Acceleration of glutathione efflux and inhibition of gamma-glutamyltranspeptidase sensitize metastatic B16 melanoma cells to endothelium-induced cytotoxicity

Highly metastatic B16 melanoma (B16M)-F10 cells, as compared with the low metastatic B16M-F1 line, have higher GSH content and preferentially overexpress BCL-2. In addition to its anti-apoptotic properties, BCL-2 inhibits efflux of GSH from B16M-F10 cells and thereby may facilitate metastatic cell resistance against endothelium-induced oxidative/nitrosative stress. Thus, we investigated in B16M-F10 cells which molecular mechanisms channel GSH release and whether their modulation may influence metastatic activity. GSH efflux was abolished in multidrug resistance protein 1 knock-out (MRP-/-1) B16M-F10 transfected with the Bcl-2 gene or in MRP-/-1 B16M-F10 cells incubated with l-methionine, which indicates that GSH release from B16M-F10 cells is channeled through MRP1 and a BCL-2-dependent system (likely related to an l-methionine-sensitive GSH carrier previously detected in hepatocytes). The BCL-2-dependent system was identified as the cystic fibrosis transmembrane conductance regulator, since monoclonal antibodies against this ion channel or H-89 (a protein kinase A-selective inhibitor)-induced inhibition of cystic fibrosis transmembrane conductance regulator gene expression completely blocked the BCL-2-sensitive GSH release. By using a perifusion system that mimics in vivo conditions, we found that GSH depletion in metastatic cells can be achieved by using Bcl-2 antisense oligodeoxynucleotide- and verapamil (an MRP1 activator)-induced acceleration of GSH efflux, in combination with acivicin-induced inhibition of gamma-glutamyltranspeptidase (which limits GSH synthesis by preventing cysteine generation from extracellular GSH). When applied under in vivo conditions, this strategy increased tumor cytotoxicity (up to approximately 90%) during B16M-F10 cell adhesion to the hepatic sinusoidal endothelium.     

Down-regulation of glutathione and Bcl-2 synthesis in mouse B16 melanoma cells avoids their survival during interaction with the vascular endothelium

B16 melanoma (B16M) cells with high GSH content show high metastatic activity. However, the molecular mechanisms linking GSH to metastatic cell survival are unclear. The possible relationship between GSH and the ability of Bcl-2 to prevent cell death was studied in B16M cells with high (F10) and low (F1) metastatic potential. Analysis of a Bcl-2 family of genes revealed that B16M-F10 cells, as compared with B16M-F1 cells, overexpressed preferentially Bcl-2 (approximately 5.7-fold). Hepatic sinusoidal endothelium-induced B16M-F10 cytotoxicity in vitro increased from approximately 19% (controls) to approximately 97% in GSH-depleted B16M-F10 cells treated with an antisense Bcl-2 oligodeoxynucleotide (Bcl-2-AS). l-Buthionine (S,R)-sulfoximine-induced GSH depletion or Bcl-2-AS decreased the metastatic growth of B16M-F10 cells in the liver. However, the combination of l-buthionine (S,R)-sulfoximine and Bcl-2-AS abolished metastatic invasion. Bcl-2-overexpressing B16M-F1/Tet-Bcl-2 and B16M-F10/Tet-Bcl-2 cells, as compared with controls, showed an increase in GSH content, no change in the rate of GSH synthesis, and a decrease in GSH efflux. Thus, Bcl-2 overexpression may increase metastatic cell resistance against oxidative/nitrosative stress by inhibiting release of GSH. In addition, Bcl-2 availability regulates the mitochondrial GSH (mtGSH)-dependent opening of the permeability transition pore complex. Death in B16M-F10 cells was sharply activated at mtGSH levels below 30% of controls values. However, this critical threshold increased to approximately 60% of control values in Bcl-2-AS-treated B16M-F10 cells. GSH ester-induced replenishment of mtGSH levels (even under conditions of cytosolic GSH depletion) prevented cell death. Our results indicate that survival of B16M cells with high metastatic potential can be challenged by inhibiting their GSH and Bcl-2 synthesis.     

Tumoricidal activity of endothelial cells. Inhibition of endothelial nitric oxide production abrogates tumor cytotoxicity induced by hepatic sinusoidal endothelium in response to B16 melanoma adhesion in vitro

The mechanism of NO- and H(2)O(2)-induced tumor cytotoxicity was examined during B16 melanoma (B16M) adhesion to the hepatic sinusoidal endothelium (HSE) in vitro. We used endothelial nitric-oxide synthetase gene disruption and N(G)-nitro-l-arginine methyl ester-induced inhibition of nitric-oxide synthetase activity to study the effect of HSE-derived NO on B16M cell viability. Extracellular H(2)O(2) was removed by exogenous catalase. H(2)O(2) was not cytotoxic in the absence of NO. However, NO-induced tumor cytotoxicity was increased by H(2)O(2) due to the formation of potent oxidants, likely ( small middle dot)OH and (-)OONO radicals, via a trace metal-dependent process. B16M cells cultured to low density (LD cells), with high GSH content, were more resistant to NO and H(2)O(2) than B16M cells cultured to high density (HD cells; with approximately 25% of the GSH content found in LD cells). Resistance of LD cells decreased using buthionine sulfoximine, a specific GSH synthesis inhibitor, whereas resistance increased in HD cells using GSH ester, which delivers free intracellular GSH. Because NO and H(2)O(2) were particularly cytotoxic in HD cells, we investigated the enzyme activities that degrade H(2)O(2). NO and H(2)O(2) caused an approximately 75% (LD cells) and a 60% (HD cells) decrease in catalase activity without affecting the GSH peroxidase/GSH reductase system. Therefore, B16M resistance to the HSE-induced cytotoxicity appears highly dependent on GSH and GSH peroxidase, which are both required to eliminate H(2)O(2). In agreement with this fact, ebselen, a GSH peroxidase mimic, abrogated the increase in NO toxicity induced by H(2)O(2).     

Bcl-2 and Mn-SOD antisense oligodeoxynucleotides and a glutamine-enriched diet facilitate elimination of highly resistant B16 melanoma cells by tumor necrosis factor-alpha and chemotherapy

Mitochondrial glutathione (mtGSH) depletion increases sensitivity of Bcl-2-overexpressing B16 melanoma (B16M)-F10 cells (high metastatic potential) to tumor necrosis factor-alpha (TNF-alpha)-induced oxidative stress and death in vitro. In vivo, mtGSH depletion in B16M-F10 cells was achieved by feeding mice (where the B16M-F10 grew as a solid tumor in the footpad) with an L-glutamine (L-Gln)-enriched diet, which promoted in the tumor cells an increase in glutaminase activity, accumulation of cytosolic L-glutamate, and competitive inhibition of GSH transport into mitochondria. L-Gln-adapted B16M-F10 cells, isolated using anti-Met-72 monoclonal antibodies and flow cytometry-coupled cell sorting, were injected into the portal vein to produce hepatic metastases. In l-Gln-adapted invasive (iB16M-Gln+) cells, isolated from the liver by the same methodology and treated with TNF-alpha and an antisense Bcl-2 oligodeoxynucleotide, viability decreased to approximately 12%. iB16M-Gln+ cell death associated with increased generation of O2*- and H2O2, opening of the mitochondrial permeability transition pore complex, and release of proapoptotic molecular signals. Activation of cell death mechanisms was prevented by GSH ester-induced mtGSH replenishment. The oxidative stress-resistant survivors showed an adaptive response that includes overexpression of manganese-containing superoxide dismutase (Mn-SOD) and catalase activities. By treating iB16M-Gln+ cells with a double anti- antisense therapy (Bcl-2 and SOD2 antisense oligodeoxynucleotides) and TNF-alpha, metastatic cell survival decreased to approximately 1%. Chemotherapy (taxol plus daunorubicin) easily removed this minimum percentage of survivors. This contribution identifies critical molecules that can be sequentially targeted to facilitate elimination of highly resistant metastatic cells.     

Nitric oxide and oxygen radicals induced apoptosis via bcl-2 and p53 pathway in hypoxia-reoxygenated cardiomyocytes

Twotypesofcellulardemisecanoccursimultaneouslyintissuesorculturedcellbynecrosisandapoptosis.Lossofmembraneintegrity,celledemaandbreak,andthecellcomponentsre-leasedoutarethecharacteristicsofnecrosis.Whilethecellapoptosisisaprogramcelldeathcodedbygeneandactivatedseriousendogenousenzymes[1].Recentstudieshavedemonstratedthatmyocardialischemia-reperfusioninjuryresultedinapoptoticcelldeathinadditiontotissuenecrosis[2—4].Oxygenstressisoneofthereasonsthatcausedcellapoptosisandtheoxygenradicalsinthest…     

Chinonin, a novel drug against cardiomyocyte apoptosis induced by hypoxia and reoxygenation

The inhibitory effects of Chinonin, a natural antioxidant extracted from a Chinese medicine, on apoptotic and necrotic cell death of cardiomyocytes in hypoxia-reoxygenation process were observed in this study. The possible mechanisms of Chinonin on scavenging reactive oxygen species and regulating apoptotic related genes bcl-2 and p53 were also investigated. Neonatal rat cardiomyocytes were subjected to 24-h hypoxia and 4-h reoxygenation. Cell death was evaluated by DNA electrophoresis on agarose gel, cell death ELISA and annexin-V-FLUOS/propidium iodide (PI) double staining cytometry. Hypoxia caused the increase of apoptotic rates and the release of lactate dehydrogenase (LDH), while reoxygenation not only further increased the apoptotic rates and leakage of LDH, but also induced necrosis of cardiomyocytes. In addition, hypoxia increased the levels of NO(2)(-)/NO(3)(-) and thiobarbituric acid reacted substances (TBARS), while reoxygenation decreased NO(2)(-)/NO(3)(-), but further increased TBARS in the cultured media. Moreover, hypoxia up-regulated the expression levels of bcl-2 and p53 proteins, while reoxygenation down-regulated bcl-2 and further up-regulated p53. Chinonin significantly decreased the rates of apoptotic and necrotic cardiomyocytes, and inhibited the leakage of LDH. It also diminished NO(2)(-)/NO(3)(-) and TBARS, down-regulated the expression level of p53 protein, and up-regulated bcl-2 protein, respectively. The results suggest that Chinonin has preventive effects against apoptotic and necrotic cell death and its protective mechanisms are related to the antioxidant properties of scavenging nitric oxide and oxygen free radicals, and the modulating effects on the expression levels of bcl-2 and p53 proteins.     

Nitric oxide and oxygen radicals induced apoptosis via bcl-2 and p53 pathway in hypoxia-reoxygenated cardiomyocytes

Neonatal rat cardiomyocytes were subjected to 24 h of hypoxia 95%N2/5%CO2 and 24 h of hypoxia plus 4 h of reoxygenation 95%O2/5%CO2. 24 h of hypoxia increased the levels of NO, TBARS and LDH. 24 h of hypoxia plus 4 h of reoxygenation decreased the levels of NO, but further increased TBARS and LDH. The hypoxia up-regulated the expression of bcl-2, p53 and p21/waf1/cip1 but the reoxygenation down-regulated the expression of bcl-2, and further up-regulated p53 and p21/waf1/cip1. The hypoxia increased cell apoptosis and reoxygenation further increased both apoptotic and necrotic cell death. NO, TBARS, DNA fragmentation and cell apoptosis were enhanced by SNP and inhibited by L-NAME respectively. In addition, SOD/catalase down-regulated the expression of p53, p21/wafl/cipl and TBARS but up-regulated bcl-2 and increased indirectly the level of NO, and inhibited DNA fragmentation. The results suggest that hypoxia-induced cell death is associated with the activation of NO, bcl-2 and p53 pathway, while hypoxia-reoxygenation induced cell death via the generation of reactive oxygen species and activation of p53 pathway. The present study clarified that NO may be an initiative signal to apoptotic cell death and the activation of bcl-2, p53 and p21/waf1/cip1 pathway in hypoxic and hypoxia-reoxygenated cardiomyocytes.     

Regulation of A1/Bfl-1 expression in peripheral splenic B cells

We analyzed here the expression of the prosurvival Bcl-2 homologue A1 in peripheral B cell compartment. We observed that A1 mRNA are highly expressed in peripheral B cells as compared with other anti-apoptotic genes of the Bcl-2 family such as bcl-xl and bcl-2 itself. The expression of A1 is up-regulated in immature B cells at the transition between transitional type 1 (T1) and type 2 (T2) cells, and remained highly expressed in mature (M) B cells. We, therefore, analyzed the effect of B cell antigen receptor (BCR) and BAFF receptor (BAFF-R) engagement on the regulation of A1 in total B220(+) cells but also FACS-sorted immature T1, T2 and M B cells. We demonstrated that only BCR engagement up-regulated the expression of A1 mRNA and protein. These results suggest that A1 may play a key role in antigen-dependent signals that are required for survival and/or proliferation of peripheral B cells.     

Trolox enhances curcumin's cytotoxicity through induction of oxidative stress

Curcumin, a natural polyphenol in the spice turmeric, has been found to exhibit anticancer activity. Although curcumin is generally considered an antioxidant, it is also able to elicit apoptosis through the generation of ROS, thereby functioning as a pro-oxidant in cancer cells. The present study investigated the effects of antioxidant pretreatment on curcumin-induced cytotoxicity in the human cancer cell lines A2780, MCF-7, and MDA-MB-231. Cytotoxicity was enhanced by trolox, vitamin C or vitamin E; trolox, a water soluble vitamin E derivative, was the most potent. The combination of curcumin (10 μM) and trolox (10-50 μM) induced apoptosis of cancer cells as evidenced by PARP cleavage and caspase-3 activation. Furthermore, expression of the pro-apoptotic protein Bad was up-regulated and expression of the anti-apoptotic proteins Bcl-2 and Bcl-xl was down-regulated in cells that had been treated with trolox plus curcumin. ROS generation was detected in curcumin-treated cells and was significantly enhanced when cells were treated with trolox plus curcumin. Exogenous catalase or SOD1 did not alter cytotoxicity, while over-expression of either catalase or SOD1 did, pointing to the importance of intracellular hydrogen peroxide generation in cell killing. In conclusion, we demonstrated for the first time that antioxidants such as trolox can potentiate cancer cell killing by curcumin, a finding which may help in the development of novel drug combination therapies.     

Tumor cytotoxicity by endothelial cells. Impairment of the mitochondrial system for glutathione uptake in mouse B16 melanoma cells that survive after in vitro interaction with the hepatic sinusoidal endothelium

High GSH content associates with high metastatic activity in B16-F10 melanoma cells cultured to low density (LD B16M). GSH homeostasis was investigated in LD B16M cells that survive after adhesion to the hepatic sinusoidal endothelium (HSE). Invasive B16M (iB16M) cells were isolated using anti-Met-72 monoclonal antibodies and flow cytometry-coupled cell sorting. HSE-derived NO and H(2)O(2) caused GSH depletion and a decrease in gamma-glutamylcysteine synthetase activity in iB16M cells. Overexpression of gamma-glutamylcysteine synthetase heavy and light subunits led to a rapid recovery of cytosolic GSH, whereas mitochondrial GSH (mtGSH) further decreased during the first 18 h of culture. NO and H(2)O(2) damaged the mitochondrial system for GSH uptake (rates in iB16M were approximately 75% lower than in LD B16M cells). iB16M cells also showed a decreased activity of mitochondrial complexes II, III, and IV, less O(2) consumption, lower ATP levels, higher O(2) and H(2)O(2) production, and lower mitochondrial membrane potential. In vitro growing iB16M cells maintained high viability (>98%) and repaired HSE-induced mitochondrial damages within 48 h. However, iB16M cells with low mtGSH levels were highly susceptible to TNF-alpha-induced oxidative stress and death. Therefore depletion of mtGSH levels may represent a critical target to challenge survival of invasive cancer cells.     

Epigallocatechin gallate up-regulation of miR-16 and induction of apoptosis in human cancer cells

Epigallocatechin gallate (EGCG) is a major type of green tea polyphenols and is known to have cancer prevention effect. MicroRNAs (miRNAs) are 19 to 25 nucleotides and are believed to be important in gene regulation. In the present study, the influence of EGCG on the expressions of miRNAs in human cancer cells was investigated as this has not yet been reported. By miRNA microarray analysis, EGCG treatment was found to modify the expressions of some of the miRNAs in human hepatocellular carcinoma HepG2 cells, 13 were up-regulated and 48 were down-regulated. miR-16 is one of the miRNAs up-regulated by EGCG and one of its target genes is confirmed to be the anti-apoptotic protein Bcl-2. EGCG treatment induced apoptosis and down-regulated Bcl-2 in HepG2 cells. Transfection with anti-miR-16 inhibitor suppressed miR-16 expression and counteracted the EGCG effects on Bcl-2 down-regulation and also induction of apoptosis in cells. Results from the present study confirm the role of miR-16 in mediating the apoptotic effect of EGCG and also support the importance of miRNAs in the regulation of the biological activity of EGCG.     

A-Myb up-regulates Bcl-2 through a Cdx binding site in t(14;18) lymphoma cells

In follicular lymphoma, bcl-2 is translocated to the immunoglobulin heavy chain locus leading to deregulation of bcl-2 expression. We examined the role of Myb proteins in the regulation of bcl-2 expression in lymphoma cells. We showed that A-Myb up-regulates bcl-2 promoter activity. Northern and Western analyses demonstrated that A-Myb was expressed in the DHL-4 t(14;18) cell line. In t(14;18) cells and mature B cells, A-Myb up-regulated bcl-2 expression, whereas B- and c-Myb had little effect on bcl-2 gene expression. Deletion analysis of the bcl-2 5'-region identified a region responsive to A-Myb in t(14;18) cells. A potential binding site for the Cdx homeodomain proteins was located in this sequence. Analysis of the A-Myb-responsive region by UV cross-linking experiments revealed that a 32-kDa protein formed a complex with this region, but direct binding by Myb proteins could not be demonstrated. A-Myb could be recovered along with Cdx2 when nuclear extracts were passed over the Cdx site. Mutagenesis of the Cdx binding site abolished binding by the 32-kDa protein and significantly reduced the ability of A-Myb to induce bcl-2 expression. A strong induction of bcl-2 P2 promoter activity was observed in cotransfection studies of DHL-4 cells with the A-Myb and Cdx2 expression vectors, and increased endogenous Bcl-2 protein expression was observed in B cells transfected with A-Myb and/or Cdx2 expression constructs.     

In vitro induction of apoptosis by isosclerone from marine-derived fungus Aspergillus fumigatus

In the present study, we investigated the apoptotic effects of isosclerone from marine-derived fungi on human breast cancer MCF-7 cells. Treatment with isosclerone exhibited a characteristic feature of apoptosis including significant cytotoxicity and DNA fragmentation in cancer cells. In addition, The apoptosis induction abilities of the isosclerone was studied by analyzing the expression of caspase-3, -8 and -9, Bcl-2 family, NF-κ-B P50, P65, and IKK proteins. Western blot and RT-PCR analysis have indicated that isosclerone induce cancer cells apoptosis through down-regulated Bcl-2 family and up-regulated caspases, and activating the NF-κ-B signaling pathway. Our data demonstrate that isosclerone specifically binds to crystal structure of apoptosis regulator BCL-2 and pseudo-activated procaspase-3 proteins through down-regulated Bcl-2 family and up-regulated caspases, and activating the NF-κ-B signaling pathway. Our proof-of-principle study should have a positive impact on future drug discovery.     

cDNA expression array analysis of gene expression in human hepatocarcinoma Hep3B cells induced by BNIPL-1

Bcl-2/adenovirus E1B 19 kDa interacting protein 2 like-1 （BNIPL-1） is a novel human protein identified in our laboratory, which can interact with Bcl-2 and Cdc42GAP and induce apoptosis via the BNIP-2 and Cdc42GAP homology （BCH） domain. In the present study, we established the Hep3B-Tet-on stable cell line in which expression of BNIPL-1 can be induced by doxycycline. The cell proliferation activity assay showed that the overexpression of BNIPL-1 suppresses Hep3B cell growth in vitro. The differential expression profiles of 588 known genes from BNIPL-1-transfected Hep3B-Tet-on and vector control cells were determined using the Atlas human cDNA expression array. Fifteen genes were differentially expressed between these two cell lines, among which seven genes were up-regulated and eight genes were down-regulated by BINPL-1. Furthermore, the differential expression result was confirmed by semiquantitative RT-PCR. Among these differentially expressed genes, p16^INK4, IL-12, TRAIL and the lymphotoxin β gene involved in growth suppression or cell apoptosis were up-regulated, and PTEN involved in cell proliferation was down-regulated by BNIPL-1. These results suggest that BNIPL-1 might inhibit cell growth though cell cycle arrest and/or apoptotic cell death pathway（s）.     

Preconditioning regulation of bcl-2 and p66shc by human NOS1 enhances tolerance to oxidative stress.

Preconditioning stress induced by a transient ischemia may increase brain tolerance to oxidative stress, and the underlying neuroprotective mechanisms are not well understood. In a series of experiments, we found that endogenous nitric oxide (NO), S-nitrosoglutathione (GSNO), and antioxidants blocked serum deprivation-induced oxidative stress and apoptosis in human neuroblastoma cells. Similar to nuclear redox factor-1 (Ref-1), mRNA of human neuronal nitric oxide synthase (hNOS1) was maximally up-regulated within 2 h after oxidative stress and down-regulated by NO/GSNO and hydroxyl radical (OH) scavenger. A brief preconditioning stress induced by serum deprivation for 2 h caused a delayed increase in the expression of hNOS1 protein and the associated formation of NO and cGMP, which in turn decreased OH generation and stress-related cell death. In addition to inhibiting caspase-3 through a dithiothreitol-sensitive S-nitrosylation process, preconditioning stress concomitantly up-regulated the expression of the anti-apoptotic bcl-2 protein and down-regulated the p66shc adaptor protein. This beneficial cytoprotective process of preconditioning stress is mediated by newly synthesized NO because it can be suppressed by the inhibition of hNOS1 and guanylyl cyclase. Therefore, the constitutive isoform of hNOS1 is dynamically redox-regulated to meet both functional and compensatory demands of NO for gene regulation, antioxidant defense, and tolerance to oxidative stress.     

Silencing SOCS3 could inhibit TNF-α induced apoptosis in 3T3-L1 and mouse preadipocytes

Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine involved in the apoptosis of many types of cells. In this study we demonstrated the effect of (suppressor of cytokine signalling-3) SOCS3 siRNA on TNF-α induced apoptosis in 3T3-L1 preadipocytes and mouse preadipocytes. 3T3-L1 preadipocytes and mouse preadipocytes were transfected with SOCS3 siRNA, and then the cells were treated with TNF-α at 100 ng/mL for 24 h. We used fluorescence microscope to observe morphological changes during apoptosis after Hoechst 33258 and PI staining. Quantitative PCR and Western blotting were used to measure the expression of apoptosis-associated gene c-myc, survivin, mcl-1, bcl-2, bax, NF-κB, and the key genes of the JAK/STAT3 pathway including SOCS1, SOCS2, JAK2, STAT3. Compared with control group, the number of cells apoptosis was decreased remarkably in SOCS3 siRNA group (P < 0.01). The expression of apoptotic suppressor genes c-myc, survivin, mcl-1, bcl-2 and NF-κB were up-regulated markedly (P < 0.01); in contrast, apoptotic gene bax was down-regulated (P < 0.05). Western blotting showed that the protein expressions of bcl-2 and NF-κB were increased remarkably (P < 0.01), while the protein expression of bax was decreased remarkably (P < 0.05). The expression of the JAK/STAT3 pathway key gene SOCS1 mRNA was down-regulated markedly (P < 0.05), but the key protein p-STAT3 was up-regulated (P < 0.05). Taken together, our data established that silenced SOCS3 can regulate the expression of apoptosis-associated genes via the JAK/STAT3 pathway, and effectively inhibit TNF-α induced apoptosis in 3T3-L1 preadipocytes and mouse preadipocytes.     

Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.     

Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes.

Cell death is a prominent feature of B cell development. For example, a large population of B cells dies at the pre-B cell stage presumably due to the failure to express a functional immunoglobulin receptor. In addition, developing B cells expressing antigen receptors for self are selectively eliminated at the immature B cell stage. The molecular signals that control B cell survival are largely unknown. The product of the bcl-2 proto-oncogene may be involved as its overexpression inhibits apoptotic cell death in a variety of biological systems. However, the physiological role of the endogenous Bcl-2 protein during B cell development is undetermined. Here we show a striking developmental regulation of the Bcl-2 protein in B lymphocytes. Bcl-2 is highly expressed in CD43+ B cell precursors (pro-B cells) and mature B cells but downregulated at the pre-B and immature B cell stages of development. We found that Bcl-2 expressed by B cells is a long-lived protein with a half-life of approximately 10 h. Importantly, susceptibility to apoptosis mediated by the glucocorticoid hormone dexamethasone is stage-dependent in developing B cells and correlates with the levels of Bcl-2 protein. Furthermore, expression of a bcl-2 transgene rescued pre-B and immature B cells from dexamethasone-induced cell death, indicating that Bcl-2 can inhibit the apoptotic cell death of progenitors and early B cells. Taken together, these findings argue that Bcl-2 is a physiological signal controlling cell death during B cell development.