Edmond Auzenne

Metronomic Activity of CD44-Targeted Hyaluronic Acid-Paclitaxel in Ovarian Carcinoma

Purpose: Most primary human ovarian tumors and peritoneal implants, as well as tumor vascular endothelial cells, express the CD44 family of cell surface proteoglycans, the natural ligand for which is hyaluronic acid. Metronomic dosing, the frequent administration of chemotherapeutics at substantially lower than maximum tolerated doses (MTD), has been shown to result in reduced normal tissue toxicity and to minimize “off-treatment” exposure resulting in an improved therapeutic ratio. Experimental Design: We tested the hypothesis that hyaluronic acid (HA) conjugates of paclitaxel (TXL; HA-TXL) would exert strong antitumor effects with metronomic (MET) dosing and induce antiangiogenic effects superior to those achieved with MTD administration or with free TXL. Female nude mice bearing SKOV3ip1 or HeyA8 ovarian cancer cells were treated intraperitoneally (i.p.) with MET HA-TXL regimens (or MTD administration) to determine therapeutic and biologic effects. Results: All MET HA-TXL–treated mice and the MTD group revealed significantly reduced tumor weights and nodules compared with controls (all P values < 0.05) in the chemotherapy-sensitive models. However, the MTD HA-TXL–treated mice showed significant weight loss compared with control mice, whereas body weights were not affected in the metronomic groups in HeyA8-MDR model, reflecting reduced toxicity. In the taxane-resistant HeyA8-MDR model, significant reduction in tumor weight and nodule counts was noted in the metronomic groups whereas the response of the MTD group did not achieve significance. While both MTD and metronomic regimens reduced proliferation (Ki-67) and increased apoptosis (TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling), only metronomic treatment resulted in significant reductions in angiogenesis (CD31, microvessel density). Moreover, metronomic treatment resulted in substantial increases in thrombospondin-1 (Tsp-1), an inhibitor of angiogenesis. Conclusions: This study showed that MET HA-TXL regimens have substantial antitumor activity in ovarian carcinoma, likely via a predominant antiangiogenic mechanism. Clin Cancer Res; 18(15); 4114–21. ©2012 AACR.

Characterization of cytotoxicity induced by sphingolipids in multidrug-resistant leukemia cells

Certain sphingolipids (SLs) exert fundamental roles in differentiative, growth-inhibitory and apoptotic pathways induced by a number of agents in leukemia cells. Multidrug-resistance (MDR) is a major cause of therapeutic failure in leukemia. SLs are among the diverse substrates for the MDR p-170 glycoprotein drug-efflux pump. We tested the hypothesis that expression of MDR would thereby block the cytotoxicity induced by the SLs sphingosine, sphinganine and N-hexanoyl-sphingosine. An MDR-expressing subline of murine P388 leukemia cells demonstrated an ED50 value > or = 2 log10 higher than the parental line in response to doxorubicin. In contrast, the ED50 values for each of the SLs were only approximately 1.5 to two-fold higher in the MDR line than in the parental; induction of DNA damage by SLs was comparable or actually greater in MDR compared to parental cells. Therefore, expression of MDR does not significantly affect the cytotoxic function of these SLs, nor do these SLs likely contribute to MDR.

Hyperthermia engages the intrinsic apoptotic pathway by enhancing upstream caspase activation to overcome apoptotic resistance in MCF‐7 breast adenocarcinoma cells

Febrile hyperthermia enhanced TNF‐stimulated apoptosis of MCF‐7 cells and overcame resistance in a TNF‐resistant, MCF‐7 variant (3E9), increasing their TNF‐sensitivity by 10‐ and 100‐fold, respectively. In either cell line, the hyperthermic potentiation was attributable to increased apoptosis that was totally quenched by caspase inhibition. In MCF‐7 cells, hyperthermic potentiation of apoptosis was associated with sustained activation of upstream caspases in response to TNF and more prominent engagement of the intrinsic apoptotic pathway. Apoptotic enhancement by hyperthermia was primarily mediated by caspase‐8 activation, as the specific inhibitor, Z‐IETD, blocked cell death, whereas direct engagement of the intrinsic apoptotic pathway (with doxorubicin) was not affected. In 3E9 cells, hyperthermia alone induced activation of caspase‐8, and was further enhanced by TNF. In 3E9 cells, hyperthermia caused TNF‐dependent loss of mitochondrial membrane potential and activation of capspase‐9 that was initiated and dependent on upstream caspases. MCF‐7 and 3E9 cells were equally sensitive to exogenous C6‐ceramide, but mass spectroscopic analysis of ceramide species indicated that total ceramide content was not enhanced by TNF and/or hyperthermia treatment, and that the combination of TNF and hyperthermia caused only modest elevation of one species (dihydro‐palmitoyl ceramide). We conclude that febrile hyperthermia potentiates apoptosis of MCF‐7 cells and overcomes TNF‐resistance by sustained activation of caspase‐8 and engagement of the intrinsic pathway that is independent of ceramide flux. This report provides the first evidence for regulation of caspase‐dependent apoptosis by febrile hyperthermia. J. Cell. Biochem. 98: 356–369, 2006.

Febrile and acute hyperthermia enhance TNF-induced necrosis of murine L929 fibrosarcoma cells via caspase-regulated production of reactive oxygen intermediates.

Previous studies have demonstrated the essential role of TNF‐induced reactive oxygen intermediates (ROI) in the necrosis of L929 cells. We investigated the molecular basis for the interaction of hyperthermia and TNF in these cells. Hyperthermia, both febrile (40.0–40.5°C) and acute (41.5–41.8°C), strongly potentiated TNF killing, and sensistization was significantly quenched by the antioxidant, BHA. The broad‐spectrum caspase inhibitor, Z‐VAD, has been shown to markedly increase the TNF sensitivity of L929 cells at 37°C; we observed that hyperthermia would also enhance the sensitivity of L929 cells to TNF + Z− VAD and that BHA could significantly quench the response, as well. The basis for hyperthermic potentiation was unlikely thermally‐increased sensitivity to ROI, as treatment with hydrogen peroxide for 24 h killed L929 cells essentially equivalently, whether incubated continuously at 37°C or at 40.0–40.5°C, or for 2 h at 41.5–41.8°C. However, febrile and acute hyperthermia markedly increased TNF‐induced production of ROI, with or without Z‐VAD. Hyperthermia dramatically accelerated the onset of this production, as well as the onset of necrotic death, as determined by oxidation of dihydro‐rhodamine and propidium iodide staining, respectively, both of which were significantly quenchable with BHA. We conclude that hyperthermia potentiates TNF‐mediated killing in this cell model primarily by increasing the afferent, and not the efferent, phase of TNF‐induced necrosis.

Inhibition of tumor cell mitochondrial respiration by macrophage cytotoxic mediators distinct from interferon-gamma.

Macrophage‐mediated inhibition of mitochondrial respiration in EMT‐6 murine mammary adenocarcinoma cells can be mimicked in vitro by treatment of the cells with interferon‐γ (IFN‐γ) in combination with tumor necrosis factor, interleukin‐1, or lipopolysaccharide. Conditioned supernatants obtained from activated macrophages appear to contain interferon‐γ, suggesting that inhibition of mitochondrial respiration in tumor cells was caused by synergy of IFN‐γ with other cytokines. To further characterize monokines that cause inhibition of mitochondrial respiration in tumor cells, EA13.5 macrophage‐like cells were isolated and selected for inhibition of mitochondrial respiration in EMT‐6 tumor cells. After stimulation with IFN‐γ and lipopolysaccharide, the EA13.5 cells released into conditioned supernatants a cytotoxic mediator that induced nitric oxide synthesis and caused lesions in the electron transport chain of EMT‐6 cells similar to the lesions caused by activated peritoneal macrophages. Enzyme‐linked immunosorbent assay demonstrated that the conditioned supernatants produced by EA13.5 macrophage cells did not contain IFN‐γ. Treatment of the EA13.5 cell‐conditioned supernatants with neutralizing antibody against IFN‐γ did not abrogate the inhibition of mitochondrial respiration in EMT‐6 cells caused by these conditioned supernatants. This study demonstrated that unidentified macrophage cytotoxic mediators distinct from IFN‐γ are involved in the induction of nitric oxide synthesis and inhibition of mitochondrial respiration in tumor cells.

MRP- and BCL-2-mediated drug resistance in human SCLC: Effects of apoptotic sphingolipids in vitro

Multidrug-resistance-associated protein (MRP) and BCL-2 contribute to drug resistance expressed in SCLC. To establish whether MRP-mediated drug resistance affects sphingolipid (SL)-induced apoptosis in SCLC, we first examined the human SCLC cell line, UMCC-1, and its MRP over-expressing, drug-resistant subline, UMCC-1/VP. Despite significantly decreased sensitivity to doxorubicin (Dox) and to the etoposide, VP-16, the drug-selected line was essentially equally as sensitive to treatment with exogenous ceramide (Cer), sphingosine (Sp) or dimethyl-sphingosine (DMSP) as the parental line. Next, we observed that high BCL-2-expressing human H69 SCLC cells, that were approximately 160-fold more sensitive to Dox than their combined BCL-2 and MRP-over-expressing (H69AR) counterparts, were only approximately 5-fold more resistant to DMSP. Time-lapse fluorescence microscopy of either UMCC cell line treated with DMSP-Coumarin revealed comparable extents and kinetics of SL uptake, further ruling out MRP-mediated effects on drug uptake. DMSP potentiated the cytotoxic activity of VP-16 and Taxol, but not Dox, in drug-resistant UMCC-1/VP cells. However, this sensitization did not appear to involve DMSP-mediated effects on the function of MRP in drug export; nor did DMSP strongly shift the balance of pro-apoptotic Sps and anti-apoptotic Sp-1-Ps in these cells. We conclude that SL-induced apoptosis markedly overcomes or bypasses MRP-mediated drug resistance relevant to SCLC and may suggest a novel therapeutic approach to chemotherapy for these tumors.

N,N-Dimethylsphingosine conjugates of poly-l-glutamic acid: Synthesis, characterization, and initial biological evaluation

Poly-L-glutamic acid (PGA) has previously been demonstrated to be an effective backbone for creating a hydrophilic prodrug of the established anti-tumor agent, paclitaxel, the active agent in Taxol; this approach has obviated the need for the toxic Cremophor excipient, used to enhance the solubility of paclitaxel in the clinical formulation. In order to form hydrophilic prodrugs of the hydrophobic pro-apoptotic sphingolipid, N,N-dimethylsphingosine (DMSP), PGA was condensed with DMSP, previously modified with coumarin to allow spectroscopic detection during conjugate synthesis, to yield PGA-DMSP. Conjugates with different loadings of DMSP were prepared and evaluated for in vitro cytotoxicity against two human breast adenocarcinoma cell lines. Time- and loading-dependent expression of cytotoxicity was observed, such that endpoints essentially equivalent to those observed with free-DMSP were achieved, but in a more protracted manner, consistent with prodrug behavior. PGA-DMSP was initially evaluated for toxicity in female nude mice, and administration of high net levels of DMSP, exceeding those achievable with free-DMSP, was well-tolerated. We propose that PGA-DMSP conjugates merit evaluation for anti-tumor efficacy in pre-clinical tumor models.

Abstract B8: Selective inhibition of Stat3 Tyr705 phosphorylation inhibits angiogenesis in breast cancer xenografts.

Signal transducer and activator of transcription 3 (Stat3) is recruited, via its SH2 domain, to phosphotyrosine residues on growth factor and IL-6 family receptors at which time it is phosphorylated on Tyr705 by Janus kinases, Src, or the kinase activity of the receptor. Phosphorylation of Tyr705 (pStat3) leads to dimerization, translocation to the nucleus, and transcription of downstream genes. Stat3 is activated in several human cancers and is considered a drug target. Starting with pTyr-Leu-Pro-Gln, we have been developing phosphopeptide mimics to target the SH2 domain with the goal of blocking recruitment to receptors to uncouple Stat3 from its signaling role. We have developed cell-permeable, phosphatase-stable prodrugs that inhibit constitutive and IL-6-stimulated Tyr705 phosphorylation at concentrations of 0.5 − 1 μM in a variety of human cancer cell lines. They are selective for Stat3 over Stat1, Stat5, Src, and PI3K. At 5 μM they are not cytotoxic to a panel of human cancer cell lines. Higher concentrations lead to off-target effects and cytotoxicity. Recent reports indicate that Ser727 phosphorylated Stat3 participates in electron transfer in the mitochodria and unphosphorylated Stat3 participates with NF-κB-mediated transcription. As opposed to Stat3 knockdown techniques which cannot discriminate the multiple roles of Stat3, our prodrugs are potential tools to selectively probe Stat3 phosphorylation. Both intra-tumoral and intra-peritoneal administration of our lead prodrug, PM-73G, resulted in reduction of orthotopic breast tumor xenografts in mice. There was no evidence of apoptosis, reduction of cyclin D1 or survivin. Treated tumors exhibited significant reduction in microvessel density. Thus, selectively inhibiting Tyr705 phosphorylation is a potential antiangiogenic treatment modality. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B8.