Dietary prevention of malignant glioma aggressiveness, implications in oxidant stress and apoptosis.

Our study explored the influence of diet on gliomagenesis and associated systemic effects (SE) in rats. The experimental diet contained various ingredients supposed to interfere with carcinogenesis, mainly phytochemicals (PtcD for phytochemical diet) and its effects were compared to those of the same diet without the phytochemicals (BD for basal diet). Glioma was induced by ethylnitrosourea to pregnant females fed the diets from the start of gestation until the moment of sacrifice of the offpsrings. In male rats fed the PtcD or the BD the incidence of gliomas was markedly reduced compared to rats fed a standard diet (StD). In females this effect was weaker and was limited to the PtcD. A significant proportion of rats with brain tumors and fed the StD exhibited SE evidenced by weight loss, a shorter survival, reduction in liver weight and an increased proportion of liver mitochondria, effects that were not observed in their counterpart fed PtcD. Comparison of the expression of genes involved in the balance proliferation/apoptosis and in the response to oxidative stress in male brain tumors showed that the prevention of SE was associated with an increase in bcl-2 and catalase and a decrease in ki-67, sod-1 and sod-2 transcripts. These results show that the degree of agressiveness of gliomas can be modulated by dietary interventions and suggest that some phytochemicals with antioxidant properties could participate to the mechanism.

Nonredundant role of Bax and Bak in Bid-mediated apoptosis.

Animal models suggest that Bax and Bak play an essential role in the implementation of apoptosis and as a result can hinder tumorigenesis. We analyzed the expression of these proteins in 50 human glioblastoma multiforme (GBM) tumors. We found that all the tumors expressed Bak, while three did not express Bax. In vitro, Bax-deficient GBM (BdGBM) exhibited an important resistance to various apoptogenic stimuli (e.g., UV, staurosporine, and doxorubicin) compared to the Bax-expressing GBM (BeGBM). Using an antisense strategy, we generated Bak(-) BeGBM and Bak(-) BdGBM, which enabled us to show that the remaining sensitivity of the BdGBM to apoptosis was due to the overexpression of Bak. Bax/Bak single or double deficiency had no influence on either the clonogenicity or the growth of tumors in Swiss nude mice. Of note, Bak(-) BeGBM cells were resistant to apoptosis induced by caspase 8 (C8) but not to that induced by granzyme B (GrB). Cells lacking both Bax and Bak (i.e., Bak(-) BdGBM) were completely resistant to all stimuli including the microinjection of C8 and GrB. We show that GrB-cleaved Bid and C8-cleaved Bid differ in size and utilize preferentially Bax and Bak, respectively, to promote cytochrome c release from mitochondria. Our results suggest that Bax deficiency is compensated by an increase of the expression of Bak in GBM and show, for the first time in human cancer, that the double Bax and Bak deficiency severely impairs the apoptotic program.

Changes in liver mitochondrial plasticity induced by brain tumor.

BACKGROUND: Accumulating data suggest that liver is a major target organ of systemic effects observed in the presence of a cancer. In this study, we investigated the consequences of the presence of chemically induced brain tumors in rats on biophysical parameters accounting for the dynamics of water in liver mitochondria. METHODS: Tumors of the central nervous system were induced by intraveinous administration of ethylnitrosourea (ENU) to pregnant females on the 19th day of gestation. The mitochondrial crude fraction was isolated from the liver of each animal and the dynamic parameters of total water and its macromolecule-associated fraction (structured water, H2Ost) were calculated from Nuclear Magnetic Resonance (NMR) measurements. RESULTS: The presence of a malignant brain tumor induced a loss of water structural order that implicated changes in the physical properties of the hydration shells of liver mitochondria macromolecules. This feature was linked to an increase in the membrane cholesterol content, a way to limit water penetration into the bilayer and then to reduce membrane permeability. As expected, these alterations in mitochondrial plasticity affected ionic exchanges and led to abnormal features of mitochondrial biogenesis and caspase activation. CONCLUSION: This study enlightens the sensitivity of the structured water phase in the liver mitochondria machinery to external conditions such as tumor development at a distant site. The profound metabolic and functional changes led to abnormal features of ion transport, mitochondrial biogenesis and caspase activation.

Profiling dendritic cell maturation with dedicated microarrays.

Dendritic cell (DC) maturation is the process by which immature DC in the periphery differentiate into fully competent antigen-presenting cells that initiate the T cell response. However, DC respond to many distinct maturation stimuli, and different types of mature DC induce qualitatively different T cell responses. As DC maturation involves the coordinated regulation of hundreds of genes, comprehensive assessment of DC maturation status would ideally involve monitoring the expression of all of these transcripts. However, whole-genome microarrays are not well-suited for routine phenotyping of DC, as the vast majority of genes represented on such chips are not relevant to DC biology, and their cost limits their use for most laboratories. We therefore developed a DC-dedicated microarray, or "DC Chip", incorporating probes for 121 genes up-regulated during DC maturation, 93 genes down-regulated during maturation, 14 DC-specific genes, and 90 other genes with known or probable immune functions. These microarrays were used to study the kinetics of DC maturation and the differences in maturation profiles among five healthy donors after stimulation with tumor necrosis factor-alpha + polyI:C. Results obtained with the DC Chip were consistent with flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction, as well as previously published data. Furthermore, the coordinated regulation of a cluster of genes (indoleamine dioxygenase, kynureninase, kynurenine monoxygenase, tryptophanyl tRNA synthetase, and 3-hydroxyanthranilate 3,4-dioxygenase) involved in tryptophan metabolism was observed. These data demonstrate the use of the DC Chip for monitoring the molecular processes involved in the orientation of the immune response by DC.

Increased vaccination efficiency with apoptotic cells by silica-induced, dendritic-like cells.

We have demonstrated previously the ability of apoptotic cells to prime a functional immune response using an i.p. vaccination protocol with apoptotic cells and interleukin 2, before injecting a lethal dose of tumor cells into syngeneic rats. This protocol resulted in a survival rate of 33%. To elucidate the nature and the activity of the phagocytes involved in the clearance of apoptotic cells in vivo, we modulated the peritoneal cavity environment by administrating either thioglycollate or silica i.p. before injecting the apoptotic cells. Our results showed that thioglycollate abrogated vaccination efficiency, because none of the rats survived under these conditions. In fact, thioglycollate treatment induced a massive recruitment and activation of inflammatory macrophages that efficiently engulfed apoptotic cells, bypassing induction of specific immune responses. In contrast, silica treatment enhanced the vaccination efficiency of apoptotic cells plus interleukin 2 up to 66%. We distinguished a population of dendrite-like cells among the cells derived from the silica-treated peritoneal cavity both by their phenotype (MHC II(+)/CD80(+)/CD86(+)) and by their ability to induce the proliferation of allogeneic T cells in a mixed leukocyte reaction. Our results demonstrate the different roles of macrophages and dendritic-like cells in the physiological clearance of dead tumor cells and their implication in the design of immunomodulating vaccines.

Induction of leukemia-specific cytotoxic response by cross-presentation of late-apoptotic leukemic blasts by autologous dendritic cells of nonleukemic origin.

Acute myeloid leukemias (AMLs) are monoclonal proliferations of undifferentiated myeloid progenitors in blood and bone marrow. Long-term remissions are achieved in <50% of patients. There is hope that activation of specific antileukemic immune responses could efficiently eliminate minimal residual disease at the end of chemotherapy and decrease the frequency of relapses. It was demonstrated that AML leukemic blasts can acquire the morphology and phenotype of dendritic cells (DCs), i.e., differentiate into leukemic DCs. However, this method has limitations as a potential immunotherapy. The alternative approach for the induction of leukemia-specific cytotoxicity we explored in this study consisted of using DCs of nonleukemic origin, pulsed with autologous apoptotic leukemic blasts. We show that mature pulsed nonleukemic DCs were successfully generated from remission samples of all tested patients with minimal interindividual differences. Mature pulsed DCs were used as antigen-presenting cells for leukemia-specific CTL induction. Specific cytotoxic activity against autologous AML blasts was demonstrated. Tumor lysis was autologous blast specific, with no killing activity against allogeneic leukemic cells or autologous mature unpulsed DCs and was MHC class I and class II restricted. In one patient, autologous CTLs stimulated by leukemic DCs or pulsed nonleukemic DCs showed similar significant cytotoxic activity against autologous AML cells. These findings demonstrate the induction of leukemia-specific cytotoxic response by nonleukemic mature DCs cross-presenting apoptotic leukemic blasts and offer a complementary approach to the use of leukemic DCs. We believe that this strategy permits the generation of DC vaccines for the majority of patients with hematological malignancies.

Caspase-3 can be pseudo-activated by a Ca2+-dependent proteolysis at a non-canonical site.

We have shown previously that calcium could trigger nuclear fragmentation, which was associated with a caspase 3 (C3)-like activity [Juin, P., Pelletier, M., Oliver, L., Tremblais, K., Gregoire, M., Meflah, K. and Vallette, F.M. (1998) Induction of a caspase-3-like activity by calcium in normal cytosolic extracts triggers nuclear apoptosis in a cell-free system. J. Biol. Chem. 273, 17559]. Here, we report that this activation is associated with a non-canonical truncation of C3, which induces a weak DEVDase activity. The cleavage of C3 via calcium-dependent proteolysis is independent of caspase 9; lysate exposure to calcium prevents further cleavage and activation by the cytochrome c and dATP pathway. Altogether, our data suggest that calcium could favour a necrotic mechanism by inducing the generation of a form of C3 insensitive to mitochondrial activation.

Impact of pH on Bax alpha conformation, oligomerisation and mitochondrial integration.

The change in the conformation of Bax at the onset of apoptosis is a determinant for the execution of this cell death programme. However, very few models can account for this modification and the factors involved in this process remain elusive. We have analysed the modifications in the conformation induced by a variation in pH using a cell-free assay. We show that a moderate basic or acidic pH can induce apoptotic-like changes in the conformation of Bax, such as the exposure of the N-terminal or the BH3 domain. These changes in the conformation are associated with the binding of Bax to mitochondria and an enhanced Bax homo- and oligomerisation. Our results suggest that variations in the pH, in a range consistent with that often observed during apoptosis, are sufficient to trigger Bax translocation to mitochondria and the subsequent release of apoptogenic factors from this organelle.

Opposite role of Bax and BCL-2 in the anti-tumoral responses of the immune system.

BACKGROUND: The relative role of anti apoptotic (i.e. Bcl-2) or pro-apoptotic (e.g. Bax) proteins in tumor progression is still not completely understood. METHODS: The rat glioma cell line A15A5 was stably transfected with human Bcl-2 and Bax transgenes and the viability of theses cell lines was analyzed in vitro and in vivo. RESULTS: In vitro, the transfected cell lines (huBax A15A5 and huBcl-2 A15A5) exhibited different sensitivities toward apoptotic stimuli. huBax A15A5 cells were more sensitive and huBcl-2 A15A5 cells more resistant to apoptosis than mock-transfected A15A5 cells (pCMV A15A5). However, in vivo, in syngenic rat BDIX, these cell lines behaved differently, as no tumor growth was observed with huBax A15A5 cells while huBcl-2 A15A5 cells formed large tumors. The immune system appeared to be involved in the rejection of huBax A15A5 cells since i) huBax A15A5 cells were tumorogenic in nude mice, ii) an accumulation of CD8+ T-lymphocytes was observed at the site of injection of huBax A15A5 cells and iii) BDIX rats, which had received huBax A15A5 cells developed an immune protection against pCMV A15A5 and huBcl-2 A15A5 cells. CONCLUSIONS: We show that the expression of Bax and Bcl-2 controls the sensitivity of the cancer cells toward the immune system. This sensitization is most likely to be due to an increase in immune induced cell death and/or the amplification of an anti tumour immune response

Impact of proapoptotic proteins Bax and Bak in tumor progression and response to treatment.

The cell death program, apoptosis, is currently viewed as the ultimate obstacle in cancer therapy. Inhibition of apoptosis is thought to be involved in both tumorigenesis and resistance to chemo- and radiotherapy. Considerable efforts are underway to design new tools capable of overcoming this inhibition. In this review, the current understanding of mechanisms of apoptosis in normal and tumor cells as well as possible or existing strategies designed to induce specific and efficient cell death in cancers are summarized.

Relevance of both individual risk factors and occupational exposure in cancer survival studies: the example of intestinal type sinonasal adenocarcinoma.

OBJECTIVES/HYPOTHESIS: Wood dust is a well-established risk factor for intestinal type sinonasal adenocarcinoma. The 5-year overall survival has varied from 20% to 80% according T1-T4 stages; 5-year survival according to histologic subtype has varied from 20% to 50%. To date, no study has evaluated whether environmental, occupational, and personal risk factors have any impact on both overall and cancer-specific survival. We aimed to determine whether exposure to carcinogenic risk factors besides wood exposure can influence the survival of patients with sinonasal ethmoid carcinoma. STUDY DESIGN: Retrospective cohort study of the association of survival data and occupational and personal carcinogenic risk factors. METHODS: All patients hospitalized for ethmoid adenocarcinoma at the Nantes University Hospital between 1988 and 2004 were included . Data concerning TNM classification, histology, type and quality of tumor resection at the macro- and microscopic level, and occupational and personal exposure to carcinogens were collected. Statistical analysis was conducted using univariate and multivariate linear regression. RESULTS: A total of 98 patients were included with a response rate of 98%. Data showed 86% of patients had been exposed to wood dust. The 5-year survival was 62%. We first identified four factors that independently influenced overall survival: diplopia (P = .0159), spread to the orbit (P = .0113), bilateral involvement (P = .0134), TNM stage (P < .001). When the analysis included all occupational environmental factors (wood dust, solvent, and metals exposure) as well as personal risk factors, the length of exposure to metals (P = .0307) and tobacco exposure (P = .0031) also were found to influence 5-year overall survival. We identified high prevalence of colon cancer (4%) and double cancer (18%). CONCLUSIONS: We showed exposure to both environmental (tobacco) and occupational (metal dust) factors could influence survival in the diagnosis of a cancer. Our study suggests that screening for colon cancer should be offered to wood dust workers. A prospective multicentric study should be necessary to confirm our results.

Large intestine intraepithelial lymphocytes from Apc+/+ and Apc+/Min mice and their modulation by indigestible carbohydrates: the IL-15/IL-15R alpha complex and CD4+ CD25+ T cells are the main targets.

We have shown recently that some indigestible carbohydrate (short-chain fructo-oligosaccharides [sc-FOS]) reduced colon tumor incidence in Apc+/Min mice, and that this effect depended on a functional local immune system. In addition, IL-15 mRNA was concomitantly modulated in the mucosa. Since intraepithelial lymphocytes (IELs) are in close contact with intestinal epithelial cells, these cells are the candidates most likely to be involved in early cancer immunosurveillance. The present study documents the effects of sc-FOS on large intestine IELs (LI-IELs) from Apc+/+ or Apc+/Min mice by analyzing markers related to their phenotype, their activation status, and the cell surface IL-15/IL-5R alpha. In the colons of Apc+/Min mice, fewer LI-IELs expressed surface IL-15/IL-15R alpha. In addition, a lower number of CD4+ LI-IELs expressed CD25, although more LI-IELs expressed CD69, as compared to normal mice. The sc-FOS enriched diet caused a decrease in the proportion of CD25+ LI-IELs and an increase in the percentage of LI-IELs bearing surface IL-15/IL-15R alpha, independently of the Apc gene status. The IL-15/IL-15R alpha increase was, however, higher in Min mice, and returned to a level very similar to that of Apc+/+ mice when the latter mice were fed a low-fiber diet. The sc-FOS-enriched diet specifically induced an increase in CD69+ cells in Apc+/+ mice, and a decrease in the proportion of CD4+ CD25+ LI-IELs in Apc+/Min mice. Some of these modulations could contribute to the development of a better immune anticancer response in the early steps of cancer development.

Distinct domains control the addressing and the insertion of Bax into mitochondria.

The translocation of Bax from the cytosol into the mitochondrial outer membrane is a central event during apoptosis. We report that beyond the addressing step, which involves its first alpha-helix (halpha1), the helices alpha5 and alpha6 (halpha5alpha6) are responsible for the insertion of Bax into mitochondrial outer membrane bilayer. The translocation of Bax to mitochondria is associated with specific changes in the conformation of the protein that are under the control of two prolines: Pro-13, which controls the unfolding of halpha1, and Pro-168, a proline located immediately before the hydrophobic carboxyl-terminal end (i.e. helix alpha9, halpha9), which controls the disclosure of halpha5alpha6. An additional step, the disruption of an electrostatic bond formed between Asp-33 (halpha1) and Lys-64 (BH3), allows the mitochondria addressing of Bax. We conclude that, although the intramolecular interactions of halpha1 with the BH3 region control the addressing of Bax to mitochondria, the Pro-168 is involved in the control of its membrane insertion through halpha5alpha6.

The N-terminal end of Bax contains a mitochondrial-targeting signal.

The translocation of Bax alpha, a pro-apoptotic member of the BCL-2 family from the cytosol to mitochondria, is a central event of the apoptotic program. We report here that the N-terminal (NT) end of Bax alpha, which contains its first alpha helix (Eta alpha 1), is a functional mitochondrial-addressing signal both in mammals and in yeast. Similar results were obtained with a newly described variant of Bax called Bax psi, which lacks the first 20 amino acids of Bax alpha and is constitutively associated with mitochondria. Deletion of Eta alpha 1 impairs the binding of Bax psi to mitochondria, whereas a fusion of the N terminus of Bax alpha, which contains Eta alpha 1 with a cytosolic protein, results in the binding of the chimeric proteins to mitochondria both in a cell-free assay and in vitro. More importantly, the mitochondria-bound chimeric proteins inhibit the interaction of Bax psi with mitochondria as well as Bax-apoptogenic properties. The mutations of the Eta alpha 1, which inhibit Bax alpha and Bax psi translocation to mitochondria, also block the subsequent activation of the execution phase of apoptosis. Conversely, a deletion of the C terminus does not appear to influence Bax alpha and Bax psi mitochondrial addressing. Taken together, our results suggest that Bax is targeted to mitochondria by its NT and thus through a pathway that is unique for a member of the BCL-2 family.

The p18 truncated form of Bax behaves like a Bcl-2 homology domain 3-only protein.

p21(Bax) is a pro-apoptotic member of the Bcl-2 family and is converted by calpain into a truncated form called p18(Bax). This proteolysis enhanced the apoptogenic properties of Bax by a mechanism not yet elucidated. We have shown recently that the first alpha helix (Halpha1) of p21(Bax) contained a mitochondrial addressing sequence, which appeared to be necessary for p21(Bax)-induced apoptosis (Cartron, P. F., Priault, M., Oliver, L., Meflah, K., Manon, S., and Vallette, F. M. (2003) J. Biol. Chem. 278, 11633-11641). This feature is in contradiction with the high apoptogenic profile of p18(Bax), because the Halpha1 is lost during the calpain cleavage of p21(Bax). We investigated the role of p18(Bax) in apoptosis and found that its activity required the presence of p21(Bax). In addition, p18(Bax) exhibited a higher affinity for Bcl-Xl than p21(Bax) did, a property that seems to be essential for the fulfillment of its pro-apoptotic role. In conclusion, calpain proteolysis converts the multi-domain p21(Bax) into a Bcl-2 homology 3-like protein capable of overcoming the inhibition of apoptosis due to Bcl-Xl.

Increased expression of inducible HSP70 in apoptotic cells is correlated with their efficacy for antitumor vaccine therapy.

Apoptosis is a physiologic process in normal development, tissue remodeling and cell turnover. This cell death is noninflammatory and nonimmunogenic, but when associated with a danger signal, it can activate the immune system. However, the capacity of apoptotic cells to activate the immune system is not clearly established, although dead tumor cells have been largely exploited as a source of TAA in cellular therapy against cancer. From these cellular preparations, contradictory results have been reported on the effect of apoptotic cells as an effective source of TAA and their immunologic properties. These conflicting data strongly suggest that the optimal preparation of apoptotic cells derived from tumor cells remains to be determined. In this work, we studied and compared the efficacy of antitumor immune responses derived from repeated injections using different preparations of apoptotic cells. We investigated the importance of HSP70 and TGF-beta expression in apoptotic cells used in the treatment of an established and nonimmunogenic rat carcinoma. UVB-mediated apoptosis did not affect TGF-beta expression in tumor cells, whereas HS treatment sharply downregulated it. Thus, downregulation of TGF-beta permits normal DC activation and maturation and the induction of tumor immunity. We conclude that HS followed by UVB irradiation is a superior source of tumor antigen for the treatment of established tumors. Future work will determine whether HS independently upregulates HSP70, thereby suppressing expression of active TGF-beta, or whether the 2 are linked via a still undefined mechanism.

Transient exposure of dendritic cells to maturation stimuli is sufficient to induce complete phenotypic maturation while preserving their capacity to respond to subsequent restimulation.

Dendritic cells (DC) are activated by pathogens, cytokines and activated T cells. We investigated the impact of a transient initial DC stimulation on the kinetics of maturation using a combination of double-stranded RNA and TNFalpha and subsequent restimulation by T cell-derived stimuli. Transient stimulation of DC was sufficient to start an irreversible program of phenotypic maturation which proceeded in the absence of the initial stimulus. Transiently stimulated DC secreted lower amounts of IL-12 during the 48-h period of the first stimulation than cells activated for 48 h. Although both DC preparations expressed the same level of maturation-associated markers at 48 h, DC stimulated for shorter periods preserved higher sensitivity to boosting upon subsequent stimulation by T cell-derived signals. We showed that DC initially stimulated for shorter periods were more potent stimulators of T lymphocytes and they induced a more polarized Th1 response. These results indicate that short exposure of DC to maturation stimuli enables an efficient defensive immune response induction by differentially regulating phenotypic maturation and cytokine production of DC.

Butyrate restores motile function and actin cytoskeletal network integrity in apc mutated mouse colon epithelial cells.

Loss of function of the Apc gene product is an early and frequent event in colorectal carcinogenesis. Altered migration of intestinal epithelial cells has been described in vivo in the Min mouse Apc+/Min model. Using cell lines established from this model we show in vitro that Apc+/Min cells are less motile than Apc+/+ cells and exhibit a disordered actin cytoskeletal network. This would increase the probabilities of the initiated cell to acquire additional genetic alterations leading to neoplasia. Butyrate, a product of indigestible carbohydrate fermentation by the colonic flora, is able to restore both motility and actin cytoskeletal organization. This feature may contribute to explain the protective effect exerted by butyrogenic diets on colon carcinogenesis in animal models.

Caspase 3 activation is controlled by a sequence located in the N-terminus of its large subunit.

We report that the induction and completion of the apoptotic program is delayed in a doxorubicin-resistant cell line (HL60/ADR). This hindrance to cell death occurred downstream of the multidrug-resistant protein (mrp), a transmembrane transporter. In vitro studies showed that these cells were incapable of correctly activating procaspase 3 (pC3), the main executioner of apoptosis. Sequencing of HL60/ADR pC3 revealed point mutations in a sequence located in the N-terminal region of the large subunit of caspase 3 (C3, amino acids 31-37; i.e., immediately after the propeptide). We called this particular form of C3, the C3 N-terminal modified (C3-NTM), and show that it is partially active when transfected into MCF-7 cells shown to have little or no endogenous pC3. As a deletion of the amino acids 31-37 in wild-type C3 leads to the same phenotype, we conclude that this sequence is involved in C3 activation during apoptosis.