Purpose: Angiogenesis is required for tumor progression and represents a rational target for therapeutic intervention. Histone deacetylase (HDAC) inhibitors have been shown to have activity against various tumor cell types by inhibiting proliferation and inducing apoptosis both in vitro and in vivo. HDAC inhibitors have also been reported to inhibit angiogenesis. The goal of this study was to characterize the antiangiogenic and antitumor activity of a recently developed HDAC inhibitor, the hydroxamic derivative LBH589. Materials and Methods:To evaluate the antiangiogenesis activity of LBH589, we did cell cycle analysis, cell proliferation, tube formation, invasion assays in vitro, and Matrigel plug assay in vivo. To determine the antitumor activity of LBH589, we established human prostate carcinoma cell PC-3 xenografts in vivo.To evaluate the effect of LBH589 on endothelial signaling pathways, gene expression, and protein acetylation, we didWestern blots and reverse transcription-PCR in human umbilical vein endothelial cells (HUVEC). Immunohistochemical analysis was done to evaluate new blood vessel formation in vivo. Results: LBH589 induced acetylation of histone H3 and a-tubulin protein in HUVECs. Histone and nonhistone protein acetylation correlated with induction of G 2 -M cell cycle arrest, inhibition of HUVEC proliferation, and viability. Noncytotoxic concentrations of LBH589 inhibited endothelial tube formation, Matrigel invasion, AKT, extracellular signal-regulated kinase 1/2 phosphorylation, and chemokine receptor CXCR4 expression. In vivo dosing of mice with LBH589 (10 mg/kg/d) reduced angiogenesis and PC-3 tumor growth. Conclusion: This study provides evidence that LBH589 induces a wide range of effects on endothelial cells that lead to inhibition of tumor angiogenesis. These results support the role of HDAC inhibitors as a therapeutic strategy to target both the tumor and endothelial compartment and warrant the clinical development of these agents in combination with angiogenesis inhibitors.

Chromatin remodeling agents such as histone deacetylase inhibitors have been shown to modulate gene expression in tumor cells and inhibit tumor growth and angiogenesis. Vascular endothelial growth factor (VEGF) and VEGF receptors represent critical molecular targets for antiangiogenesis therapy. In this study, we investigated the biological effect of the histone deacetylase inhibitor NVP-LAQ824 in combination with the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 on tumor growth and angiogenesis. We report that treatment with NVP-LAQ824 affected tumor and endothelial cells and was associated with increased histone acetylation, p21 up-regulation, and growth inhibition. In addition, NVP-LAQ824 treatment inhibited the expression of angiogenesis-related genes such as angiopoietin-2, Tie-2, and survivin in endothelial cells and down-regulated hypoxia-inducible factor 1-␣ and VEGF expression in tumor cells. Combination treatment with NVP-LAQ824 and PTK787/ZK222584 was more effective than single agents in inhibiting in vitro and in vivo VEGF-induced angiogenesis. Endothelial cell proliferation, tube formation, and invasion into the Matrigel plugs were reduced. In mouse models with established subcutaneous prostate (PC3) and orthotopic breast tumors (MDA-MB321), this combination treatment induced 80 to 85% inhibition of tumor growth without overt toxicity. These results suggest that the combination of histone deacetylase inhibitors and VEGF receptor inhibitors may target multiple pathways in tumor progression and angiogenesis and represents a novel therapeutic approach in cancer treatment.

The lipophilic yeast Malassezia globosa is one of the major constituents of the mycoflora of the skin of patients with atopic dermatitis (AD). We compared the genotypes of M. globosa colonizing the skin surface of 32 AD patients and 20 healthy individuals for polymorphism of the intergenic spacer (IGS) 1 region of the rRNA gene. Sequence analysis demonstrated that M. globosa was divided into four major groups, which corresponded to the sources of the samples, on the phylogenetic tree. Of the four groups, two were from AD patients and one was from healthy subjects. The remaining group included samples from both AD patients and healthy subjects. In addition, the IGS 1 region of M. globosa contained short sequence repeats: (CT) n , and (GT) n . The number of sequence repeats also differed between the IGS 1 of M. globosa from AD patients and that from healthy subjects. These findings suggest that a specific genotype of M. globosa may play a significant role in AD, although M. globosa commonly colonizes both AD patients and healthy subjects.Malassezia species are lipophilic yeasts that are part of the normal human cutaneous commensal flora; they are isolated from sebaceous gland-rich areas of the skin, particularly on the chest, back, and head. They are also associated with several cutaneous diseases, including atopic dermatitis (AD), folliculitis, pityriasis versicolor, and seborrheic dermatitis (1, 7). In a taxonomic revision in 1996, the genus Malassezia was classified into seven different species: M. furfur, M. globosa, M. restricta, M. obtusa, M. pachydermatis, M. slooffiae, and M. sympodialis (9). Recently, we described an eighth species, M. dermatis, which was isolated from Japanese patients with AD (27). Since the taxonomic revision of the genus Malassezia, several studies have examined the distribution of the newly defined species of Malassezia on healthy human skin and lesions of skin diseases (2, 10, 21). However, culture media or sampling techniques often affect analyses of the Malassezia microflora. In a previous study, we used a nonculture method as an alternative to fungal culture to analyze the distribution of cutaneous Malassezia species (25). M. globosa and M. restricta were detected in approximately 90% of AD patients, and M. furfur and M. sympodialis were detected in approximately 40% of the subjects. In healthy subjects, M. globosa, M. restricta, and M. sympodialis were detected in approximately 40 to 60% of the subjects; M. furfur was found in only 4% of the subjects; and no other Malassezia species were detected. Therefore, these four species are common inhabitants of the skin of both AD patients and healthy individuals. In addition, while anti-Malassezia immunoglobulin E (IgE) antibody was detected in more than 90% of AD patients, no antibody was found in healthy subjects. Based on these results, M. globosa and M. restricta are thought to play a significant mycological role in AD. M. globosa is also part of the major microflora on the skin of healthy individuals. We used the intergenic space...

Purpose: High-dose interleukin 2 (IL-2) is a Food and Drug Administration^approved regimen for patients with metastatic renal cell carcinoma. However, the toxicity and limited clinical benefit associated with IL-2 has hampered its use. Histone deacetylase (HDAC) inhibitors have been shown to have antitumor activity in different tumor models including renal cell carcinoma, and to have immunomodulatory properties. In our study, we tested the effectiveness of combination therapy of IL-2 with the HDAC inhibitor MS-275 in a murine renal cell carcinoma (RENCA) model. Experimental Design: RENCA luciferase^expressing cells were implanted in the left kidney of BALB/C mice. Animals were randomly divided into four groups and treated with either vehicle, 150,000 IU of IL-2 twice daily by i.p. injections (twice weekly), 5 mg/kg of MS-275 daily by oral gavage (5 d/wk), or its combination.Treatment was started either 3 or 9 days following tumor cell injection. Results: Weekly luciferase images and tumor weight after 2 weeks of treatment showed significant tumor inhibition (>80%) in the combination treatment as compared with the IL-2 (no significant inhibition) or MS-275 (f40% inhibition) treatment groups. Spontaneous lung metastases were also inhibited in the combination treatment (>90% inhibition) as compared with the single treatment group. Kaplan-Meier analyses showed statistically significant increased survival in the combination group as compared with controls and single agents. Splenocytes from mice treated with combination treatment showed greater lysis of RENCA cells than splenocytes from mice treated with single agents. The percentage of CD4 + CD25+ Tcells and Foxp3 + T cells (T regulatory cells) was increased or reduced, respectively, in lymph nodes from tumor-bearing animals treated with the combination of MS-275 and IL-2 as compared with control and single agents. Depletion of CD8 + T cells abrogated the survival benefit from MS-275 + IL-2 combination. Conclusions: These results show that the combination of IL-2 and MS-275 has a synergistic antitumor effect in vivo in an immunocompetent murine model of renal cell carcinoma. The antitumor effect was associated with the decreased number of T regulatory cells and the increased antitumor cytotoxicity by splenocytes. In conclusion, these preclinical data provide the rationale for clinical testing of the combination of IL-2 and HDAC inhibitors in the treatment of patients with renal cell carcinoma.

Purpose: Histone deacetylase (HDAC) inhibitors have been shown to reverse epigenetic repression of certain genes, including retinoic acid receptor B2 (RARb2). In this study, we examined whether RARb2 expression is repressed in human renal cell carcinoma (RCC) and whether the HDAC inhibitor MS-275 may revert its epigenetic repression. Experimental Design: Six human tumor RCC cell lines were analyzed for RARb2 gene expression and for methylation and acetylation status at the promoter level. Modulation of RARb2 expression and correlation with antitumor activity by combination of MS-275 with 13-cis-retinoic acid (CRA) was assessed in a RARb2-negative RCC cell line. Results: RARb2 expression was either strongly present, weakly expressed, or absent in the RCC cell lines analyzed. Methylation-specific PCR indicated that the RARb2 promoter was partially methylated in three of the cell lines. CRA treatment did not inhibit clonogenic growth in the RARb2-negative cell line RCC1.18, whereas MS-275 induced a dose-dependent inhibitory effect. A greater inhibitory effect was observed with combination treatment (MS-275 + CRA).Treatment with MS-275 was associated with histone acetylation at the promoter level and synergistic gene reexpression of RARb2 in combination with CRA. RARb2 reexpression was associated with synergistic induction of the retinoid-responsive gene HOXA5. In vivo, single-agent CRA treatment showed no significant effect, whereas MS-275 and the combination induced a regression of RCC1.18 tumor xenografts. Discontinuation of treatment produced tumor recurrence in MS-275-treated mice, whereas animals treated with the combination remained tumor free. Conclusion: The HDAC inhibitor MS-275 seems to revert retinoid resistance due to epigenetic silencing of RARb2 in a human RCC model and has greater antitumor activity in combination with CRA compared with single agents. Thus, the combination of HDAC inhibitors and retinoids may represent a novel therapeutic approach in patients with RCC.

Cancers display distinct patterns of organ-specific metastasis. Comparative analysis of a broad array of cell membrane molecules on a liver-metastasizing subline of B16 melanoma versus the parental B16-F0 revealed unique up-regulation of integrin α2. The direct role of integrin α2 in hepatic metastasis was shown by comparison of high versus low-expressing populations, antibody blockade, and ectopic expression. Integrin α2–mediated binding to collagen type IV (highly exposed in the liver sinusoids) and collagen type IV–dependent activation of focal adhesion kinase are both known to be important in the metastatic process. Analysis of primary colorectal cancers as well as coexisting liver and lung metastases from individual patients suggests that integrin α2 expression contributes to liver metastasis in human colorectal cancer. These findings define integrin α2 as a molecule conferring selective potential for formation of hepatic metastasis, as well as a possible target to prevent their formation.

Resistance to chemotherapy is a major hurdle in the treatment of malignant melanoma. Histone deacetylase (HDAC) inhibitors have been shown to have antitumor activity in different tumor types, including melanoma, and to reverse epigenetic repression of tumor suppressor genes, such as retinoic acid receptor b (RARb). In this study, we tested the antitumor effect of the HDAC inhibitor LAQ824 in combination with 13-cis-retinoic acid (CRA) on two human melanoma cell lines both in vitro and in vivo. Treatment of LAQ824 showed a dose-dependent inhibitory effect on A2058 and HMV-I cell lines in a clonogenic assay. These cell lines were relatively resistance to CRA. On treatment with combination of LAQ824 and CRA, a greater inhibitory effect (up to 98%) was achieved compared with single agents. Lack of RARb2 gene expression was associated with histone acetylation and gene methylation at the promoter level. Treatment with LAQ824 restored retinoid sensitivity by reverting RARb2 epigenetic silencing. The biological effect of LAQ824 was associated with p21 induction in both cell lines but G 2 cell cycle arrest in A2058 and apoptosis in HMV-I cell line. The induction of apoptosis by LAQ824 was associated with increased reactive oxygen species and induction of SM22 gene expression in HMV-I but not in A2058 cell line. Administration of the free radical scavenger L-N -acetylcysteine blocked LAQ824 + CRAmediated apoptosis in HMV-I cells, suggesting a primary role for reactive oxygen species generation in LAQ824 + CRA -associated lethality. Combination treatment showed 61% and 82% growth inhibition in A2058 and HMV-I tumors, respectively. Greater induction of in vivo apoptosis was observed in the HMV-I but not in the A2058 tumors treated with combination therapy compared with single agents. These results suggest that the HDAC inhibitor LAQ824 has a greater antitumor activity in combination with CRA in melanoma tumors but the degree of induced apoptosis may vary. Combination of HDAC inhibitors and retinoids represents a novel therapeutic approach for malignant melanoma that warrants clinical testing. [Mol Cancer Ther 2007;6(1):70 -81]

Taken together, these data support the clinical testing of MS-275 for the treatment of prostate cancer.