further investigation as a biomarker for PCa.
Inhibitors of the mammalian target of rapamycin (mTOR) have improved the treatment of renal cell carcinoma (RCC). However, chronic drug exposure may trigger resistance, limiting the utility of these agents. The metastatic behavior of RCC cells, susceptible (RCC(par)) or resistant (RCC(res)) to the mTOR inhibitor temsirolimus, was investigated. Adhesion to vascular endothelium or immobilized collagen and fibronectin was quantified. Chemotactic motility was evaluated with a modified Boyden chamber assay. Integrin α and β subtype receptors were analyzed by flow cytometry and Western blot analysis. The physiological relevance of the integrins was then determined by blocking studies and small interfering RNA knockdown. Adhesion to endothelial cells and to fibronectin (not to collagen) and chemotaxis were enhanced in RCC(res) compared to RCC(par). RCC(res) detached from fibronectin and motile activity further increased under retreatment with low-dosed temsirolimus. α5 integrin was diminished inside the cell and at the cell surface, whereas the β3 subtype was reduced intracellularly but elevated at the plasma membrane. In RCC(par), blocking α5 surface receptors enhanced RCC-collagen but reduced RCC-fibronectin interaction, whereas the opposite was true for RCC(res). Chemotaxis of RCC(par) but not of RCC(res) was strongly diminished by the α5 antibody. Blocking β3 significantly lowered chemotaxis with stronger effects on RCC(res), compared to RCC(par). Importantly, β3 knockdown reduced chemotaxis of RCC(par) but upregulated the motile behavior of RCC(res). Temsirolimus resistance is characterized by quantitative alterations of integrin α5 and β3 expression, coupled to functional changes of the integrin molecules, and forces a switch from RCC adhesion to RCC migration.
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.
Renal cell carcinoma alters endothelial receptor expression responsible for leukocyte adhesion
(2016)
Renal cell carcinoma (RCC) escapes immune recognition. To elaborate the escape strategy the influence of RCC cells on endothelial receptor expression and endothelial leukocyte adhesion was evaluated. Human umbilical vein endothelial cells (HUVEC) were co-cultured with the RCC cell line, Caki-1, with and without tumor necrosis factor (TNF)-alpha. Intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial (E)-selectin, standard and variants (V) of CD44 were then analysed in HUVEC, using flow cytometry and Western blot analysis. To determine which components are responsible for HUVEC-Caki-1 interaction causing receptor alteration, Caki-1 membrane fragments versus cell culture supernatant were applied to HUVECS. Adhesion of peripheral blood lymphocytes (PBL) and polymorphonuclear neutrophils (PMN) to endothelium was evaluated by co-culture adhesion assays. Relevance of endothelial receptor expression for adhesion to endothelium was determined by receptor blockage. Co-culture of RCC and HUVECs resulted in a significant increase in endothelial ICAM-1, VCAM-1, E-selectin, CD44 V3 and V7 expression. Previous stimulation of HUVECs with TNF-alpha and co-cultivation with Caki-1 resulted in further elevation of endothelial CD44 V3 and V7 expression, whereas ICAM-1, VCAM-1 and E-selectin expression were significantly diminished. Since Caki-1 membrane fragments also caused these alterations, but cell culture supernatant did not, cell-cell contact may be responsible for this process. Blocking ICAM-1, VCAM-1, E-selectin or CD44 with respective antibodies led to a significant decrease in PBL and PMN adhesion to endothelium. Thus, exposing HUVEC to Caki-1 results in significant alteration of endothelial receptor expression and subsequent endothelial attachment of PBL and PMN.
Relevance of the natural HDAC inhibitor sulforaphane as a chemopreventive agent in urologic tumors
(2018)
Due to an increased understanding of molecular biology and the genomics of cancer, new and potent agents have been approved by the Food and Drug Administration (FDA) to fight this disease. However, all of these drugs cause severe side effects and resistance inevitably develops, re-activating tumor growth and dissemination. For this reason, patients turn to natural compounds as alternative or complementary treatment options, since it has been found that natural plant products may block, inhibit, or reverse cancer development. The present review focusses on the role of the natural compound sulforaphane (SFN) as an anti-tumor agent in urologic cancer. SFN is a natural compound found in cruciferous vegetables from the Brassicaceae family such as broccoli, cauliflower and cabbage. Several epidemiologic and clinical studies have documented chemopreventive properties of SFN, making it an interesting candidate for additive cancer treatment. SFN shows remarkable anti-tumor effects in vitro and in vivo without exerting toxicity. The review summarizes the current understanding of SFN and provides insights into its molecular mode of action with particular emphasis on epigenetic tumor control.