Altered expression of inflammatory cytokine receptors in response to LPS challenge through interaction between intestinal epithelial cells and lymphocytes of Peyer's patch.

The intense innate immunological activities occurring at the enteric mucosal surface involve interactions between intestinal epithelial cells and immune cells. Our previous studies have indicated that Peyer's patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact as well as cytokine signaling. The present study was undertaken using the established co-culture system of Caco-2 epithelial cells with lymphocytes of Peyer's patch to investigate the expression of IL-8 and IL-6 cytokines and cytokine receptors in the co-culture system after challenge with Shigella F2a-12 lipopolysaccharide (LPS). The human colonic epithelial cell line Caco-2 was co-cultured with freshly isolated lymphocytes from the murine Peyer's patch either in the mixed or separated (isolated but permeable compartments) co-culture configuration, and was challenged with Shigella F2a-12 LPS for 8 h. The level of mRNA expressions of human interleukin-8 (hIL-8), human interleukin-8 receptor (hIL-8R), mouse interleukin-8 receptor (mIL-8R), mouse interleukin-6 (mIL-6), mouse interleukin-6 receptor (mIL-6R) and human interleukin-6 receptor (hIL-6R) was examined by semi-quantitative PCR. In both co-culture groups, hIL-8 expression of Caco-2 cells was decreased, and hIL-8R expression was increased compared to the Caco-2 alone group. Upon LPS challenge, hIL-8 expression from the Caco-2 cells of both co-culture groups was higher than in the Caco-2 control group. The increased hIL-8 expression of Caco-2 cells in the separated co-culture group is correlated with a decreased hIL-8R expression and an increased mIL-8R expression. In the mixed co-culture group, the increased expression of hIL-8 was associated with the upregulated hIL-8R expression on Caco-2 cells and downregulated mIL-8R on murine Peyer's patch lymphocytes (PPL). mIL-6 expression from mouse PPL was also upregulated by LPS in mixed co-culture. However, upon the treatment with LPS, hIL-6R expression of Caco-2 cells was decreased in the mixed co-culture, but increased in separated co-culture. The data suggest that release of hIL-8 from epithelial cells may act on lymphocytes through a paracrine pathway, but it may also act on the epithelial cells themselves via an autocrine pathway. The data also suggest that the release of mIL-6 from Peyer's patch lymphocytes affects epithelial cells in a paracrine fashion.

A suppressor of multiple extracellular matrix-degrading proteases and cancer metastasis.

Cancer metastasis remains the most poorly understood process in cancer biology. It involves the degradation of extracellular matrix (ECM) proteins by a series of 'tumour-associated' proteases. Here we report the identification of a novel protease suppressor, NYD-SP8, which is located on human chromosome 19q13.2. NYD-SP8 encodes a 27 kD GPI-anchored cell surface protein, which shows structural homology to urokinase plasminogen activator receptor (uPAR). Co-immunoprecipitation experiments showed that NYD-SP8 binds to uPA/uPAR complexes and interfere with active uPA production. Overexpression of NYD-SP8 results in reducing activities of the three major classes of proteases known to be involved in ECM degradation, including uPA, matrix metalloproteinases (MMPs) and cathepsin B, leading to suppression of both in vitro and in vivo cancer cell invasion and metastasis. These data demonstrate an important role of NYD-SP8 in regulating ECM degradation, providing a novel mechanism that modulates urokinase signalling in the suppression of cancer progression.

Interaction between enteric epithelial cells and Peyer's patch lymphocytes in response to Shigella lipopolysaccharide: effect on nitric oxide and IL-6 release.

AIM: To investigate the effect of interaction between enteric epithelial cells and lymphocytes of Peyer's patch on the release of nitric oxide (NO) and IL-6 in response to Shigella lipopolysaccharide (LPS). METHODS: Human colonic epithelial cells (Caco-2) were mixed cocultured with lymphocytes of Peyer's patch from wild-type (C57 mice) and inducible NO synthase knockout mice, and challenged with Shigella F2a-12 LPS. Release of NO and mIL-6 was measured by Griess colorimetric assay and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: In the absence of LPS challenge, NO was detected in the culture medium of Caco-2 epithelial cells but not in lymphocytes of Peyer's patch, and the NO release was further up-regulated in both cocultures with lymphocytes from either the wild-type or iNOS knockout mice, with a significantly higher level observed in the coculture with iNOS knockout lymphocytes. After Shigella F2a-12 LPS challenge for 24-h, NO production was significantly increased in both Caco-2 alone and the coculture with lymphocytes of Peyer's patch from the wild-type mice but not from iNOS knockout mice. LPS was found to stimulate the release of mIL-6 from lymphocytes, which was suppressed by coculture with Caco-2 epithelial cells. The LPS-induced mIL-6 production in lymphocytes from iNOS knockout mice was significantly greater than that from the wild-type mice. CONCLUSION: Lymphocytes of Peyer's patch maintain a constitutive basal level of NO production from the enteric epithelial cell Caco-2. LPS-induced mIL-6 release from lymphocytes of Peyer's patch is suppressed by the cocultured epithelial cells. While no changes are detectable in NO production in lymphocytes from both wild-type and iNOS knockout mice before and after LPS challenge, NO from lymphocytes appears to play an inhibitory role in epithelial NO release and their own mIL-6 release in response to LPS.

Anti-apoptotic activity of Bak Foong Pills and its ingredients on 6-hydroxydopamine-induced neurotoxicity in PC12 cells.

Bak Foong Pills (BFP), a traditional Chinese medicine used for centuries for the enhancement of women's health, was shown to display neuro-protective activity in the 1-methyl-4-phenyl-1,2,4,6,-tetrahydro-pyridine (MPTP)-induced mouse model in a previous study. In order to elucidate its mechanism of action, we investigated the anti-apoptotic properties of Bak Foong Pills and its main ingredients, including Panax ginseng, Angelica sinensis, Glycyrrhiza uralensis, and Ligusticum chuanxiong, in the 6-hydroxydopamine (6-OHDA)-treated PC12 cell model. The addition of the neurotoxin could cause significant cell death and reduction of cell proliferation, as shown in the results determined by MTT assay, nitric oxide (NO) measurement and flow cytometric propidium iodine (PI) staining analysis, while pre-treatment of PC12 cell with either BFP or its main ingredients prevented the toxicity to some degree. In addition, the neurotoxin caused an elevated activation of caspase-3, the key enzyme for activation of the cellular apoptotic cascade, whereas BFP or its main ingredients inhibited the activation of caspase-3. These results strongly indicate that BFP and its main ingredients may provide a useful therapeutic strategy for the treatment of neurodegenerative diseases, such as Parkinson's disease.

Estrogen-induced abnormally high cystic fibrosis transmembrane conductance regulator expression results in ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) remains one of the most life-threatening and potentially fatal complications of assisted reproduction treatments, arising from excessive stimulation of the ovaries by exogenous gonadotropins administrated during in vitro fertilization procedures, which is characterized by massive fluid shift and accumulation in the peritoneal cavity and other organs, including the lungs and the reproductive tract. The pathogenesis of OHSS remains obscure, and no definitive treatments are currently available. Using RT-PCR, Western blot, and electrophysiological techniques we show that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel expressed in many epithelia, is involved in the pathogenesis of OHSS. Upon ovarian hyperstimulation, rats develop OHSS symptoms, with up-regulated CFTR expression and enhanced CFTR channel activity, which can also be mimicked by administration of estrogen, but not progesterone, alone in ovariectomized rats. Administration of progesterone that suppresses CFTR expression or antiserum against CFTR to OHSS animals results in alleviation of the symptoms. Furthermore, ovarian hyperstimulation does not induce detectable OHSS symptoms in CFTR mutant mice. These findings confirm a critical role of CFTR in the pathogenesis of OHSS and may provide grounds for better assisted reproduction treatment strategy to reduce the risk of OHSS and improve in vitro fertilization outcome.

Modulation of human enteric epithelial barrier and ion transport function by Peyer's patch lymphocytes.

AIM: To investigate the role of Peyer's patch lymphocytes in the regulation of enteric epithelial barrier and ion transport function in homeostasis and host defense. METHODS: Mouse Peyer's patch lymphocytes were co-cultured with human intestinal epithelial cell line Caco-2 either in the mixed or separated (isolated but permeable compartments) culture configuration. Barrier and transport functions of the Caco-2 epithelial monolayers were measured with short-circuit current (Isc) technique. Release of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and cytokine mRNA expression was analyzed by semi-quantitative RT-PCR. Barrier and iontransport functions of both culture conditions following exposure to Shigella lipopolysaccharide (LPS) were also examined. RESULTS: The transepithelial resistance (TER) of the epithelial monolayers co-cultured with Peyer's patch lymphocytes was maintained whereas that of the Caco-2 monolayers alone significantly decreased after eight days in culture. The forskolin-induced anion secretion, in either absence or presence of LPS, was significantly suppressed in the both co-cultures as compared with the Caco-2 cells alone. Furthermore, only the mixed co-culture condition induced the expression and release of mIL-6 from Peyer's patch lymphocytes, which could be further enhanced by LPS. However, both co-culture conditions suppressed expression and release of epithelial hIL-8 under the unstimulated conditions, while the treatment with LPS stimulated their hIL-8 expression and release. CONCLUSION: Peyer's patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact and cytokine signaling.

Differential mode of regulation of the checkpoint kinases CHK1 and CHK2 by their regulatory domains.

CHK1 and CHK2 are key mediators that link the machineries that monitor DNA integrity to components of the cell cycle engine. Despite the similarity and potential redundancy in their functions, CHK1 and CHK2 are unrelated protein kinases, each having a distinctive regulatory domain. Here we compare how the regulatory domains of human CHK1 and CHK2 modulate the respective kinase activities. Recombinant CHK1 has only low basal activity when expressed in cultured cells. Surprisingly, disruption of the C-terminal regulatory domain activates CHK1 even in the absence of stress. Unlike the full-length protein, C-terminally truncated CHK1 displays autophosphorylation, phosphorylates CDC25C on Ser(216), and delays cell cycle progression. Intriguingly, enzymatic activity decreases when the entire regulatory domain is removed, suggesting that the regulatory domain contains both inhibitory and stimulatory elements. Conversely, the kinase domain suppresses Ser(345) phosphorylation, a major ATM/ATR phosphorylation site in the regulatory domain. In marked contrast, CHK2 expressed in either mammalian cells or in bacteria is already active as a kinase against itself and CDC25C and can delay cell cycle progression. Unlike CHK1, disruption of the regulatory domain of CHK2 abolishes its kinase activity. Moreover, the regulatory domain of CHK2, but not that of CHK1, can oligomerize. Finally, CHK1 but not CHK2 is phosphorylated during the spindle assembly checkpoint, which correlates with the inhibition of the kinase. The mitotic phosphorylation of CHK1 requires the regulatory domain, does not involve Ser(345), and is independent on ATM. Collectively, these data reveal the very different mode of regulation between CHK1 and CHK2.

DNA damage during the spindle-assembly checkpoint degrades CDC25A, inhibits cyclin-CDC2 complexes, and reverses cells to interphase.

Cell cycle checkpoints that monitor DNA damage and spindle assembly are essential for the maintenance of genetic integrity, and drugs that target these checkpoints are important chemotherapeutic agents. We have examined how cells respond to DNA damage while the spindle-assembly checkpoint is activated. Single cell electrophoresis and phosphorylation of histone H2AX indicated that several chemotherapeutic agents could induce DNA damage during mitotic block. DNA damage during mitotic block triggered CDC2 inactivation, histone H3 dephosphorylation, and chromosome decondensation. Cells did not progress into G1 but seemed to retract to a G2-like state containing 4N DNA content, with stabilized cyclin A and cyclin B1 binding to Thr14/Tyr15-phosphorylated CDC2. The loss of mitotic cells was not due to cell death because there was no discernible effect on caspase-3 activation, DNA fragmentation, or viability. Extensive DNA damage during mitotic block inactivated cyclin B1-CDC2 and prevented G1 entry when the block was removed. The mitotic DNA damage responses were independent of p53 and pRb, but they were dependent on ATM. CDC25A that accumulated during mitosis was rapidly destroyed after DNA damage in an ATM-dependent manner. Ectopic expression of CDC25A or nonphosphorylatable CDC2 effectively inhibited the dephosphorylation of histone H3 after DNA damage. Hence, although spindle disruption and DNA damage provide conflicting signals to regulate CDC2, the negative regulation by the DNA damage checkpoint could overcome the positive regulation by the spindle-assembly checkpoint.

X-linked inhibitor of apoptosis (XIAP) blocks Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis of prostate cancer cells in the presence of mitochondrial activation: sensitization by overexpression of second mitochondria-derived activator of caspase/direct IAP-binding protein with low pl (Smac/DIABLO).

The resistance to Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis could be overcome by treatment with subtoxic concentrations of actinomycin D (Act D) in prostate tumor cells. Furthermore, the sensitization to Apo2L/TRAIL-mediated apoptosis by Act D positively correlated with selective down-regulation of X-linked inhibitor of apoptosis (XIAP). In this study, we examined whether second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pl (Smac/DIABLO), a known inhibitor of apoptosis (IAP)-neutralizing protein, sensitizes resistant prostate tumor cells to Apo2L/TRAIL-mediated apoptosis. The prostate tumor cell line CL-1 was treated with Apo2L/TRAIL, Act D, or a combination of the two. The apoptosis-mediated signaling pathway was examined by Western blotting and flow cytometry. Furthermore, CL-1 cells transfected with the anti-IAP inhibitor Smac/DIABLO were examined for sensitivity to Apo2L/TRAIL. Whereas Apo2L/TRAIL induced the release of cytochrome c and endogenous Smac/DIABLO in the CL-1 tumor cells, the cytosolic levels of both molecules were not sufficient to induce apoptosis. Transient transfectants with a Smac/DIABLO cDNA encoding a neutralizing inhibitor of IAPs were sensitized to Apo2L/TRAIL-mediated apoptosis. The sensitization to Apo2L/TRAIL by Smac/DIABLO overexpression was a result of synergistic activation of caspases-3, -9, and -8. Treatment of the Smac/DIABLO transient transfectant with Apo2L/TRAIL enhanced the release of Smac/DIABLO from mitochondria and led to reduction of IAP family proteins (XIAP, c-IAP1, and c-IAP2). These results show that Smac/DIABLO can sensitize CL-1 tumor cells to Apo2L/TRAIL-mediated apoptosis. Thus, up-regulation of Smac/DIABLO and sensitization to Apo2L/TRAIL-mediated apoptosis are of potential clinical application in the immunotherapy of drug-/Apo2L/TRAIL-resistant tumors.

Synergy is achieved by complementation with Apo2L/TRAIL and actinomycin D in Apo2L/TRAIL-mediated apoptosis of prostate cancer cells: role of XIAP in resistance.

BACKGROUND: Tumors have an inherent immunogenicity that can be exploited by immunotherapy. However, often tumors develop mechanisms that render them resistant to most immunologic cytotoxic effector mechanisms. This study examines the underlying mechanism of resistance to Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-mediated apoptosis. METHODS: We studied prostate tumor cell lines for their sensitivity to Apo2L/TRAIL-mediated apoptosis in the presence and absence of the sensitizing agent actinomycin D (Act D). Apoptosis was determined by flow cytometry and signaling for apoptosis by Western blot. RESULTS: Treatment with subtoxic concentrations of Act D significantly sensitizes the tumor cells (CL-1, DU-145, and PC-3 prostate tumor cells) to Apo2L/TRAIL-mediated apoptosis. The cytotoxicity of Act D-sensitized prostate tumor cells was a result of synergistic activation of caspases (caspase-3, -9, and -8), detectable after 6 hr of treatment. Treatment with Apo2L/TRAIL alone, although it was insufficient to induce apoptosis, resulted in the loss of mitochondrial membrane potential and release of cytochrome c from the mitochondria into the cytoplasm in the absence of significant caspases activation. These findings suggested that a major apoptosis resistance factor blocking the Apo2L/TRAIL apoptotic signaling events is present downstream of the mitochondrial activation. The expression of receptors and anti-apoptotic proteins were examined in Act D-sensitized CL-1 cells. The earliest and the most pronounced change induced by Act D was down-regulation of X-linked inhibitor of apoptosis (XIAP) and up-regulation of Bcl-xL/-xS proteins. The role of XIAP in resistance was demonstrated by overexpression of Smac/DIABLO, which inhibited inhibitors of apoptosis (IAPs) and sensitized the cells to Apo2L/TRAIL. Apo2L/TRAIL receptors (DR4, DR5, DcR1, and DcR2), c-FLIP, Bcl-2, and other IAP members (c-IAP1 and c-IAP2) were marginally affected at later times in the cells sensitized by Act D. CONCLUSION: This study suggests that the combination of Act D-induced down-regulation of XIAP (Signal I) and Apo2L/TRAIL-induced release of cytochrome c (Signal II) leads to the reversal of resistance to Apo2L/TRAIL-mediated apoptosis in the tumor cells. The sensitization of tumor cells to Apo2L/TRAIL by Act D is of potential clinical application in the immunotherapy of drug/Apo2L/TRAIL refractory tumors.

A new challenge for successful immunotherapy by tumors that are resistant to apoptosis: two complementary signals to overcome cross-resistance.

Tumor resistance to conventional therapies is a major problem in cancer treatment. While tumors initially respond to radiation or chemotherapies, subsequent treatments with these conventional modalities are ineffective against relapsed tumors. The problem of tumor resistance to chemotherapy and radiation has led to the development of immunotherapy and gene-based therapies. These alternative therapeutic approaches are intensely explored because they are supposed to be more tumor specific and better tolerated than the conventional therapies. Recent advances in apoptosis have revealed that resistance to apoptosis is one of the major mechanisms of tumor resistance to conventional therapies. Resistance to apoptosis is a naturally acquired characteristic during oncogenesis and is selected for after successive rounds of conventional therapies. Resistance to apoptosis involves dysregulation and/or mutation of apoptotic signaling molecules that render tumor cells unresponsive to apoptotic stimuli. Since both immunotherapy and chemotherapy kill tumors by apoptosis and the killings are signaled through a central core apoptotic program, dysregulation of this central program and development of resistance to apoptosis in chemoresistant cells could render them cross-resistant to immunotherapy. Therefore, in order to establish an effective antitumor response and to complement immunotherapy and gene-based therapies, cross-resistance due to resistance to apoptosis must be overcome. In this review, based on prior findings and recent evidence, we put forth a model, verified experimentally, in which chemoresistant tumor cells can be sensitized to immune-mediated killing by subtoxic concentrations of chemotherapeutic drugs/factors. The model involves two complementary signals. The first signal is a sensitizing signal that regulates pro/antiapoptotic targets, thus facilitating the apoptotic signal. The second apoptotic signal initiates a partial activation of the apoptotic signaling pathway, and activation is completed by complementation with signal one. Thus, effective killing of immunoresistant cells is achieved by both signals. The two-signal approach provides a new strategy to overcome cancer cross-resistance to immunotherapy and opens new avenues for the development of more effective and selective immunosensitizing agents.

Rituximab modifies the cisplatin-mitochondrial signaling pathway, resulting in apoptosis in cisplatin-resistant non-Hodgkin's lymphoma.

PURPOSE: Rituximab (chimeric anti-CD20) can reverse the cisplatin-resistant phenotype of AIDS-related non-Hodgkin's lymphoma cell lines and results in cisplatin-mediated apoptosis. The mechanism by which apoptosis is achieved by the combination treatment was examined. EXPERIMENTAL DESIGN: The AIDS-related lymphoma (ARL) cell line 2F7 was treated with rituximab, cisplatin, and a combination of the two and analyzed by Western blot analyses for signaling proteins involved in the death receptor-mediated and mitochondrial pathways. RESULTS: Rituximab selectively inhibited the expression of Bcl-2 in the ARL cells. However, other proteins analyzed [namely, Apaf-1, Bax, Bid, caspase-3, caspase-8, caspase-9, X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2, cytochrome c, Fas, Fas ligand, FLIP, p53, and poly(ADP-ribose) polymerase] were not affected by either rituximab or cisplatin. Treatment with cisplatin induced the generation of mitochondrial reactive oxygen species, specifically intracellular peroxides. Furthermore, cisplatin alone was unable to induce the mitochondrial apoptotic events; however, the rituximab-cisplatin combination was able to synergistically induce significant apoptosis and mitochondria-mediated apoptotic events [mitochondrial membrane depolarization (DeltaPsi(m)), cytochrome c release from mitochondria, and caspase-3 and -9 activation]. The combination treatment facilitated the down-regulation of Bcl-2 by rituximab at an early time point. Decreased expression of additional proteins (Apaf-1, cIAP-1, cIAP-2, and XIAP) paralleled apoptosis detected at 24 h. CONCLUSIONS: These findings show that the selective down-regulation of Bcl-2 by rituximab leading to apoptosis in ARL cells by cisplatin is through the mitochondria-dependent caspase pathway.