Combining electrical stimulation and tissue engineering to treat large bone defects in a rat model.

Bone Tissue engineering (BTE) has recently been introduced as an alternative to conventional treatments for large non-healing bone defects. BTE approaches mimic autologous bone grafts, by combining cells, scaffold, and growth factors, and have the added benefit of being able to manipulate these constituents to optimize healing. Electrical stimulation (ES) has long been used to successfully treat non-healing fractures and has recently been shown to stimulate bone cells to migrate, proliferate, align, differentiate, and adhere to bio compatible scaffolds, all cell behaviors that could improve BTE treatment outcomes. With the above in mind we performed in vitro experiments and demonstrated that exposing Mesenchymal Stem Cells (MSC) + scaffold to ES for 3 weeks resulted in significant increases in osteogenic differentiation. Then in in vivo experiments, for the first time, we demonstrated that exposing BTE treated rat femur large defects to ES for 8 weeks, caused improved healing, as indicated by increased bone formation, strength, vessel density, and osteogenic gene expression. Our results demonstrate that ES significantly increases osteogenic differentiation in vitro and that this effect is translated into improved healing in vivo. These findings support the use of ES to help BTE treatments achieve their full therapeutic potential.

Effects of electrical stimulation on rat limb regeneration, a new look at an old model.

Limb loss is a devastating disability and while current treatments provide aesthetic and functional restoration, they are associated with complications and risks. The optimal solution would be to harness the body's regenerative capabilities to regrow new limbs. Several methods have been tried to regrow limbs in mammals, but none have succeeded. One such attempt, in the early 1970s, used electrical stimulation and demonstrated partial limb regeneration. Several researchers reproduced these findings, applying low voltage DC electrical stimulation to the stumps of amputated rat forelimbs reporting "blastema, and new bone, bone marrow, cartilage, nerve, skin, muscle and epiphyseal plate formation". In spite of these encouraging results this research was discontinued. Recently there has been renewed interest in studying electrical stimulation, primarily at a cellular and subcellular level, and studies have demonstrated changes in stem cell behavior with increased proliferation, differentiation, matrix formation and migration, all important in tissue regeneration. We applied electrical stimulation, in vivo, to the stumps of amputated rat limbs and observed significant new bone, cartilage and vessel formation and prevention of neuroma formation. These findings demonstrate that electricity stimulates tissue regeneration and form the basis for further research leading to possible new treatments for regenerating limbs.

Intensified antineoplastic effect by combining an HDAC-inhibitor, an mTOR-inhibitor and low dosed interferon alpha in prostate cancer cells.

A significant proportion of men diagnosed with prostate cancer (PCa) eventually develop metastatic disease, which progresses to castration resistance, despite initial response to androgen deprivation. As anticancer therapy has become increasingly effective, acquired drug resistance has emerged, limiting efficacy. Combination treatment, utilizing different drug classes, exemplifies a possible strategy to foil resistance development. The effects of the triple application of the histone deacetylase (HDAC) inhibitor valproic acid (VPA), the mammalian target of rapamycin inhibitor everolimus and low dosed interferon alpha (IFNα) on PCa cell growth and dissemination capacity were investigated. For that purpose, the human PCa cell lines, PC-3, DU-145 and LNCaP were treated with the combined regimen or separate single agents. Cell growth was investigated by the MTT dye reduction assay. Flow cytometry served to analyse cell cycle progression. Adhesion to vascular endothelium or immobilized collagen, fibronectin and laminin was quantified. Migration and invasion characteristics were determined by the modified Boyden chamber assay. Integrin α and ß subtypes were investigated by flow cytometry, western blotting and RT-PCR. Integrin related signalling, Epidermal Growth Factor Receptor (EGFr), Akt, p70S6kinase and extracellular signal-regulated kinases (ERK)1/2 activation were also assessed. The triple application of VPA, everolimus and low dosed IFNα blocked tumour cell growth and dissemination significantly better than any agent alone. Antitumour effects were associated with pronounced alteration in the cell cycle machinery, intracellular signalling and integrin expression profile. Combining VPA, everolimus and low dosed IFNα might be a promising option to counteract resistance development and improve outcome in PCa patients.

HDAC inhibition suppresses bladder cancer cell adhesion to collagen under flow conditions.

The influence of the histone deacetylase (HDAC)-inhibitor, valproic acid (VPA), on bladder cancer cell adhesion in vitro was investigated in this paper. TCCSUP and RT-112 bladder cancer cells were treated with VPA (0.5 or 1 mM) twice or thrice weekly for 14 days. Controls remained untreated. Tumour cell interaction with immobilized collagen was evaluated by a flow-based adhesion assay using a shear force of 2 or 4 dyne/cm(2). The effects of VPA on the integrin adhesion receptors α3, α5, ß1, ß3 and ß4 were assessed by flow cytometry to determine integrin surface expression and by western blotting to determine the cytoplasmic integrin level. VPA of 0.5 mM and 1 mM significantly prevented binding of both RT-112 and TCCSUP cells to collagen, compared with the untreated controls. Adhesion was reduced to a higher extent when RT-112 (subjected to 2 dyne/cm(2)) or TCCSUP (subjected to 2 or 4 dyne/cm(2)) tumour cells were treated with VPA three times a week, compared to the two times a week protocol. VPA caused a significant up-regulation of the integrin α3, α5, ß1, ß3 and ß4 subtypes on the TCCSUP cell surface membrane. In RT-112 cells, only integrin α5 was elevated on the cell surface following VPA exposure. Western blotting revealed an up-regulation of α3, α5, ß3 and ß4 integrins and down-regulation of the integrin ß1 protein by VPA in TCCSUP. VPA also up-regulated α5 and down-regulated ß1 integrin in RT-112 cells, but also reduced α3 and ß3 in TCCSUP. VPA exerted adhesion-blocking properties on bladder cancer cells under physiologic flow conditions. The effects were accompanied by distinct modifications of the integrin expression profile, which differ depending on the cell lines used. Application of VPA might be an innovative option to prevent bladder cancer dissemination.

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 ß 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 ß1 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.

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.

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.

BACKGROUND: Single 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. METHODS: PC-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 ß subtypes were analyzed. Finally, effects of drug treatment on cell signaling pathways were determined. RESULTS: All 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 ß 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. CONCLUSIONS: Simultaneous 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.

HDAC inhibition delays cell cycle progression of human bladder cancer cells in vitro.

Our aim was to analyze the impact of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on bladder cancer cell growth in vitro. RT-4, TCCSUP, UMUC-3, and RT-112 bladder cancer cells were treated with VPA (0.125-1 mmol/l) without and with preincubation periods of 3 and 5 days. Controls remained untreated. Tumor cell growth, cell cycle progression, and cell cycle-regulating proteins were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and western blotting, respectively. Effects of VPA on histone H3 and H4 acetylation and HDAC3 and HDAC4 were also determined. Without preincubation, no tumor cell growth reduction was observed with 0.125 and 0.25 mmol/l VPA in TCCSUP, UMUC-3, and RT-112 cells, whereas 0.5 and 1 mmol/l VPA diminished the cell number significantly. VPA (0.25 mmol/l) did exert tumor growth-blocking effects after a 3-day preincubation. To achieve antitumor effects with VPA (0.125 mmol/l), a 5-day preincubation was necessary. A 3-day or 5-day preincubation was also necessary to distinctly delay cell cycle progression, with maximum effects at VPA (1 mmol/l). After the 5-day preincubation, the cell cycle-regulating proteins cdk1, cdk2, cdk4, and cyclins B, D1, and E were reduced, whereas p27 was enhanced. Diminished HDAC3 and 4 expression induced by VPA was accompanied by elevated acetylation of H3 and H4. VPA exerted growth-blocking properties on a panel of bladder cancer cell lines, commensurate with dose and exposure time. Long-term application induced much stronger effects than did shorter application and should be considered when designing therapeutic strategies for treating bladder carcinoma.

Impact of combined HDAC and mTOR inhibition on adhesion, migration and invasion of prostate cancer cells.

The concept of molecular tumor targeting might provide new hope in the treatment of advanced prostate cancer. We evaluated metastasis blocking properties of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell lines. RAD001 or VPA were applied to PC-3 or LNCaP cells, either separately or in combination. Adhesion to vascular endothelium or to immobilized collagen, fibronectin or laminin was quantified. Migration and invasion were explored by a modified Boyden chamber assay. Integrin α and ß subtypes were analyzed by flow cytometry, western blotting and RT-PCR. Effects of drug treatment on integrin related signaling, Akt and p70S6kinase activation, histone H3 and H4 acetylation were also determined. Separate application of RAD001 or VPA distinctly reduced tumor cell adhesion, migration and invasion, accompanied by elevated Akt activation and p70S6kinase de-activation. Integrin subtype expression was altered significantly by both compounds (VPA > RAD001). When both drugs were used in concert additive effects were observed on the migratory and invasive behavior but not on tumor-endothelium and tumor-matrix interaction. Separate mTOR or HDAC inhibition slows processes related to tumor metastasis. The RAD001-VPA combination showed advantage over VPA monotreatment with particular respect to migration and invasion. Ongoing studies are required to assess the relevance of VPA monotherapy versus VPA-RAD001 combination on tumor cell motility.

Inhibitory effects of the HDAC inhibitor valproic acid on prostate cancer growth are enhanced by simultaneous application of the mTOR inhibitor RAD001.

AIMS: To analyze the combined impact of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell growth. MAIN METHODS: PC-3, DU-145 and LNCaP cells were treated with RAD001, VPA or with an RAD001-VPA combination for 3 or 5 days. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT assay, flow cytometry and Western blotting, respectively. Effects of drug treatment on cell signaling pathways were determined. KEY FINDINGS: Separate application of RAD001 or VPA distinctly reduced tumor cell growth and impaired cell cycle progression. Significant additive effects were evoked when both drugs were used in concert. Particularly, the cell cycle regulating proteins cdk1, cdk2, cdk4 and cyclin B were reduced, whereas p21 and p27 were enhanced by the RAD001-VPA combination. Signaling analysis revealed deactivation of EGFr, ERK1/2 and p70S6k. Phosphorylation of Akt was diminished in DU-145 but elevated in PC-3 and LNCaP cells. SIGNIFICANCE: The RAD001-VPA combination exerted profound antitumor properties on a panel of prostate cancer cell lines. Therefore, simultaneous blockage of HDAC and mTOR related pathways should be considered when designing novel therapeutic strategies for treating prostate carcinoma.

Critical analysis of simultaneous blockage of histone deacetylase and multiple receptor tyrosine kinase in the treatment of prostate cancer.

BACKGROUND: The concept of molecular tumor targeting might be an innovative option to treat advanced prostate cancer. We analyzed the effect of combining the multiple receptor tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) on adhesion and growth properties of prostate cancer cell lines. METHODS: PC-3, DU-145, and LNCaP cells were treated with AEE788, VPA or with an AEE788-VPA combination, and cell cycle progression investigated. Furthermore, tumor cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins was evaluated, and integrin α and ß subtypes were analyzed. Finally, effects of drug treatment on cell signaling pathways were determined. RESULTS: AEE788 moderately and VPA strongly reduced tumor cell adhesion and growth. VPA impaired cell cycle progression and altered the expression level of the cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin B, D1, cyclin E, p21, and p27. VPA also acted on the membranous, cytoplasmic, and gene expression pattern of various integrin α and ß subtypes. AEE788 acted likewise, but more moderately. Combining AEE788 and VPA did not result in an additive anti-tumor effect. Signaling analysis revealed that the EGFr downstream target Akt was similarly modified in the presence of VPA or the VPA-AEE788 combination, but not influenced by AEE788 alone. CONCLUSIONS: The AEE788-VPA combination has no advantage over VPA monotreatment in vitro. The non-responsiveness of Akt

Combined targeting of the VEGFr/EGFr and the mammalian target of rapamycin (mTOR) signaling pathway delays cell cycle progression and alters adhesion behavior of prostate carcinoma cells.

The impact of the mTOR inhibitor RAD001 combined with the EGFr/VEGFr tyrosine kinase inhibitor AEE788 on prostate tumor cell growth, adhesion and migration was analyzed in vitro. The RAD001-AEE788 combination profoundly reduced tumor-endothelium and tumor-matrix contacts, suppressing cell growth and cell cycle progression. The underlying molecular mode of action depended on the cell phenotype, since cell cycle proteins, integrin subtype expression and integrin dependent signaling were altered in a different manner in PC-3 and DU-145 versus LNCaP prostate cancer cells. Simultaneous targeting of mTOR and VEGFr/EGFr related pathways may offer a novel therapeutic strategy for prostate cancer treatment.

Effects of combined valproic acid and the epidermal growth factor/vascular endothelial growth factor receptor tyrosine kinase inhibitor AEE788 on renal cell carcinoma cell lines in vitro.

OBJECTIVE: To evaluate adhesion and growth inhibiting effects of the multiple receptor tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) on renal cell carcinoma (RCC) cells. MATERIALS AND METHODS: Caki-1 cells were treated with AEE788 and VPA, either alone or in combination, to investigate RCC cell adhesion to vascular endothelial cells or to immobilized extracellular matrix proteins. Tumour cell proliferation was examined by MTT dye reduction assay. Effects of drug treatment on cell signalling pathways were determined by Western blotting. The expression levels of integrin alpha and beta subtypes were evaluated by flow cytometry (surface expression) and Western blotting (intracellular protein expression). RESULTS: RCC cell treatment with AEE788 and VPA in combination resulted in a stronger inhibition of tumour cell proliferation than that caused by either drug alone. There were also additive effects of the combined treatment on tumour cell adhesion to endothelial cells and to immobilized laminin (but not to immobilized fibronectin and collagen). AEE788 alone or combined with VPA reduced Akt expression and histone H3 acetylation. Both compounds altered integrin alpha and beta subtype expression, in particular alpha1, alpha3 and beta4, and blocked integrin-dependent integrin-linked kinase and focal-adhesion kinase (total and phosphorylated) signalling. CONCLUSIONS: Both AEE788 and VPA profoundly block the interaction of RCC cells with endothelium and extracellular matrix and reduce tumour growth in vitro. Therefore, this combined regimen warrants further preclinical and possible clinical study for treating advanced RCC.

Tumor-endothelium cross talk blocks recruitment of neutrophils to endothelial cells: a novel mechanism of endothelial cell anergy.

Tumor cells have evolved effective strategies to escape the host immune response. The objective of this study was to determine whether tumor cells can condition endothelial cells in a specific manner to prevent subsequent adhesion of polymorphonuclear neutrophils (PMNs) and/or peripheral blood lymphocytes (PBLs). Human umbilical vein endothelial cells (HUVECs) and UKF-NB-4 neuroblastoma tumor cells were established in coculture on opposite sides of porous transwell filters. After 24 hours with and without HUVEC conditioning, PMNs or PBLs were added to the HUVEC monolayer. Adhesion to conditioned HUVEC versus adhesion to nonconditioned HUVEC was compared. Effects on endothelial CD44v4, CD44v5, CD44v7, intercellular adhesion molecule 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule 1 (VCAM-1) adhesion receptor expression were analyzed by flow cytometry, intracellular signaling proteins of the mitogen-activated protein kinase pathway and protein kinase C (PKC) subtypes quantified by Western blot analysis. Endothelial conditioning led to a distinct reduction in PMN but not in PBL adhesion to HUVEC. CD44 was significantly reduced, whereas ICAM-1, E-selectin, and VCAM-1 were not altered during HUVEC conditioning. Antibody blockade against CD44v4, CD44v5, and CD44v7 inhibited PMN but not PBL binding. The observed effects were caused by direct tumor cell-HUVEC contact because addition of isolated tumor cell membrane fragments but not of soluble cell culture supernatant to HUVEC induced the CD44 receptor loss. PKCalpha activity was strongly enhanced in conditioned HUVEC. Blocking PKC prevented the reduction in PMN binding, indicating that this protein is involved in PMN adhesion regulation. A novel tumor escape strategy is presented here. Cell contact-dependent adhesion of tumor cells to the vascular wall promotes down-regulation of endothelial CD44 receptor expression, impairing an effective neutrophil attack.

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 has now opened novel treatment options. We evaluated 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. 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. CONCLUSION: 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.

The histone deacetylase inhibitor valproic acid alters growth properties of renal cell carcinoma in vitro and in vivo.

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-alpha (IFN-alpha) 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-alpha 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.

Valproic acid blocks adhesion of renal cell carcinoma cells to endothelium and extracellular matrix.

Treatment strategies for metastatic renal cell carcinoma (RCC) have been limited due to chemotherapy and radiotherapy resistance. The development of targeted drugs has now opened novel therapeutic options. In the present study, anti-tumoral properties of the histone deacetylase inhibitor valproic acid (VPA) were tested in vitro and in vivo on pre-clinical RCC models. RCC cell lines Caki-1, KTC-26 or A498 were treated with various concentrations of VPA to evaluate tumour cell adhesion to vascular endothelial cells or to immobilized extracellular matrix proteins. In vivo tumour growth was conducted in subcutaneous xenograft mouse models. VPA was also combined with low dosed interferon-alpha (IFN-alpha) and the efficacy of the combination therapy, as opposed to VPA monotherapy, was compared. VPA significantly and dose-dependently prevented tumour cell attachment to endothelium or matrix proteins, accompanied by elevated histones H3 and H4 acetylation. VPA altered integrin-alpha and -beta subtype expression, in particular alpha(3), alpha(5) and beta(3), and blocked integrin-dependent signalling. In vivo, VPA significantly inhibited the growth of Caki-1 in subcutaneous xenografts with the 200 mg/kg being superior to the 400 mg/kg dosing schedule. VPA-IFN-alpha combination markedly enhanced the effects of VPA on RCC adhesion, and in vivo tumour growth was further reduced by the 400 mg/kg but not by the 200 mg/kg VPA dosing schedule. VPA profoundly blocked the interaction of RCC cells with endothelium and extracellular matrix and reduced tumour growth in vivo. Therefore, VPA should be considered an attractive candidate for clinical trials.

Modulation of adhesion and growth of colon and pancreatic cancer cells by the histone deacetylase inhibitor valproic acid.

Histone deacetylase (HDAC) inhibitors belong to a promising class of antineoplastic agents which affect tumor growth, differentiation and invasion. The effects of the HDAC inhibitor valproic acid (VPA) were tested in vitro on preclinical colon and pancreatic cancer models. Human colon adenocarcinoma HT-29 and pancreatic carcinoma DanG cells were treated with 1 mM VPA for different time periods during cell proliferation MTT assays, and to evaluate the tumor cell adhesion to endothelial cell monolayers. Alterations of beta1 integrin subunits alpha1-6) were analyzed by flow cytometry and RT-PCR. VPA significantly caused growth arrest in tumor cells and prevented tumor cell attachment to the endothelium. HT-29 cell adhesion was blocked to a higher extent than the adhesion of DanG cells. VPA modified membranous integrin beta1 expression, quantity and quality (up- or down-regulation) which depended on the tumor type investigated. Furthermore, VPA diminished integrin coding mRNA in HT-29 but not in DanG cells. We conclude that VPA shifts the integrin beta1 subunit balance from a 'pathological' towards a 'physiological' expression pattern leading to reduced tumor growth and invasion. Further study is required to elucidate the molecular background of the post-transcriptional modifications of VPA in order to exploit the potential of this agent in the treatment of colon and pancreatic cancer.