Flexible heteroarotinoid (Flex-Het) SHetA2 inhibits angiogenesis in vitro and in vivo

Abstract

Flexible heteroarotinoids (Flex-Hets) compounds regulate growth, differentiation and apoptosis in cancer cells. The hypothesis of this study was that the lead Flex-Het, SHetA2, inhibits angiogenesis by blocking cytokine release from cancer cells. SHetA2 altered secretion of thrombospondin-4 (TSP-4), vascular endothelial growth factor A (VEGF) and fibroblast growth factor (bFGF) proteins from normal and cancerous ovarian and renal cultures. Thymidine phosphorylase (TP) expression was inhibited in cancer, but not normal cultures. Endothelial tube formation was stimulated by conditioned media from cancer but not normal cultures, and SHetA2 reduced secretion of this angiogenic activity. SHetA2 directly inhibited endothelial cell tube formation and proliferation through G1 cell cycle arrest, but not apoptosis. Recombinant TP reversed SHetA2 anti-angiogenic activity. SHetA2 inhibition of in vivo angiogenesis was observed in Caki-1 renal cancer xenografts. In conclusion, SHetA2 inhibits angiogenesis through alteration of angiogenic factor secretion by cancer cells and through direct effects on endothelial cells.

Fig. 1

Differentiation and apoptosis in A2780 organotypic cultures used for microarrays (a). Twelve A2780 organotypic cultures were grown in the absence of drug. After 10 days of growth, six of the cultures were treated with 1 μM SHetA2 and six were treated with the same volume of solvent. After 2 weeks of treatment with drug or solvent, one untreated and one treated culture were fixed in formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). Photomicrographs representative of the entire cultures are presented in this figure (×40). The other collagen gels were digested with collagenase, the cells were pelleted, and the RNA was isolated for microarray and validation analysis. The arrows point to nuclei undergoing early apoptosis in both apoptotic clump (A) and differentiating clump (D). Regulation of TP (b) and TSP-4 (c) mRNA and protein expression. A2780, SK-OV-3, Caki-1, HK-2, and D1 cells were treated with the indicated doses of SHetA2 for 24 h or 10 μM SHetA2 for the indicated amount of time. RNA extracts from A2780 cultures were evaluated using rt-PCR. Protein extracts from the indicated cell lines were evaluated by Western blot analysis for time– and dose–response effects on TP expression by SHetA2. TSP-4 was immunoprecipitated from conditioned media and evaluated by Western blot

Fig. 2

Regulation of bFGF and VEGF mRNA and protein expression. RNA isolated from A2780 cultures treated with 10 μM SHetA2 or solvent for the indicated time was analyzed by rt-PCR for bFGF (a) and VEGF (b) expression. Results or rtPCR experiments presented are representative of two independent experiments performed in triplicate. Media conditioned by incubation with A2780 cultures and other indicated cultures were evaluated for bFGF (a) or VEGF (b) protein secretion by ELISA. ELISA results are averages of three independent experiments performed in triplicate

Fig. 3

Regulation of angiogenic factor secretion from epithelial cultures. The effects of the indicated types of conditioned media on endothelial tube branches are documented by photomicrographs (a) and quantification of endothelial branches (b and c). The results from three independent experiments performed in triplicate were averaged. Significant differences with P values <0.05 were determined by a two-tailed t test in b and by ANOVA in c

Fig. 4

Direct regulation of endothelial tube branching. Photomicrographs and quantification of branches in Eahy.926 cultures plated on Matrigel and treated with the indicated concentrations of SHetA2 (a). Photomicrographs and quantification of branches in HUVEC cultures plated on Matrigel and treated with the indicated concentrations of SHetA2 in the presence and absence of 125 mg/mL TP (b). Each endothelial tube formation graph represents the average and standard error of four independent experiments performed in triplicate. Cell cycle (c) and apoptosis (d) profiles of HUVEC cultures treated with solvent or the indicated micromolar concentrations of SHetA2 in the presence and absence of 125 mg/mL TP as determined by flow cytometric analysis

Fig. 5

Regulation of angiogenic cytokines in HUVEC cultures. HUVEC cultures were treated with 5 μM or the indicated doses of SHetA2 for 24 h. RNA isolated from the cultures was evaluated for expression of TP (a) and VEGF (c) mRNA using rt-PCR. Results represent the average and standard error of three independent experiments performed in triplicate. Protein extracts were evaluated by Western blot analysis for TP expression (b). Western blot results represent the average and standard error of two independent experiments. Data from one experiment was multiplied by a factor of 2 to normalize the results to the second experiment for presentation on a single graph. Conditioned media was evaluated by ELISA for VEGF expression (d). ELISA results represent the average and standard error of three independent experiments performed in triplicate

Fig. 6

In vivo regulation of angiogenesis in Caki-1 xenograft tumors. H&E stained sections (a) demonstrate hemorrhagic necrosis in tumors for the untreated control group and ischemic necrosis in the tumors from the group gavaged with 60 mg/kg/day SHetA2. The photographs of untreated tumors show the carcinoma invading the venous system and causing massive backup of blood and necrosis that is hemorrhagic. The last photo of this panel shows a dilated blood vessel in the midst of a large area of necrosis. The photographs of the treated tumors show an ischemic type necrosis with little or no blood. This is caused by the blood supply being interrupted and occurs almost totally in the treated animals. Trichrome staining in of the sections (b) shows blockage and increased disorganization of blood vessels in the treated tumors in comparison to the controls. In untreated tumors, there is a certain degree of vascular organization where blood vessels penetrate the tumor from the edge and go toward the center. In general, smaller blood vessels extend from larger vessels and travel parallel to the tumor surface. In SHetA2 treated tumors show marked vascular disorganization. There is a greater amount of necrosis in areas not nourished by vessels and many necrotic areas that have blood vessels running through. Immunohistochemical analysis of TP expression (c) documents up-regulation of TP expression at the end of the 30-day treatment period. Western blot analysis of protein extracts taken from the tumors (d) confirms the TP upregulation