Shijun Zhu

Novel synthetic curcumin analogues EF31 and UBS109 are potent DNA hypomethylating agents in pancreatic cancer

DNA methylation is a rational therapeutic target in pancreatic cancer. The activity of novel curcumin analogues EF31 and UBS109 as demethylating agents were investigated. MiaPaCa-2 and PANC-1 cells were treated with vehicle, curcumin, EF31 or UBS109. EF31 and UBS109 resulted in significantly higher inhibition of proliferation and cytosine methylation than curcumin. Demethylation was associated with re-expression of silenced p16, SPARC, and E-cadherin. EF31 and UBS109 inhibited HSP-90 and NF-κB leading to downregulation of DNA methyltransferase-1 (DNMT-1) expression. Transfection experiments confirmed this mechanism of action. Similar results were observed in vitro when subcutaneous tumors (MiaPaCa-2) were treated with EF31 and UBS109.

Antiangiogenic effects of a novel synthetic curcumin analogue in pancreatic cancer

Hypoxia-inducible factors (HIFs) and NF-κB play essential roles in cancer cell growth and metastasis by promoting angiogenesis. Heat shock protein 90 (Hsp90) serves as a regulator of HIF-1α and NF-κB protein. We hypothesized that curcumin and its analogues EF31 and UBS109 would disrupt angiogenesis in pancreatic cancer (PC) through modulation of HIF-1α and NF-κB. Conditioned medium from MIA PaCa-2 or PANC-1 cells exposed to curcumin and its analogues in vitro significantly impaired angiogenesis in an egg CAM assay and blocked HUVEC tube assembly in comparison to untreated cell medium. In vivo, EF31 and UBS109 blocked the vascularization of subcutaneous matrigel plugs developed by MIA PaCa-2 in mice. Significant inhibition of VEGF, angiopoietin 1, angiopoietin 2, platelet derived growth factor, COX-2, and TGFβ secretion was observed in PC cell lines treated with UBS109, EF31 or curcumin. Treatment with UBS109, EF31 or curcumin inhibited HSP90, NF-κB, and HIF-1α transcription in PC cell lines. UBS109 and EF31 inhibited HSP90 and HIF-1α expression even when elevated due to NF-κB (p65) overexpression. Finally, we demonstrate for the first time that curcumin analogues EF31 and UBS109 induce the downregulation of HIF-1α, Hsp90, COX-2 and VEGF in tumor samples from xenograft models compared to untreated xenografts. Altogether, these results suggest that UBS109 and EF31 are potent curcumin analogues with antiangiogenic activities.

Synthetic curcumin analog EF31 inhibits the growth of head and neck squamous cell carcinoma xenografts

Objectives are to examine the efficacy, pharmacokinetics, and toxicology of a synthetic curcumin analog EF31 in head and neck squamous cell carcinoma. The synthesis of EF31 was described for the first time. Solubility of EF24 and EF31 was compared using nephelometric analysis. Human head and neck squamous cell carcinoma Tu212 xenograft tumors were established in athymic nude mice and treated with EF31 i.p. once daily five days a week for about 5-6 weeks. The long term effect of EF31 on the NF-κB signaling system in the tumors was examined by Western blot analysis. EF31 at 25 mg kg(-1), i.p. inhibited tumor growth almost completely. Solubilities of EF24 and EF31 are <10 and 13 μg mL(-1) or <32 and 47 μM, respectively. The serum chemistry profiles of treated mice were within the limits of normal, they revealed a linear increase of C(max). EF31 decreased the level of phosphorylation of NF-κB p65. In conclusion, the novel synthetic curcumin analog EF31 is efficacious in inhibiting the growth of Tu212 xenograft tumors and may be useful for treating head and neck squamous cell carcinoma. The long term EF31 treatment inhibited NF-κB p65 phosphorylation in xenografts, implicating downregulation of cancer promoting transcription factors such as angiogenesis and metastasis.

Liver S9 Fraction-Derived Metabolites of Curcumin Analogue UBS109

To address the shortcomings of the natural product curcumin, many groups have created analogues that share similar structural features while displaying superior properties, particularly in anticancer drug discovery. Relatively unexplored have been the mechanisms by which such compounds are metabolized. A comprehensive in vitro study of a curcumin analogue (UBS109) in liver S9 fractions from five different species is presented. Further, we examine the cell-based bioactivity of the major metabolites. In spite of the fact that UBS109 reduces tumor growth in mice, it is quickly metabolized in vitro and 94% protein bound in mouse plasma. The primary monounsaturated metabolite is only modestly bioactive against MDA-MB-231 breast cancer cells. These observations suggest that while the α,β-unsaturated ketone common to curcumin analogues is important for bioactivity, protein binding and tissue distribution may serve to protect UBS109 from full metabolism in vivo while allowing it to exert a pharmacological effect by means of slow drug release.

Abstract 3828: Potent curcumin analogues inhibit pancreatic cancer cell growth and angiogenesis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Curcumin, a component of turmeric, has demonstrated anti-cancer properties mediated through inhibition of NF-κB in preclinical models. In clinical trials, curcumin failed to demonstrate activity against pancreatic cancer. The pharmacologic properties, low absorption, and low potency of curcumin may account for this discrepancy between preclinical and clinical results. Using the curcumin molecular model, our group synthesized over 100 analogs. Of these analogs, EF31 and UBS109 demonstrated higher potency in squamous cell cancer xenograft models as well as inhibition of NF-κB signaling pathways. Based on this preclinical data, we hypothesized that these analogs EF-31 and UBS-109 would be more effective in inhibiting growth of pancreatic cell lines than curcumin. Methods: MiaPaCa-2 and PANC-1 cell lines were used in the experiments. Cells were cultured and then treated with nothing (control), curcumin, UBS-109, EF-31, 5FU, or combination. Cell proliferation was evaluated using XTT assay. Western blot was used to evaluate effects on HSP90, HIF-1α, and VEGF levels. Angiogenic effects were measured by tube formation and chicken egg chorioallantoic membrane (Egg CAM) assays. Results: UBS-109 and EF-31 demonstrated significantly improved inhibition of cell proliferation as compared to curcumin. UBS-109 and EF-31 potentiated the growth inhibitory effects of 5FU. UBS-109 and EF-31 resulted in significant inhibition of expression of protein and mRNA for HSP90 and HIF-1α as compared to curcumin. In turn, UBS-109 and EF-31 significantly inhibited VEGF protein and mRNA expression resulting in inhibition of HUVEC tube formation in vitro and also inhibited angiogenesis in vivo using the Egg CAM assay. Conclusion: UBS-109 and EF-31 are more potent analogs of curcumin. Preliminary data demonstrate potentiation of chemotherapy effects as well as antiangiogenic effects. Evaluating UBS-109 and EF-31 in patients with pancreatic cancer is a rational approach to target HSP90 and HIF-1α in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3828. doi:1538-7445.AM2012-3828

Bioactive flavonoid p-hydroxycinnamic acid stimulates osteoblastogenesis and suppresses adipogenesis in bone marrow culture

The bioactive flavonoid p-hydroxycinnamic acid (HCA), which is an intermediate-metabolic substance in plants and fruits, is synthesized from tyrosine. The biological effect of HCA is poorly understood. Among cinnamic acid and its related compounds, HCA has a specific-anabolic effect on bone, being found to stimulate osteoblastogenesis and to inhibit osteoclastogenesis through the suppression of NF-κB signaling, thereby preventing bone loss. Bone marrow mesenchymal stem cells give rise to ostoblasts and adipocytes. HCA might therefore have effects on osteoblastogenesis and adipogenesis in bone marrow culture. This study demonstrates (1) that HCA has stimulatory effects on osteoblastogenesis and mineralization and suppressive effects on adipogenesis in mouse bone marrow culture and (2) that HCA depresses adipogenesis in mouse 3T3-L1 preadipocytes in vitro. Such effects of HCA might be involved in the differentiation of mesenchymal stem cells.

Synthetic Curcumin Analog UBS109 Inhibits the Growth of Head and Neck Squamous Cell Carcinoma Xenografts

The natural compound curcumin has been investigated as an anticancer agent in many cellular systems, in animal models and in the clinic. The overriding negative characteristics of curcumin are its low solubility, weak potency and poor bioavailability. We have examined the efficacy and mechanism of action of a synthetic curcumin analog, UBS109, in head and neck squamous cell carcinoma. By nephelometry, this analog exhibits considerably greater solubility than curcumin. Pharmacokinetic studies of a single oral dose of UBS109 in mice revealed that peak plasma concentrations were reached at 0.5 hours post-dose (Tmax) with average plasma concentrations (Cmax) of 131 and 248 ng/mL for oral doses of 50 and 150 mg/kg, respectively. The terminal elimination half-lives (T½) for these doses averaged 3.7 and 4.5 hours, respectively. In both in vitro and in vivo studies, we found that UBS109 decreased the levels of phosphorylated IKKβ and phosphorylated p65 and, unexpectedly, increased the levels of phosphorylated IκBα by Western blot analysis. These observations may suggest that UBS109 suppresses tumor growth through, in part, inhibition of NF-κB p65 phosphorylation by PKAc and not through IκBα. Finally, we demonstrate that UBS109 is efficacious in retarding the growth of Tu212 (head and neck) squamous cell carcinoma (SCC) xenograft tumors in mice and may be useful for treating head and neck SCC tumors.

Visualizing cancer and response to therapy in vivo using Cy5.5-labeled factor VIIa and anti-tissue factor antibody

Abstract We have developed a specific technique for imaging cancer in vivo using Cy5.5-labeled factor VIIa (fVIIa), clotting-deficient FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-tissue factor (TF) antibody. FVIIa is the natural ligand for TF. We took advantage of the fact that vascular endothelial cells (VECs) in cancer, but not normal tissue, aberrantly express TF due to its induction by vascular endothelial growth factor (VEGF). Under physiological conditions, TF is expressed by stromal cells and outer blood vessel layers (smooth muscle and adventitia), but not by VECs. We hypothesized that labeled fVIIa or anti-TF antibodies could be used to image the tumor vasculature in vivo. To test this, Cy5.5-labeled fVIIa, FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-TF antibody were developed and administered to athymic nude mice carrying xenografts including glioma U87EGFRviii, pancreatic cancer ASPC-1 and Mia PaCa-2, and squamous cell carcinoma KB-V1. Cy5.5 labeled with these targeting proteins specifically localized to the tumor xenografts for at least 14 days but unconjugated Cy5.5 did not localize to any xenografts or organs. This method of imaging TF in the tumor VECs may be useful in detecting primary tumors and metastases as well as monitoring in vivo therapeutic responses.

Tumor Angiogenesis Therapy Using Targeted Delivery of Paclitaxel to the Vasculature of Breast Cancer Metastases

Breast cancer aberrantly expresses tissue factor (TF) in cancer tissues and cancer vascular endothelial cells (VECs). TF plays a central role in cancer angiogenesis, growth, and metastasis and, as such, is a target for therapy and drug delivery. TF is the cognate receptor of factor VIIa (fVIIa). We have coupled PTX (paclitaxel, also named Taxol) with a tripeptide, phenylalanine-phenylalanine-arginine chloromethyl ketone (FFRck) and conjugated it with fVIIa. The key aim of the work is to evaluate the antiangiogenic effects of PTX-FFRck-fVIIa against a PTX-resistant breast cancer cell line. Matrigel mixed with VEGF and MDA-231 was injected subcutaneously into the flank of athymic nude mice. Animals were treated by tail vein injection of the PTX-FFRck-fVIIa conjugate, unconjugated PTX, or PBS. The PTX-FFRck-fVIIa conjugate significantly reduces microvessel density in matrigel (p < 0.01–0.05) compared to PBS and unconjugated PTX. The breast cancer lung metastasis model in athymic nude mice was developed by intravenous injection of MDA-231 cells expressing luciferase. Animals were similarly treated intravenously with the PTX-FFRck-fVIIa conjugate or PBS. The conjugate significantly inhibits lung metastasis as compared to the control, highlighting its potential to antagonize angiogenesis in metastatic carcinoma. In conclusion, PTX conjugated to fVIIa is a promising therapeutic approach for improving selective drug delivery and inhibiting angiogenesis.

Abstract 684: Novel potent curcumin analogues EF-31 and UBS109 inhibit DNA methylation and induce SPARC expression in pancreatic cancer.

Purpose/Objectives: DNA methylation is an essential step in epigenetic modification and carcinogenesis. HSP90 and NF-κB have a central role in DNA methylation. Curcumin down regulates HSP90 and NF-kB. UBS109 and EF-31 are potent synthetic analogues of curcumin. We tested the hypothesis that EF-31 and UBS-109 can inhibit DNA methylation and induce SPARC through inhibition of NF-kB and HSP90 in pancreatic cancer cell lines. Methods: Two pancreatic cancer cell lines were used, PANC-1 and Mia-PaCa2. Cells were either untreated (control) or treated with curcumin (20μM), EF-31 (750nM) and UBS-109 (250nM) for 24 hours. Western blot and RT-PCR analyses were carried out to determine the effect of curcumin and its analogues EF-31 and UBS-109 on different signal molecules involved in carcinogenesis. Electromobility shift assay (EMSA) was performed in pancreatic cells to evaluate the effects of ganetespib on NF-kB activity. Immunocyto and histo chemistry assays were also performed. Results: The treatment of pancreatic cancer cell lines with curcumin and its analog EF-31 and UBS-109 inhibited NF-κB and HSP90 at transcriptional and translational level resulting in inhibition of DNMT1, decrease in CpG island methylation and up-regulation of SPARC. EF-31 and UBS109 were more potent at inhibition of DNMT1 than curcumin. To confirm that the observed effects were mediated by NF-kB, a stable transfection with p65 (NF-kB) was performed. Cells with overexpression of NF-κB resulted in increased DNMT-1, HSP90 protein and decreased SPARC. Inhibition of NF-kB using EF31 or UBS109 resulted in up-regulation of SPARC and inhibition of the DNMT due to NF-κB down-regulation. Similar effects were observed in tumors from animals treated with EF-31 and UBS-109. Conclusion: UBS109, EF31 and curcumin can inhibit DNA methylation through down-regulation of DNMT-1 expression by NF-kB. Inhibition of methylation was associated with expression of silenced genes such as SPARC. UBS109 and EF31 are significantly more potent than curcumin. UBS109 and EF31 are promising agents for therapy in pancreatic cancer. Citation Format: Purnachandra Nagaraju Ganji, Shijun Zhu, Shoji Mamoru, Bassel El-Rayes. Novel potent curcumin analogues EF-31 and UBS109 inhibit DNA methylation and induce SPARC expression in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 684. doi:10.1158/1538-7445.AM2013-684