Ray M. Lee

Colchicine site tubulin inhibitors are currently developed as vascular disrupting agents (VDAs). However, they were found to have cardiotoxicity in clinical trials. To overcome the problem, we developed a stilbene derivative, cis-3, 4′, 5-trimethoxy-3′-aminostilbene (stilbene 5c), which is highly potent and has no bone marrow and cardiac toxicity in mice. Here we attempt to optimize stilbene 5c using computer-based drug design and synthesize derivatives with benzimidazole or indole group. Biological evaluation showed that they are weaker than stilbene 5c without better water solubility. Alternative approach was thus adopted to make prodrugs of stilbene 5c. A water-soluble prodrug PD7 was synthesized by addition of a morpholino group with carbamate linkage to the amino group of stilbene 5c. In vitro studies show that PD7 induces mitotic arrest and disrupts microtubule similar to stilbene 5c. The cell signaling events in Cdc2, p53, Akt, and aurora kinase are similar in cells treated with stilbene 5c, CA4 or PD7, suggesting that they share the same mechanism. Although PD7 is less effective than stilbene 5c in vitro, the biological activity of PD7 as a single agent is similar to that of stilbene 5c. Combination of PD7 with VEGF inhibitor bevacizumab significantly enhances the therapeutic efficacy of PD7 in mouse xenograft model. These data suggest that PD7 could be a good candidate for further pre-clinical and clinical development as a new VDA for cancer therapy.

Targeting tumor vasculature by colchicine site microtubule inhibitors is a new approach in cancer therapy. Here we investigate cis-3, 4′, 5-trimethoxy-3′-aminostilbene (stilbene 5c) in its effect on tumor vascular perfusion, pharmacokinetics, toxicity and therapeutic efficacy in a mouse xenograft model.

Stilbenes are a group of natural compounds with many biological activities. Two highly potent stilbenes, cis‐3,4′,5‐trimethoxy‐3′‐aminostilbene (stilbene 5c) and cis‐3,4′,5‐trimethoxy‐3′‐hydroxystilbene (stilbene 6c) induce G₂/M cell‐cycle arrest and leukemic cell death in nanomolarity range without affecting normal bone marrow progenitor cells. The mechanism of stilbenes is mediated by interfering with microtubule polymerization through the colchicine‐binding site. Docking of the stilbenes into tubulin structure confirms that stilbenes fit into the colchicine‐binding pocket. Animal studies show that stilbenes are well tolerated in mice and are capable of inducing more than 50% leukemic cell death by a single dose injection. A 5‐day treatment with low‐dose stilbenes suppresses tumor growth in mice with established tumor xenografts. No major organ damage was detected by histological section. Our results indicate that stilbene 5c is a microtubule‐interfering agent and can be potentially useful in leukemic therapy. Am. J. Hematol., 2008.

Phospholipid scramblase 3 (PLS3) is a member of the phospholipid scramblase family present in mitochondria. PLS3 plays an important role in regulation of mitochondrial morphology, respiratory function, and apoptotic responses. PLS3 is phosphorylated by PKC‐δ at Thr21 and is the mitochondrial target of PKC‐δ‐induced apoptosis. Cells with overexpression of PLS3, but not the phosphoinhibitory mutant PLS3(T21A), are more susceptible to apoptosis induced by AD198, an extranuclear targeted anthracycline that activates PKC‐δ. Here we report that the phosphomimetic mutant of PLS3(T21D) by itself can induce apoptosis in HeLa cells. Using proteoliposomes with addition of pyrene‐labeled phosphatidylcholine (PC) at the outer leaflet, we measured the lipid flip‐flop activity of PLS3 and its phosphorylation mutant. PLS3(T21D) is more potent than wild‐type PLS3 or PLS3(T21A) to transfer pyrene‐PC from the outer leaflet to the inner leaflet of liposomes. Based on our previous finding that PLS3 enhances tBid‐induced mitochondrial damages, we tested the hypothesis that PLS3 enhances cardiolipin translocation to mitochondrial surface and facilitates tBid targeting. Fluorescein‐labeled tBid(G94E) was used as a probe to quantify cardiolipin on the surface of mitochondria. Mitochondria from cells treated with AD198 or cells expressing PLS3(T21D) had a higher level of tBid‐binding capacity than control cells or cells expressing wild‐type PLS3. These findings indicate that phosphorylation of PLS3 by PKC‐δ induces PLS3 activation to facilitate mitochondrial targeting of tBid and apoptosis. J. Cell. Biochem. 101:1210–1221, 2007.

Proliferation of smooth muscle cells (SMCs) contributes to the stenosis of coronary arteries and vascular grafts. Local delivery of anti‐proliferative drugs can prevent vascular stenosis. To understand the cellular responses to anti‐proliferative agents, we investigated the signaling events in cultured human aortic SMCs (ASMCs), saphenous venous SMCs (VSMCs), and dermal fibroblasts (DFs) in response to paclitaxel or etoposide. Cellular mitochondrial and proliferative activities were examined with the methylthiazoletetrazolium (MTT) dye reduction and the bromodeoxyuridine (BrdU) incorporation assay, respectively. Cell proliferation was almost completely suppressed by paclitaxel or etoposide, but apoptosis was achieved in only about 50% of cells at the highest drug concentrations, suggesting the presence of compensatory mechanisms to prevent apoptosis. Examination of three important signaling pathways revealed significant differences between ASMCs, VSMCs, and DFs. Treatment with either paclitaxel or etoposide caused a transient phosphorylation/activation of p42 MAPK in ASMCs and DFs, but had no effect on phospho‐p42/44 MAPK in VSMCs. High‐dose etoposide enhanced p38 MAPK activation in ASMCs, but not in VSMCs. The p38 inhibitor, PD169316, partially inhibited etoposide‐induced ASMC apoptosis, but induced apoptosis in VSMCs. The effects of etoposide and paclitaxel on Akt also differed between ASMCs and VSMCs. These observations indicate that ASMCs and VSMCs differ in the response of signaling pathways to anti‐proliferative agents. In ASMCs, p42/44 MAPK appears to serve a pro‐survival role, whereas p38 MAPK is a pro‐apoptotic regulator. In contrast, p38 MAPK is an important pro‐survival regulator in VSMCs and p42/44 MAPK appears to play a minor role in responding to anti‐proliferative drugs. J. Cell. Biochem. 99: 835–844, 2006.