Curcumin analog cytotoxicity against breast cancer cells: exploitation of a redox-dependent mechanism.

A series of novel curcumin analogs, symmetrical dienones, were previously shown to possess cytotoxic, anti-angiogenic and anti-tumor activities. Analogs 1 (EF24) and 2 (EF31) share the dienone scaffold and serve as Michael acceptors. We propose that the anti-cancer effects of 1 and 2 are mediated in part by redox-mediated induction of apoptosis. In order to support this concept, 1 and 2 were treated with L-glutathione (GSH) and cysteine-containing dipeptides under mild conditions to form colorless water-soluble adducts, which were identified by LC/MS. Comparison of the cytotoxic action of 1, 2 and the corresponding conjugates, 1-(GSH)(2) and 2-(GSH)(2), illustrated that the two classes of compounds exhibit essentially identical cell killing capabilities. Compared with the yellow, somewhat light sensitive and nearly water insoluble compounds 1 and 2, the glutathione conjugates represent a promising new series of stable and soluble anti-tumor pro-drugs.

Targeting tissue factor-expressing tumor angiogenesis and tumors with EF24 conjugated to factor VIIa.

Tissue factor (TF) is aberrantly expressed on tumor vascular endothelial cells (VECs) and on cancer cells in many malignant tumors, but not on normal VECs, making it a promising target for cancer therapy. As a transmembrane receptor for coagulation factor VIIa (fVIIa), TF forms a high-affinity complex with its cognate ligand, which is subsequently internalized through receptor-mediated endocytosis. Accordingly, we developed a method for selectively delivering EF24, a potent synthetic curcumin analog, to TF-expressing tumor vasculature and tumors using fVIIa as a drug carrier. EF24 was chemically conjugated to fVIIa through a tripeptide-chloromethyl ketone. After binding to TF-expressing targets by fVIIa, EF24 will be endocytosed along with the drug carrier and will exert its cytotoxicity. Our results showed that the conjugate inhibits vascular endothelial growth factor-induced angiogenesis in a rabbit cornea model and in a Matrigel model in athymic nude mice. The conjugate-induced apoptosis in tumor cells and significantly reduced tumor size in human breast cancer xenografts in athymic nude mice as compared with the unconjugated EF24. By conjugating potent drugs to fVIIa, this targeted drug delivery system has the potential to enhance therapeutic efficacy, while reducing toxic side effects. It may also prove to be useful for treating drug-resistant tumors and micro-metastases in addition to primary tumors.

Diastereomers of dibromo-7-epi-10-deacetylcephalomannine: crowded and cytotoxic taxanes exhibit halogen bonds.

The diastereomers of dibromo-7-epi-10-deacetylcephalomannine (6 and 7) have been isolated and characterized. Cytotoxicity and microtubule assembly assays demonstrate that cephalomannine analogue 6 possesses a potency profile very similar to that of Taxol, while isomer 7 is slightly less active. Solid state, solution, and tubulin-bound conformations of the two diastereomers were probed by using X-ray crystallography, 2-D NMR experiments in conjunction with the NAMFIS analysis, and the Glide docking protocol. In the crystal, isomer 7 exhibits an intermolecular halogen bond that may contribute to self-assembly. Neither crystal structure appears in the NAMFIS solution analysis, but both diastereomers are represented in solution by a T-shaped Taxol conformer. Glide docking demonstrates the latter to best fill the tubulin binding pocket, as has been shown for the parent Taxol drug. Each model of the bound complexes for 6 and 7 presents a single well-defined halogen bond from one of the ligand's bromines to Glu22 or Asp26 near the N-terminus of beta-tubulin, respectively. This first report of a halogen bond between taxanes and tubulin may prove useful in guiding the design and synthesis of other microtubule-stabilizing agents with a similar capacity.

Synthesis of EF24-tripeptide chloromethyl ketone: a novel curcumin-related anticancer drug delivery system.

The blood coagulation cascade includes a step in which the soluble protein, factor VIIa (fVIIa), complexes with its transmembrane receptor, tissue factor (TF). The fVIIa/TF protein-protein complex is subsequently drawn into the cell by endocytosis. The observation that TF is aberrantly and abundantly expressed on many cancer cells offers an opportunity to specifically target those cells with an effective anticancer drug. Thus, we propose a new drug delivery system, drug-linker-Phe-Phe-Arg-mk-fVIIa, which can associate with TF on the surface of cancer cells, but release the cytotoxic agent in the cytoplasm. Synthetic procedures have been developed for the preparation of phenylalanine-phenylalanine-arginine chloromethyl ketone, (FFRck) followed by coupling with the cytotoxin EF24 and subsequently fVIIa to give EF-24-FFRmk-fVIIa. When breast cancer cells (MDA-MB-231) and human melanoma cells (RPMI-7951) are treated with the complex, the cells are arrested to a greater extent than EF24 alone by comparison with controls.

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

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.

Targeted delivery of paclitaxel to tumor cells: synthesis and in vitro evaluation.

We previously reported a novel drug delivery system, drug-linker-Phe-Phe-Arg-methylketone (FFR-mk)-factor VIIa (fVIIa). The method utilizes tissue factor (TF), which is aberrantly and abundantly expressed on many cancer cells. The advantage of this delivery system is its ability to furnish a potent anticancer drug specifically to the tumor vasculature and cancer cells. In this paper, we describe the synthesis of paclitaxel (PTX)-Phe-Phe-Arg-chloromethyl ketone (FFR-ck), followed by coupling with fVIIa to form PTX-FFR-mk-fVIIa. FFRck was separately linked to the OH groups at the C2' or C7 positions of PTX (C2'- or C7-PTX-FFRck), the C2' analogue exhibiting better activity against human head and neck squamous KB 3-1 cells. The activity order against PTX-sensitive KB 3-1 cells is C2'-PTX-FFRmk-fVIIa > PTX > C2'-PTX-FFRck. The C2' complex shows an IC(50) of 12 nM against the PTX-sensitive cell line and 130 nM against PTX-resistant cells.

EF24, a novel synthetic curcumin analog, induces apoptosis in cancer cells via a redox-dependent mechanism.

In this study, we show that the novel synthetic curcumin analog, EF24, induces cell cycle arrest and apoptosis by means of a redox-dependent mechanism in MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells. Cell cycle analysis demonstrated that EF24 causes a G2/M arrest in both cell lines, and that this cell cycle arrest is followed by the induction of apoptosis as evidenced by caspase-3 activation, phosphatidylserine externalization and an increased number of cells with a sub-G1 DNA fraction. In addition, we demonstrate that EF24 induces a depolarization of the mitochondrial membrane potential, suggesting that the compound may also induce apoptosis by altering mitochondrial function. EF24, like curcumin, serves as a Michael acceptor reacting with glutathione (GSH) and thioredoxin 1. Reaction of EF24 with these agents in vivo significantly reduced intracellular GSH as well as oxidized GSH in both the wild-type and Bcl-xL overexpressing HT29 human colon cancer cells. We therefore propose that the anticancer effect of a novel curcumin analog, EF24, is mediated in part by redox-mediated induction of apoptosis.