Structure-based design of Aurora A & B inhibitors.

The Aurora family of serine/threonine kinases are mitotic regulators involved in centrosome duplication, formation of the bipolar mitotic spindle and the alignment of the chromosomes along the spindle. These proteins are frequently overexpressed in tumor cells as compared to normal cells and are therefore potential therapeutic oncology targets. An Aurora A high throughput screen revealed a promising sub-micromolar indazole-benzimidazole lead. Modification of the benzimidazole portion of the lead to a C2 linker with a phenyl ring was proposed to achieve novelty. Docking revealed that a conjugated linker was optimal and the resulting compounds were equipotent with the lead. Further structure-guided optimization of substituents on the 5 & 6 position of the indazole led to single digit nanomolar potency. The homology between the Aurora A & Aurora B kinase domains is 71% but their binding sites only differ at residues 212 & 217 (Aurora A numbering). However interactions with only the latter residue may be used for obtaining selectivity. An analysis of published Aurora A and Aurora B X-ray structures reveals subtle differences in the shape of the binding sites. This was exploited by introduction of appropriately sized substituents in the 4 & 6 position of the indazole leading to Aurora B selective inhibitors. Finally we calculate the conformational energy penalty of the putative bioactive conformation of our inhibitors and show that this property correlates well with the Aurora A binding affinity.

Structural characterization of three novel rat OKL38 transcripts, their tissue distributions, and their regulation by human chorionic gonadotropin.

We previously identified a novel pregnancy-induced growth inhibitory gene, OKL38. To develop a rat model for further characterization of OKL38's role in the initiation and progression of breast and ovarian cancer, we now report the cloning and characterization of three novel rat OKL38 cDNAs that are derived through alternative splicing and differential promoter usage. These three transcripts differ in their 5' untranslated regions but share a common open reading frame that encoded for a 52-kDa protein. OKL38 is mapped to chromosome 19, spanning a region of approximately 15 kb, and contains eight exons. Differential expression of these three rat OKL38 transcripts was observed in liver, kidney, ovary, mammary gland, and uterus. In situ hybridization localized the rat OKL38 transcripts to the luminal epithelial cells of the rat mammary gland and to the granulosa cells in the rat ovary. In vivo studies showed that the RtOKL38-2.0 transcript and protein were regulated by human chorionic gonadotropin in the rat mammary gland and ovary. Importantly, overexpression of RtOKL38-enhanced green fluorescence protein fusion protein in Buffalo rat liver cells resulted in growth inhibition and cell death. Our present findings suggest that OKL38 may function as an effector for human chorionic gonadotropin protection against mammary carcinogenesis, and the availability of the three rat OKL38 cDNAs may help to elucidate the possible role of OKL38 in cellular growth, differentiation, and carcinogenesis.

Molecular cloning, characterization and isolation of novel spliced variants of the human ortholog of a rat estrogen-regulated membrane-associated protein, UO-44.

We have previously reported the characterization of an estrogen-regulated rat uterine-ovarian-specific complementary DNA (UO-44). To understand the involvement of this protein in the initiation and progression of human ovarian and uterine cancers, we now report the cloning and characterization of the human ortholog (HuUO-44). HuUO-44 is mapped to chromosome 10q26.13 and contains nine exons. Multiple tissue Northern blot detected two HuUO-44 transcripts of approximately 2 and 3 kb in the pancreas. RT-PCR demonstrated that HuUO-44 undergoes a complex series of alternative splicing events between exons 2 and 6 that yielded four novel splice variants, HuUO-44A, HuUO-44B, HuUO-44C and HuUO-44D. Putative functional motifs identified in HuUO-44 are two CUB domains and a zona-pellucida domain. Transfection studies demonstrated the membrane-associated nature of HuUO-44. By immunohistochemistry, HuUO-44 was located to the normal ovarian and ovarian tumor epithelial cells; in NIH-OVCAR3 ovarian cancer cells, HuUO-44 was detected only at the leading edge of the dividing cells. Most importantly, a marked loss in cell attachment and proliferation was observed in NIH-OVCAR3 cells cultured in the presence of a polyclonal HuUO-44 antiserum. These findings suggest the potential role of HuUO-44 in cell motility, cell-cell interactions and/or interactions with the extracellular matrices.

Reduction of CWR22 prostate tumor xenograft growth by combined tamoxifen-quercetin treatment is associated with inhibition of angiogenesis and cellular proliferation.

Combination chemotherapy is increasingly practiced for the treatment of malignant prostate cancers. The aim of this study was to evaluate the in vivo efficacy of combined tamoxifen and quercetin in prostate tumor xenografts. Severe combined immune deficient (SCID) mice inoculated with CWR22 prostate tumor cells were treated with either tamoxifen (10 mg/kg/week), quercetin (200 mg/kg/day) or combined tamoxifen-quercetin for 28 days. Tamoxifen or quercetin alone exhibited a moderate antitumor activity. Tamoxifen decreased the Ki-67 index by 52.4%, reduced the vascular endothelial growth factor (VEGF) 121 and VEGF165 mRNA by 18.6 and 21.8%, respectively, and suppressed the blood vessel formation, while quercetin modulated the expression and phosphorylation of cdc-2 and cyclin B1, and inhibited the Ki-67 index by 66.0%. Combined tamoxifen-quercetin effectively delayed the appearance of tumors, inhibited the final tumor volume by 73.3% and reduced the endpoint tumor weight by 67.1% (p<0.05). The Ki-67 index, VEGF121, VEGF165 mRNA and microvessel density (MVD) were decreased by 66.9, 22.1, 40.1 and 59.0%, respectively, by the combined treatment. These findings indicate that tamoxifen inhibits CWR22 prostate tumor by modulating the angiogenesis and its antineoplastic effects can be potentiated by combined use with quercetin.

Quercetin-induced growth inhibition and cell death in nasopharyngeal carcinoma cells are associated with increase in Bad and hypophosphorylated retinoblastoma expressions.

Nasopharyngeal carcinoma is a common cancer in South-East Asia, especially among people of Chinese origin. In this report, we investigate the effects of quercetin on the growth of wild-type and mutant p53 nasopharyngeal carcinoma cell lines, HK1 and CNE2 respectively. The wild-type p53 HK1 was more susceptible to growth inhibition by quercetin than the mutant p53 CNE2. The ID50 values for HK1 and CNE2 were 35.0 and 54.5 microM respectively. Cell growth arrest was initiated by the up-regulation of retinoblastoma gene expression, resulting in cell cycle arrest in either the G2/M or G0/G1 phase at 14.8 and 52.1 microM quercetin respectively regardless of the p53 status. Flow cytometry experiments revealed that quercetin-induced apoptosis during the first 24 h followed by necrosis in both HK1 and CNE2. Western blot experiments confirmed that cytotoxic killing of HK1 and CNE2 by quercetin was mediated by the up-regulation of pro-apoptotic protein Bad, caspase-3 and -7, resulting in cell death by apoptosis. Our study demonstrates that quercetin inhibits cell growth of nasopharyngeal carcinoma cell lines HK1 and CNE2 by inhibiting cell cycle progression to S phase. Quercetin is also able to induce apoptosis and necrosis in these cells regardless of the p53 status.

Genomic structure of human OKL38 gene and its differential expression in kidney carcinogenesis.

We previously demonstrated the growth inhibitory property of OKL38 and its possible roles in mammary carcinogenesis. To further understand the regulation and roles of OKL38 in tumorigenesis we proceeded to clone and characterize the human OKL38 gene and three of its variants with transcripts of 1.9, 2.2, and 2.4 kb. The human OKL38 gene spans approximately 18 kb and contains 8 exons and 7 introns with exon size ranging from 92 to 1270 bp. RT-PCR and sequence analysis suggest that different transcripts were arrived through differential promoter usage and alternate splicing. Multiple Tissue Expression array (MTE) and Multiple Tissue Northern blot (MTN) indicated that OKL38 was ubiquitously expressed in all tissues with high expression in liver, kidney, and testis. The cancer profiling array (CPA) of paired normal/tumor cDNA showed that OKL38 mRNA was down-regulated in 70% (14 of 20) of kidney tumors. Western analysis revealed that the OKL38 protein was undetectable in 78% (7 of 9 pairs) of kidney tumor tissues. Immunohistological analysis showed that 64% (14 of 22) of kidney tumors were either lost or underexpressed OKL38 protein compared with the adjacent normal tissue. A transfection study using OKL38-eGFP recombinant construct showed that overexpression of the 52 kDa OKL38 protein in A498 cells resulted in growth inhibition and cell death. This study demonstrates the complex genomic structure of the OKL38 gene and its growth inhibitory and cytotoxic properties. Our data suggest the potential use of OKL38 in diagnosis, prognosis, and/or treatment of kidney cancer.