Uridine abrogates the adverse effects of antiretroviral pyrimidine analogues on adipose cell functions.

OBJECTIVES: Side effects of antiretroviral treatment such as lipoatrophy have been mainly attributed to mitochondrial toxicity of nucleoside reverse transcriptase inhibitors (NRTIs). We assessed whether uridine can abrogate the adverse effects of NRTIs on adipocyte functions. METHODS: 3T3-F442A preadipocytes were exposed to stavudine (d4T; 10 microM), zidovudine (ZDV; 1 microM), zalcitabine (ddC; 0.2 microM) or didanosine (ddl; 10 microM) in the absence or presence of uridine 21 days prior to and 7 days after induction of differentiation. Then, lipid accumulation (oil red staining), apoptosis (flow cytometry, PARP-cleavage), mitochondrial mass (Mitotracker) and DNA (mtDNA), cytochrome c oxidase (COX) subunits and mitochondrial membrane potential (JC-1) were quantified. RESULTS: Whereas ddl had no effects, d4T, ZDV and ddC significantly decreased cellular lipid accumulation (by 32%, 46% and 24%, respectively), increased apoptosis and induced mitochondrial depolarization. d4T, ZDV and ddC decreased adipocyte mtDNA (by 64%, 53% and 46%, respectively) and reduced the mtDNA encoded COX II subunit. Uridine (200 microM) had no intrinsic effect, but prevented all adverse effects of d4T, ZDV and ddC on adipocyte morphology, lipid staining, apoptosis, mtDNA depletion (partial prevention with ZDV), mitochondrial mass and membrane potential. The effects of uridine were concentration-dependent. Uridine also fully reverted established d4T toxicities despite continued d4T exposure. CONCLUSIONS: Uridine supplementation protects adipocytes from the adverse effects of d4T, ZDV and ddC on lipid accumulation, cell survival and mitochondrial functions, suggesting that the toxic effects could be linked to intracellular depletion of uridine or its metabolites. Uridine is an interesting candidate in the prevention of NRTI-induced lipoatrophy in vivo.

The c-kit tyrosine kinase inhibitor STI571 for colorectal cancer therapy.

The c-kit tyrosine kinase inhibitor STI571 exhibits a substantial therapeutic activity in patients with chronic myeloid leukemia and gastrointestinal stromal tumors respectively associated with constitutive activation of the BCR-ABL and c-kit tyrosine kinases. Human colorectal tumors also express the c-kit proto-oncogene. The present study focuses on the anticancer activity of STI571 in human colorectal tumor cells in vitro and in vivo. The c-kit receptor was identified as a M(r) 145,000 immunoreactive band in human colon cancer cells HT29, HCT8/S11, and HCT116. Cellular invasion induced by 10 ng/ml stem cell factor (EC(50) = 3 ng/ml) in HT29 cells was blocked by 1 micro M STI571 (IC(50) = 56 nM) and pharmacological inhibitors of several oncogenic signaling pathways, namely, phosphatidylinositol 3-kinase (LY294002), Rho GTPases (Clostridium botulinum exoenzyme C3 transferase), and Rho-kinase (Y27632). STI571 inhibited HT29 cell proliferation (IC(50) = 6 micro M) and induced apoptosis in vitro. These cellular effects were associated with a decrease in tumor growth. We also demonstrated that stem cell factor is a proangiogenic factor in vivo and in vitro. These encouraging results warrant further preclinical investigations and clinical trials on the use of the c-kit inhibitor STI571 as a chemotherapeutic agent in colon cancer prevention and in treatment of advanced colorectal cancers associated with liver metastases.

GSK-3beta reactivation with LY294002 sensitizes hepatoma cells to chemotherapy-induced apoptosis.

Constitutive activation of phosphatidylinositol 3-kinase (PI3K) confers resistance to apoptotic stimuli induced by chemotherapeutic agents in a variety of cancer cells. Therefore, the comprehension of mechanisms whereby PI3K downregulation interferes with chemotherapy is of major clinical interest for the elaboration of combined anticancer treatment modalities. Here, we examined the molecular mechanisms whereby the PI3K inhibitor LY294002 sensitized p53- and Fas-deficient hepatoma cells to etoposide and camptothecin. LY294002 increased Hep3B cell susceptibility to chemotherapy-induced apoptosis by enhancing the expression of DR4 and DR5 and the activation of caspase-8 and -3. Moreover, LY294002-mediated sensitization to chemotherapy involved mitochondrial Bax translocation and cytosolic cytochrome c accumulation. In Hep3B cells, LY294002 led to the reactivation of glycogen synthase kinase-3beta (GSK-3beta) by promoting its dephosphorylation on the serine 9 residue independently from Akt inhibition. The transient transfection of a constitutively active and non-phosphorylable S9AGSK-3beta mutant sensitized cells to etoposide cytotoxic effects while cell treatment with the small GSK-3beta inhibitor SB-415286 repressed the sensitizing effect of LY294002 on chemotherapy-induced apoptosis and caspase-8 activation. Altogether, our results show that LY294002 sensitizes hepatoma cells to chemotherapy-induced apoptosis via death receptor and mitochondria signalling pathways and that GSK-3beta reactivation is involved in this process. Therefore, PI3K-mediated GSK-3beta inhibition could be a mechanism by which cancer cells escape from chemotherapy-induced apoptosis.

Increased anticancer activity of the thymidylate synthase inhibitor BGC9331 combined with the topoisomerase I inhibitor SN-38 in human colorectal and breast cancer cells: induction of apoptosis and ROCK cleavage through caspase-3-dependent and -independent mechanisms.

The folate analogue BGC9331 is a new thymidylate synthase (TS) inhibitor showing a broad spectrum of cyto-toxic activity against several human solid tumors, including colorectal cancer. In this study, we investigated the anticancer activity of BGC9331 either alone or combined with 5-fluorouracil (5-FU), MTA (multi-target antifolate), oxali-platin and SN-38, the active metabolite of the topoisomerase I inhibitor CPT-11. The antiproliferative activity of each drug and BGC9331-based combinations was investigated in the HT-29 human colorectal cancer cell line and its HT-29/5-FU counterparts selected for resistance to 5-FU. BGC9331 combined with MTA or SN-38 induced synergistic responses in HT-29 cells. Treatment of HT-29 cells with either BGC9331 or SN-38 increased caspase-3 activity and the percentage of apoptotic cells from 3 to 13%. Both drugs also augmented the proteolytic cleavage of the Rho-kinase ROCK-1 that was attenuated by the caspase-3 pathway inhibitor z-DEVD-fmk. BGC9331 combined with SN-38 further increased the percentage of apoptotic cells to 25%, and inhibited cell cycle progression and cell proliferation by 65%. This was accompanied by proteolytic activation of ROCK-1, through both caspase-3-dependent and -independent mechanisms, as shown in caspase-3-deficient MCF-7 breast cancer cells. These encouraging results warrant further preclinical investigations and clinical trials on the use of BGC9331 combined with SN-38/CPT-11 in treatment of patients with advanced colorectal or gastric cancers.

Some HIV protease inhibitors alter lamin A/C maturation and stability, SREBP-1 nuclear localization and adipocyte differentiation.

OBJECTIVES: To study whether HIV protease inhibitors could induce nuclear lamina alterations in adipocytes as observed in a genetic form of lipodystrophy due to lamin A/C mutation. DESIGN: We have previously observed that indinavir (IDV) impairs adipocyte differentiation and sterol regulatory element-binding protein-1 (SREBP-1) nuclear localization in 3T3-F442A adipocytes. We compared here the effects of IDV with that produced by two other PIs, nelfinavir (NFV) and amprenavir (APV) on adipose conversion, cellular localization of SREBP-1, nuclear morphology, and maturation and stability of the lamina network. RESULTS: IDV and NFV, but not APV, altered adipose cell differentiation, as shown by lipid staining and protein expression of SREBP-1, CAAAT/enhancer binding protein (C/EBP)alpha and fatty acid synthase (FAS). In IDV-treated cells, 50-60 % of the nuclei could not accumulate SREBP-1. Twenty percent of these SREBP-negative nuclei were grossly dysmorphic, with blebs and prominent herniations, and showed an altered distribution of lamin A/C and lamin B. In IDV-treated cells, nuclear fragilization was shown by the abnormal extractibility of lamina proteins and SREBP-1, and the accumulation of prelamin A. NFV similarly altered lamin A/C maturation whereas APV was almost ineffective. CONCLUSIONS: We show in an adipose cell line that IDV and NFV induced alterations at the nuclear level by promoting defects in lamin A/C maturation, organization and stability. We suggest that these lamina network alterations might be responsible for SREBP-1 nuclear mislocalization therefore resulting in altered adipocyte differentiation.

The HIV-1 nucleoside reverse transcriptase inhibitors stavudine and zidovudine alter adipocyte functions in vitro.

OBJECTIVES: Nucleoside analogues are suspected of playing a role in peripheral fat loss in patients during long-term treatment with antiretroviral drugs. DESIGN AND METHODS: We compared the long-term effects of stavudine (10 microM), zidovudine (1 muM), didanosine (10 microM), abacavir (4 microM), lamivudine (10 microM), and tenofovir (1 microM), near their maximum concentration values, on the differentiation, lipid accumulation, survival and mitochondrial function of differentiating 3T3-F442A and differentiated 3T3-L1 adipocytes. RESULTS: None of the nucleoside reverse transcriptase inhibitors (NRTI) markedly altered the differentiation of 3T3-F442A cells, as shown by the unmodified percentage of cells with lipid droplets on day 7 and the expression of the early differentiation markers CCAAT/enhancer binding protein (C/EBP) beta (on day 2) and sterol regulatory element-binding protein. However, stavudine and zidovudine altered the lipid phenotype, decreasing the lipid content and expression of markers involved in lipid metabolism, namely C/EBPalpha, peroxisome proliferator-activated receptor gamma, adipocyte lipid binding protein 2, fatty acid synthase and acetyl-coenzyme A carboxylase. Stavudine and zidovudine, contrary to the other NRTI, drove 5-10% of 3T3-F442A cells towards apoptosis, and reduced the lipid content and survival of differentiated 3T3-L1 adipocytes. Stavudine and zidovudine increased mitochondrial mass by two to fourfold, and lowered the mitochondrial membrane potential (JC-1 stain) as did zalcitabine (0.2 microM). Co-treatment with zidovudine plus lamivudine, or zidovudine plus lamivudine and abacavir, did not increase the effect of zidovudine on cell viability or apoptosis. CONCLUSION: The thymidine analogues stavudine and zidovudine decreased lipid content, mitochondrial activity, and adipocyte survival in vitro.

Antiretroviral drugs with adverse effects on adipocyte lipid metabolism and survival alter the expression and secretion of proinflammatory cytokines and adiponectin in vitro.

OBJECTIVE: The lipodystrophy syndrome is a major adverse effect of highly active antiretroviral therapy (HAART), associated with altered circulating levels and adipose tissue mRNA expression of proinflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor (TNF)alpha, and adiponectin. Proinflammatory cytokines and adiponectin, which are secreted by adipose tissue, regulate fat metabolism, insulin sensitivity and adipose cell apoptosis. We examined the direct effects of individual antiretrovirals on lipid metabolism and cytokine and adiponectin production by cultured adipocytes. METHODS: Differentiating 3T3-F442A cells and differentiated 3T3-L1 adipocytes were treated for 12 or 4 days, respectively, with protease inhibitors (PIs) indinavir, nelfinavir, amprenavir, lopinavir and ritonavir, or nucleoside reverse transcriptase inhibitors (NRTIs) stavudine and zidovudine, at near-Cmax concentrations. Lipid metabolism was estimated by Oil Red O staining of intracellular lipids, mRNA expression of fatty acid synthase and adipocyte lipid binding protein 2, and insulin activation of lipogenesis. Apoptosis was estimated by flow cytometry. The expression and secretion of proinflammatory cytokines (IL-6, TNFalpha and IL-1beta) and adiponectin were evaluated by real-time reverse transcription PCR and ELISA. RESULTS: Chronic treatment of 3T3-F442A differentiating adipocytes and differentiated 3T3-L1 adipocytes with PIs and NRTIs reduced lipid accumulation, mRNA expression of lipid markers and insulin-induced lipogenesis. IL-6, TNFalpha, IL-1beta and adiponectin expression and secretion were markedly altered in differentiating 3T3-F442A adipocytes. PIs had either no effect on differentiated 3T3-L1 adipocytes (TNFalpha expression and sucretion) or their effect was less marked than in 3T3-F442A cells. Indinavir and amprenavir did not alter cytokine secretion and expression by mature adipocytes. The effects of stavudine and zidovudine on differentiating and mature adipocytes were similar, despite the difference in treatment procedure. The drugs with the strongest effect on TNFalpha expression also increased adipocyte apoptosis, in contrast to the drugs that only moderately increased TNFalpha expression. CONCLUSIONS: These results suggest that increased cytokine and decreased adiponectin secretion and expression induced by some PIs and NRTIs may contribute to the adipose tissue loss (via apoptosis and lipid leakage) and insulin resistance associated with the lipodystrophy syndrome.

Glycogen synthase kinase-3 inhibitors augment TRAIL-induced apoptotic death in human hepatoma cells.

Hepatocellular carcinoma (HCC) displays a striking resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Therefore, the characterization of pharmacological agents that overcome this resistance may provide new therapeutic modalities for HCC. Here, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors could restore TRAIL sensitivity in hepatoma cells. To this aim, the effects of two GSK-3 inhibitors, lithium and SB-415286, were analyzed on TRAIL apoptotic signaling in human hepatoma cell lines in comparison with normal hepatocytes. We observed that both inhibitors sensitized hepatoma cells, but not normal hepatocytes, to TRAIL-induced apoptosis by enhancing caspase-8 activity and the downstream recruitment of the mitochondrial machinery. GSK-3 inhibitors also stabilized p53 and the down-regulation of p53 by RNA interference abolished the sensitizing effect of lithium on caspase-3 activation. Concomitantly, GSK-3 inhibitors strongly activated c-Jun N-terminal kinases (JNKs). The pharmacological inhibition of JNKs with AS601245 or SP600125 resulted in an earlier and stronger induction of apoptosis indicating that activated JNKs transduced protective signals and provided an anti-apoptotic balance to the pro-apoptotic effects of GSK-3 inhibitors. These findings demonstrate that GSK-3 exerts a negative and complex constraint on TRAIL apoptotic signaling in hepatoma cells, which can be greatly alleviated by GSK-3 inhibitors. Therefore, GSK-3 inhibitors may open new perspectives to enhance the anti-tumor activity of TRAIL in HCC.

[Inhibition of phosphatidylethanolamine and phosphatidylinositol synthesis, alteration of the G0 and G1 cell cycle phases and spontaneous apoptosis in lymphocytes from patients under immunosuppressive treatment]. / Inhibition de la synthèse de la phosphatidylethanolamine et du phosphatidylinositol, altération des phases G0 et G1 et apoptose spontanée chez les lymphocytes de sujets sous traitement immunosuppresseur.

Lymphocytes from patients treated with immunosuppressive agents were cultured for 48 hours either with or without concanavalin A. Phospholipid synthesis was then studied using 32p pulse-incorporation (5-hours pulses). Phosphatidylethanolamine synthesis was strongly decreased under immunosuppressive treatment: 1.5-fold in resting lymphocytes and 3- to 4-fold in concanavalin A-stimulated lymphocytes. Phosphatidylinositol synthesis also decreased about 2-fold in stimulated lymphocytes. These results indicate a loss of sensitivity of immunodeficient lymphocytes to the mitogen and an alteration of the G0 and the late G1 cell cycle phases. In parallel, but after a 72-hours incubation, lymphocytes were analysed by flow-cytofluorimetry with propidium iodide. Under concanavalin A-triggered stimulation, the entry into the S phase was much lower in immunodeficient lymphocytes as compared to standard. The characteristics of the G0-G1 population of lymphocytes were also modified. More importantly, after incubation in the culture medium in the absence of mitogen, we observed, among the immunodeficient lymphocytes, a high level of apoptotic cells, about 20 to 30%. This susceptibility to spontaneous apoptosis seems inherent to the status of immunodeficiency itself, whatever its origin. It may be related to the inhibition of phosphatidylethanolamine synthesis in the G0 phase.

Butyrate-treated colonic Caco-2 cells exhibit defective integrin-mediated signaling together with increased apoptosis and differentiation.

We previously reported that the enterocytic differentiation of human colonic Caco-2 cells correlated with alterations in integrin signaling. We now investigated whether differentiation and apoptosis of Caco-2 cells induced by the short-chain fatty acid butyrate (NaBT) was associated with alterations in the integrin-mediated signaling pathway with special interest in the expression and activity of focal adhesion kinase (FAK), of the downstream phosphatidylinositol 3'-kinase (PI 3-kinase)-Akt pathway and in the role of the nuclear factor kappaB (NF-kappaB). NaBT increased the level of sucrase. It induced apoptosis as shown by: (1) decreased Bcl-2 and Bcl-X(L) proteins and increased Bax protein; (2) activation of caspase-3; and (3) increased shedding of apoptotic cells in the medium. This effect was associated with defective integrin-mediated signaling as shown by: (1) down-regulation of beta1 integrin expression; 2) decreased FAK expression and tyrosine phosphorylation; (3) concerted alterations in cytoskeletal and structural focal adhesions proteins (talin, ezrin); and (4) decreased FAK ability to associate with PI 3-kinase. However, in Caco-2 cells, beta1-mediated signaling failed to be activated downstream of FAK and PI 3-kinase at the level of Akt. Transfection studies show that NaBT treatment of Caco-2 cells promoted a significant activation of the NF-kappaB which was probably involved in the NaBT-induced apoptosis. Our results indicate that the prodifferentiating agent NaBT induced apoptosis of Caco-2 cells probably through NF-kappaB activation together with a defective beta1 integrin-FAK-PI 3-kinase pathways signaling.

GSK-3beta inhibition by lithium confers resistance to chemotherapy-induced apoptosis through the repression of CD95 (Fas/APO-1) expression.

Lithium exerts neuroprotective actions that involve the inhibition of glycogen synthase kinase-3beta (GSK-3beta). Otherwise, recent studies suggest that sustained GSK-3beta inhibition is a hallmark of tumorigenesis. In this context, the present study was undertaken to examine whether lithium modulated cancer cell sensitivity to apoptosis induced by chemotherapy agents. We observed that, in different human cancer cell lines, lithium significantly reduced etoposide- and camptothecin-induced apoptosis. In HepG2 cells, lithium repressed drug induction of CD95 expression and clustering at the cell surface as well as caspase-8 activation. Lithium acted through deregulation of GSK-3beta signaling since (1) it provoked a rapid and sustained phosphorylation of GSK-3beta on the inhibitory serine 9 residue; (2) the GSK-3beta inhibitor SB-415286 mimicked lithium effects by repressing drug-induced apoptosis and CD95 membrane expression; and (3) lithium promoted the disruption of nuclear GSK-3beta/p53 complexes. Moreover, the overexpression of an inactivated GSK-3beta mutant counteracted the stimulatory effects of etoposide and camptothecin on a luciferase reporter plasmid driven by a p53-responsive sequence from the CD95 gene. In conclusion, we provide the first evidence that lithium confers resistance to apoptosis in cancer cells through GSK-3beta inhibition and subsequent repression of CD95 gene expression. Our study also highlights the concerted action of GSK-3beta and p53 on CD95 gene expression.