A novel hybrid drug between two potent anti-tubulin agents as a potential prolonged anticancer approach.

We report the design, synthesis and biological characterisation of a novel hybrid drug by conjugation of two tubulin inhibitors, a hemiasterlin derivative A (H-Mpa-Tle-Aha-OH), obtained by condensation of three non-natural amino acids, and cis-3,4',5-trimethoxy-3'aminostilbene (B). As we have previously demonstrated synergy between A and B, we used a monocarbonyl derivative of triethylene glycol as linker (L) to synthesise compounds A-L and A-L-B; via HPLC we analysed the release of its potential hydrolysis products A, A-L, B and B-L in physiological fluids: the hybrid A-L-B undergo hydrolysis in rat whole blood of the ester bond between A and L (half-life=118.2±9.5min) but not the carbamate bond between B and L; the hydrolysis product B-L was further hydrolyzed, but with a slower rate (half-life=288±12min). The compound A-L was the faster hydrolyzed conjugate (half-life=25.4±1.1min). The inhibitory activity of the compounds against SKOV3 ovarian cancer cell growth was analysed. The IC50 values were 7.48±1.27nM for A, 40.3±6.28nM for B, 738±38.5nM for A-L and 37.9±2.11nM for A-L-B. The anticancer effect of A-L-B was evidenced to be obtained via microtubule dynamics suppression. Finally, we stated the expression of the active efflux transporters P-gp (ABCB1) and MRP1 (ABCC1) in the human normal colon epithelial NCM460 cell line by reverse-transcription PCR. Via permeation studies across NCM460 monolayers we demonstrate the poor aptitude of A to interact with active efflux transporters (AET): indeed, the ratio between its permeability coefficients for the basolateral (B)→apical (A) and B→A transport was 1.5±0.1, near to the ratio of taltobulin (1.12±0.06), an hemiasterlin derivative able to elude AETs, and significantly different form the ratio of celiprolol (3.4±0.2), an AET substrate.

Hemiasterlin derivative (R)(S)(S)-BF65 and Akt inhibitor MK-2206 synergistically inhibit SKOV3 ovarian cancer cell growth.

We reported previously that a hemiasterlin derivative BF65 is a potent anticancer agent that can inhibit microtubule assembly. Here we show that a more potent stereospecific diastereomer (R)(S)(S)-BF65 can synergize with an allosteric Akt inhibitor MK-2206 to suppress the growth of SKOV3 ovarian cancer cells with constitutively active Akt. (R)(S)(S)-BF65 induced mitotic arrest and MK-2206 caused G0/G1 arrest, while the combination of both induced simultaneous G0/G1 and G2/M cell cycle arrest. (R)(S)(S)-BF65 induced phosphorylation and inactivation of Bcl-2, and downregulated Mcl-1, consequently may lead to apoptosis. (R)(S)(S)-BF65 inhibited mitogen-activated protein kinases (MAPKs), which may stimulate cell proliferation upon activation. (R)(S)(S)-BF65 also induced DNA damage after long-term treatment. MK-2206 is known to inhibit phosphorylation and activation of Akt and suppress cancer cell growth. The combination of (R)(S)(S)-BF65 and MK-2206 also inhibited the Akt pathway. Interestingly, MK-2206 upregulated Bcl-2 and induced activation of MAPKs in SKOV3 cells; however, when combined with (R)(S)(S)-BF65, these prosurvival effects were reversed. The combination also more significantly decreased Mcl-1 protein, increased PARP cleavage, and induced γ-H2AX, a DNA damage marker. Remarkably, MK-2206 enhanced the microtubule depolymerization effect of (R)(S)(S)-BF65. The combination of (R)(S)(S)-BF65 and MK-2206 also markedly inhibited cell migration. Thus, MK-2206 synergizes with (R)(S)(S)-BF65 to inhibit SKOV3 cell growth via downregulating the Akt signaling pathway, and enhancing the microtubule disruption effect of (R)(S)(S)-BF65. (R)(S)(S)-BF65 in turn suppresses Bcl-2 and MAPKs induced by MK-2206. (R)(S)(S)-BF65 and MK-2206 compensate each other leading to increased apoptosis and enhanced cytotoxicity, and may also suppress cancer cell invasion.

Development of hemiasterlin derivatives as potential anticancer agents that inhibit tubulin polymerization and synergize with a stilbene tubulin inhibitor.

Hemiasterlins are cytotoxic tripeptides with antimicrotubule activity originally isolated from marine sponges. We have developed new hemiasterlin derivatives BF65 and BF78 that are highly potent to induce cancer cell death in the low nanomolar range. Examination of their mechanisms of cell cycle arrest and disruption of microtubules revealed an unusual characteristic in addition to anti-tubulin effect. Immunofluorescence staining revealed that A549 lung carcinoma cells treated with BF65 or BF78 exhibited both monopolar and multipolar mitotic spindles. Centrosomes were separated with short spindle microtubules in cells with multipolar spindles. In vitro tubulin polymerization assay confirmed that both BF65 and BF78 were highly potent to inhibit tubulin polymerization. These two compounds induced the formation of monoastral spindles suggesting that they might be inhibitors of mitotic kinesins such as KSP/Eg5. However, kinetic measurement of microtubule activated kinesin ATPase activity demonstrated that unlike the positive control monastrol, neither BF65 nor BF78 suppressed KSP/Eg5 activity. Hence the effect may be a variant form of tubulin inhibition. Similar to vinca alkaloids, BF compounds synergized with a colchicine site microtubule inhibitor stilbene 5c both in vitro and in vivo, which may provide a potential drug combination in the future clinical application.

Versatile synthesis of new cytotoxic agents structurally related to hemiasterlins.

A representative series of structural analogues of the antimitotic tripeptides hemiasterlins have been synthesized. The key-step of this synthetic strategy consists of an Ag(2)O-promoted nucleophilic substitution on a common precursor, a chiral non-racemic 2-bromoacyl derivative. Simple variation of nucleophile substituents allows a rapid and stereocontrolled development of new series of derivatives. Some reported compounds showed potent biological activity as growth inhibitors of cancer cell lines and tubulin polymerization inhibitors.

Hydropathic analysis and biological evaluation of stilbene derivatives as colchicine site microtubule inhibitors with anti-leukemic activity.

The crucial role of the microtubule in cell division has identified tubulin as a target for the development of therapeutics for cancer; in particular, tubulin is a target for antineoplastic agents that act by interfering with the dynamic stability of microtubules. A molecular modeling study was carried out to accurately represent the complex structure and the binding mode of a new class of stilbene-based tubulin inhibitors that bind at the alphabeta-tubulin colchicine site. Computational docking along with HINT (Hydropathic INTeractions) score analysis fitted these inhibitors into the colchicine site and revealed detailed structure-activity information useful for inhibitor design. Quantitative analysis of the results was in good agreement with the in vitro antiproliferative activity of these derivatives (ranging from 3 nM to 100 muM) such that calculated and measured free energies of binding correlate with an r(2) of 0.89 (standard error +/- 0.85 kcal mol(-1)). This correlation suggests that the activity of unknown compounds may be predicted.

Hypermetabolism, hyperphagia, and reduced adiposity in tankyrase-deficient mice.

OBJECTIVE: Tankyrase (TNKS) is a Golgi-associated poly-ADP-ribose polymerase that is implicated in the regulation of GLUT4 trafficking in 3T3-L1 adipocytes. Its chromosomal locus 8p23.1 is linked to monogenic forms of diabetes in certain kindred. We hypothesize that TNKS is involved in energy homeostasis in mammals. RESEARCH DESIGN AND METHODS: Gene-trap techniques were used to ablate TNKS expression in mice. Homozygous and wild-type littermates maintained on standard chow were compared. RESULTS: Wild-type mice express the TNKS protein abundantly in adipose tissue, the brain, and the endocrine pancreas but scarcely in the exocrine pancreas and skeletal muscle. TNKS-deficient mice consume increased amounts of food (by 34%) but have decreased plasma leptin levels and a >50% reduction in epididymal and perirenal fat pad size. Their energy expenditure is increased as assessed by metabolic cage studies and core body temperatures. These changes are not attributable to an increase in physical activity or uncoupled respiration (based on oxygraph analyses of mitochondria isolated from brown fat and skeletal muscle). The heightened thermogenesis of TNKS-deficient mice is apparently fueled by increases in both fatty acid oxidation (based on muscle and liver gene expression analyses and plasma ketone levels) and insulin-stimulated glucose utilization (determined by hyperinsulinemic-euglycemic clamps). Although TNKS deficiency does not compromise insulin-stimulated GLUT4 translocation in primary adipocytes, it leads to the post-transcriptional upregulation of GLUT4 and adiponectin in adipocytes and increases plasma adiponectin levels. CONCLUSIONS: TNKS-deficient mice exhibit increases in energy expenditure, fatty acid oxidation, and insulin-stimulated glucose utilization. Despite excessive food intake, their adiposity is substantially decreased.

Mitochondrial kinases and their molecular interaction with cardiolipin.

Mitochondrial isoforms of creatine kinase (MtCK) and nucleoside diphosphate kinase (NDPK-D) are not phylogenetically related but share functionally important properties. They both use mitochondrially generated ATP with the ultimate goal of maintaining proper nucleotide pools, are located in the intermembrane/cristae space, have symmetrical oligomeric structures, and show high affinity binding to anionic phospholipids, in particular cardiolipin. The structural basis and functional consequences of the cardiolipin interaction have been studied and are discussed in detail in this review. They mainly result in a functional interaction of MtCK and NDPK-D with inner membrane adenylate translocator, probably by forming proteolipid complexes. These interactions allow for privileged exchange of metabolites (channeling) that ultimately regulate mitochondrial respiration. Further functions of the MtCK/membrane interaction include formation of cardiolipin membrane patches, stabilization of mitochondria and a role in apoptotic signaling, as well as in case of both kinases, a role in facilitating lipid transfer between two membranes. Finally, disturbed cardiolipin interactions of MtCK, NDPK-D and other proteins like cytochrome c and truncated Bid are discussed more generally in the context of apoptosis and necrosis.

Design, synthesis and biological evaluation of novel stilbene-based antitumor agents.

A series of novel stilbene derivatives has been synthesized and studied with the main goal to investigate SAR of the amino compound 1a, as well as to improve its water solubility, a potentially negative aspect of the molecule that could be a serious obstacle for a pre-clinical development. We have obtained derivatives with good cytotoxic activity, in particular, the derivatives 5c and 6b could represent two novel leads for further investigation. Compound 8b, a morpholino-carbamate derivative, prodrug of 1a, has a very good solubility in water, and is active in suppressing growth of tumor cells at a concentration of 5000 nM, which is a concentration 100 times higher than the parent stilbene 1a.

cis-3, 4', 5-Trimethoxy-3'-aminostilbene disrupts tumor vascular perfusion without damaging normal organ perfusion.

PURPOSE: 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. METHODS: Tumor xenograft model was established with subcutaneous injection of UCI-101 ovarian cancer cells into nude mice. Tumor blood perfusion was investigated by dynamic contrast-enhanced (DCE) MRI studies. Pharmacokinetic studies were performed by LC/MS/MS to quantify the concentrations of stilbene 5c in plasma. Tumor size was measured by the long and short axes of tumor to calculate tumor volume. Mouse cardiac function study was determined by Doppler echocardiography using the Vevo770TM imaging system. Microvascular density was determined by CD34 staining of tissue sections. RESULTS: Stilbene 5c selectively suppresses tumor perfusion without damaging normal organ perfusion in DCE-MRI studies. Histological sections of normal organs treated with stilbene 5c do not reveal any major toxicity in H&E staining. Microvascular density determined by CD34 staining is unchanged in normal organs, but significantly decreased in tumor after stilbene 5c treatment. Biodistribution study shows that stilbene 5c is not detectable in heart and lung, rapidly decreased in brain, liver, and kidney, but remains high in tumor for more than 3 h after IV injection of stilbene 5c, suggesting preferential accumulation in tumor. Mice treated with 5 days of stilbene 5c had negligible cardiac toxicity based on their normal left ventricular ejection fraction. In vivo efficacy study of stilbene 5c showed that it only suppresses tumor growth by 40% if used alone, but combination with bevacizumab is significantly better. CONCLUSION: Stilbene 5c is a useful vascular disrupting agent and combination with bevacizumab could be a promising therapy for cancer.

Development of water soluble derivatives of cis-3, 4', 5-trimethoxy-3'-aminostilbene for optimization and use in cancer therapy.

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.

Mechanism of cell death induced by cis-3, 4', 5-trimethoxy-3'-aminostilbene in ovarian cancer.

OBJECTIVE: Stilbene derivative, cis-3, 4', 5-trimethoxy-3'-aminostilbene (stilbene 5c), is highly potent to induce cell death in ovarian cancer cells. This study is to investigate its mechanism to induce cell death. METHODS: UCI101 ovarian cancer cells were used for this study. Cell death was analyzed by Alamar blue staining. Cell cycle was analyzed by flow cytometry after PI staining. Mitochondrial potential and reactive oxygen species were determined by MitoTracker green and DCF-DA, respectively. Immunofluorescent staining was done with tubulin antibody following by confocal microscope examination. Cell lysates were collected after treatment with stilbene 5c for Western blotting analysis of various cell cycle regulators and signal transduction mediators. RESULTS: Stilbene-treated cells die in both cell cycle-dependent and -independent pathways. Low concentration (30 nM) induces cell death without cell cycle arrest. This process involves disruption of mitochondrial potential and production of ROS by a Bcl-2-independent pathway. Higher concentration of stilbene 5c arrests cell cycle in G(2)/M phase, which is supported by dephosphorylation of Cdc2 and Cdc25C, and transiently elevation of spindle checkpoint BubR1. Although phosphorylation of Chk1 and Chk2 both increases after treatment, loss of Chk1 suppresses, whereas loss of Chk2 enhances, stilbene 5c-induced cell death. Phosphorylation of Akt and Stat3, but not MAPK, is suppressed after stilbene 5c treatment. CONCLUSION: These studies provide a mechanistic insight in using stilbenes in ovarian cancer. Stilbenes could be potentially useful agents for ovarian cancer therapy and induce cell death through mitochondrial damage and cell cycle arrest.

Role of phospholipid scramblase 3 in the regulation of tumor necrosis factor-alpha-induced apoptosis.

In tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis, tBid is targeted to mitochondria and causes cytochrome c release. We investigated the regulation of tBid-induced cytochrome c release and apoptosis by phospholipid scramblase 3 (PLS3). Overexpression of PLS3 enhanced, whereas downregulation of PLS3 delayed, TNF-alpha-induced apoptosis and targeting of tBid to mitochondria. On the basis of the theory that tBid targets mitochondrial cardiolipin, we hypothesize that PLS3 enhances translocation of cardiolipin to the mitochondrial surface to facilitate tBid targeting. NAO, a cardiolipin binding dye, was first used to quantify the distribution of cardiolipin. Overexpression of PLS3 increases, whereas downregulation of PLS3 decreases, the percentage of cardiolipin on the mitochondrial surface. Determination of the tBid binding capacity on the mitochondrial surface by FITC-labeled tBid(G94E) also confirmed that tBid binding capacity increased upon PLS3 overexpression and decreased with downregulation of PLS3. PLS3 activity, determined by a lipid flip-flop assay, was activated by calcium and tBid but inhibited by Bcl-2. Mutation of the calcium binding motif abolishes the lipid flip-flop activity of PLS3. PLS3 and tBid may form a bidirectional positive feedback loop that is antagonized by Bcl-2. Overexpression of PLS3 does not affect mitochondrial potential but does interfere with mitochondrial respiration and production of reactive oxygen species. These studies thus establish PLS3 as an important downstream effector of Bcl-2 and tBid in apoptosis.

Stilbene derivatives that are colchicine-site microtubule inhibitors have antileukemic activity and minimal systemic toxicity.

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.

Phosphorylation of mitochondrial phospholipid scramblase 3 by protein kinase C-delta induces its activation and facilitates mitochondrial targeting of tBid.

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-delta at Thr21 and is the mitochondrial target of PKC-delta-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-delta. 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-delta induces PLS3 activation to facilitate mitochondrial targeting of tBid and apoptosis.

Phospholipid scramblase-3 regulates cardiolipin de novo biosynthesis and its resynthesis in growing HeLa cells.

PLS3 (phospholipid scramblase-3) is a new member of the family of phospholipid scramblases and transports CL (cardiolipin) from the inner to the outer mitochondrial membrane. In the present paper we examined whether changing the levels of functional PLS3 in HeLa cells altered de novo CL biosynthesis and its resynthesis. HeLa cells overexpressing PLS3 or expressing a disrupted PLS3 (F258V) or control were incubated with [1,3-3H]glycerol and radioactivity incorporated into CL was determined. CL biosynthesis from [1,3-3H]glycerol was increased 1.8-fold in PLS3 cells and 2.1-fold in F258V cells compared with control. This was due to a 64% (P<0.05) and 2.6-fold (P<0.05) elevation in CL synthase activity in PLS3 and F258V cells respectively, compared with control, and not due to changes in phosphatidylglycerolphosphate synthase activity. The increase in CL synthase activity in these cells was due to an increase in its mRNA expression. In contrast, resynthesis of CL from [1-14C]linoleic acid was reduced 52% (P<0.05) in PLS3 and 45% (P<0.05) in F258V cells compared with control and this was due to a reduction in mitochondrial monolysocardiolipin acyltransferase activity. Although protein levels of mitochondrial monolysocardiolipin acyltransferase were unaltered, activity and mRNA expression of endoplasmic reticulum monolysocardiolipin acyltransferase was upregulated in PLS3 and F258V cells compared with controls. These data indicate that the CL resynthesis in HeLa cells is sensitive to the mitochondrial localization of CL and not the level of the reacylating enzymes. Alterations in functional PLS3 levels in PLS3 or F258V cells did not affect the mitochondrial decarboxylation of phosphatidylserine to phosphatidylethanolamine indicating that the biosynthetic changes to CL were specific for this mitochondrial phospholipid. We hypothesize that the cardiolipin resynthesis machinery in the cell 'senses' altered levels of CL on mitochondrial membranes and that de novo CL biosynthesis is up-regulated in HeLa cells as a compensatory mechanism in response to altered movement of mitochondrial CL. The results identify PLS3 as a novel regulator of CL de novo biosynthesis and its resynthesis.

Tankyrase recruitment to the lateral membrane in polarized epithelial cells: regulation by cell-cell contact and protein poly(ADP-ribosyl)ation.

PARsylation [poly(ADP-ribosyl)ation] of proteins is implicated in the regulation of diverse physiological processes. Tankyrase is a molecular scaffold with this catalytic activity and has been proposed as a regulator of vesicular trafficking on the basis, in part, of its Golgi localization in non-polarized cells. Little is known about tankyrase localization in polarized epithelial cells. Using MDCK (Madin-Darby canine kidney) cells as a model, we found that E-cadherin-mediated intercellular adhesion recruits tankyrase from the cytoplasm to the lateral membrane (including the tight junction), where it stably associates with detergent-insoluble structures. This recruitment is mostly completed within 8 h of calcium-induced formation of cell-cell contact. Conversely, when intercellular adhesion is disrupted by calcium deprivation, tankyrase returns from the lateral membrane to the cytoplasm and becomes more soluble in detergents. The PARsylating activity of tankyrase promotes its dissociation from the lateral membrane as well as its ubiquitination and proteasome-mediated degradation, resulting in an apparent protein half-life of approximately 2 h. Inhibition of tankyrase autoPARsylation using H2O2-induced NAD+ depletion or PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide hydrochloride] treatment results in tankyrase stabilization and accumulation at the lateral membrane. By contrast, stabilization through proteasome inhibition results in tankyrase accumulation in the cytoplasm. These data suggest that cell-cell contact promotes tankyrase association with the lateral membrane, whereas PARsylating activity promotes translocation to the cytosol, which is followed by ubiquitination and proteasome-mediated degradation. Since the lateral membrane is a sorting station that ensures domain-specific delivery of basolateral membrane proteins, the regulated tankyrase recruitment to this site is consistent with a role in polarized protein targeting in epithelial cells.

Activation of dual apoptotic pathways in human melanocytes and protection by survivin.

Apoptosis resistance in melanoma is a primary cause of treatment failure. Apoptotic pathways in melanocytes, from which melanoma arises, are poorly characterized. Human melanocytes were susceptible to apoptosis following exposure to UV radiation (UVB, 24-48 hours), 4-tert-butylphenol (4-TBP, 1-4 hours), and cisplatin (24-48 hours). These responses were associated with Bid cleavage, caspase activation (caspases 3, 8, and 9), mitochondrial depolarization and release of cytochrome c, Smac/DIABLO, and apoptosis-inducing factor (AIF), but not endonuclease G. The apoptotic responses and AIF release were caspase-independent, as they were not blocked by zVal-Ala-Asp(OMe)-fluoromethyl ketone (zVAD-fmk). While RNA interference-mediated knockdown of AIF protected melanocytes against apoptosis induced by serum withdrawal, apoptotic responses to UVB, cisplatin, and 4-TBP were not compromised by AIF knockdown, even in the presence of zVAD-fmk. Finally, adenoviral-mediated expression of Survivin, an inhibitor of apoptosis expressed in melanoma but not melanocytes, protected melanocytes against UVB-induced apoptosis. Survivin expression in melanocytes partially blocked caspase activation and release of mitochondrial release of AIF, cytochrome c, and Smac induced by UVB. These data indicate that multiple stimuli can activate both caspase-dependent and caspase-independent apoptotic pathways in melanocytes, and that endogenous expression of Survivin in melanoma may contribute to apoptosis resistance by multiple mechanisms.

Different signaling responses to anti-proliferative agents in human aortic and venous smooth muscle cells.

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.

N-benzyladriamycin-14-valerate (AD198) induces apoptosis through protein kinase C-delta-induced phosphorylation of phospholipid scramblase 3.

Phospholipid scramblase 3 (PLS3) is an enzyme that plays a critical role in mitochondrial morphology, functions, and apoptotic response. During apoptosis, activated protein kinase C-delta (PKC-delta) translocates to mitochondria and phosphorylates PLS3. Here, we utilize an extranuclear-targeted anthracycline N-benzyladriamycin-14-valerate (AD198), a PKC-delta activator, to investigate the mechanism of PLS3 phosphorylation by PKC-delta. Overexpression of PLS3 enhanced, whereas down-regulation of PLS3 by small interfering RNA decreased, the sensitivity of AD198-induced apoptosis. Overexpression of PKC-delta, but not the kinase-defective PKC-delta, and AD198 treatment enhanced threonine phosphorylation of PLS3. The phosphorylated threonine was mapped to Thr21 of PLS3. Mutation of Thr21 to alanine did not affect mitochondrial localization of PLS3 but abolished threonine phosphorylation by PKC-delta in vitro and AD198-induced PLS3 phosphorylation in vivo. Expression of PLS3(T21A) in cells could not enhance AD198-induced apoptosis compared with expression of the wild-type PLS3. Using benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone and cyclosporine A, we also showed that AD198-induced PLS3 phosphorylation occurs upstream of caspase activation and independent of mitochondrial permeability transition. These studies establish that AD198-activated PKC-delta induces phosphorylation of mitochondrial PLS3 at Thr21 and that PLS3 is a critical downstream effector of PKC-delta in AD198-induced apoptosis.

Tankyrase-1 overexpression reduces genotoxin-induced cell death by inhibiting PARP1.

Poly(ADP-ribose) polymerases or PARPs are a family of NAD(+)-dependent enzymes that modify themselves and other substrate proteins with ADP-ribose polymers. The founding member PARP 1 is localized predominantly in the nucleus and is activated by binding to DNA lesions. Excessive PARP 1 activation following genotoxin treatment causes NAD(+) depletion and cell death, whereas pharmacological PARP 1 inhibition protects cells from genotoxicity. This study investigates whether cellular viability and NAD(+) metabolism are regulated by tankyrase-1, a PARP member localized predominantly in the cytosol. Using a tetracycline-sensitive promoter to regulate tankyrase-1 expression in Madin-Darby canine kidney (MDCK) cells, we found that a 40-fold induction of tankyrase-1 (from 1,500 to 60,000 copies per cell) lowers steady-state NAD(+) levels but does not affect basal cellular viability. Moreover, the induction confers protection against the oxidative agent H(2)O(2) and the alkylating agent MNNG, genotoxins that kill cells by activating PARP 1. The cytoprotective effect of tankyrase-1 is not due to enhanced scavenging of oxidants or altered expression of Mcl-1, an anti-apoptotic molecule previously shown to be down-regulated by tankyrase-1 in CHO cells. Instead, tankyrase-1 appears to protect cells by preventing genotoxins from activating PARP 1-mediated reactions such as PARP 1 automodification and NAD(+) consumption. Our findings therefore indicate a cytoprotective function of tankyrase-1 mediated through altered NAD(+) homeostasis and inhibition of PARP 1 function.