Ambrosio Hernandez

The role of NF-κB/IκB proteins in cancer: implications for novel treatment strategies

Abstract The nuclear factor-kappaB (NF- κ B) family of transcription factors are involved in multiple cellular processes, including cytokine gene expression, cellular adhesion, cell cycle activation, apoptosis and oncogenesis. Constitutive activation of NF- κ B has been described in a number of solid tumors and this activation appears to affect cancer cell survival. Inhibition of NF- κ B has been shown to enhance the sensitivity of some cancer cell lines to antineoplastic- or radiation-induced apoptosis. Furthermore, suppression of NF- κ B results in attenuation of cancer cachexia in a mouse tumor model. Studies are underway to further delineate the role of NF- κ B in cancer cell survival, growth and resistance to standard chemotherapy and radiation regimens. Moreover, the effects of novel therapeutic agents which specifically target NF- κ B proteins are currently being assessed in experimental models of cancer cell growth both in vitro and in vivo. In this review, we discuss the possible involvement of NF- κ B in the growth of various solid tumors and potential future treatment strategies based on NF- κ B inhibition.

Sensitization of human colon cancer cells to TRAIL-mediated apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL), a novel member of the tumor necrosis factor (TMF) family, is thought to induce apoptosis preferentially in cancer cells; however, increasing evidence suggests that a number of cancers are resistant to TRAIL treatment. FLICE-like inhibitory protein (FLIP), which structurally resembles caspase-8, can act as an inhibitor of apoptosis when expressed at high levels in certain cancer cells. The purpose of our present study was to determine whether human colon cancer cells are sensitive to TRAIL treatment and, if not, to identify potential mechanisms of resistance. Colon cancer cells of different metastatic potential (KMlZC, KML4A, and KM20) were found to be resistant to the effects of TRAIL when used as a single agent. FLIP expression levels were increased in all three KM cell lines. Treatment with either actinomycin D (Act D;10 μ/ml) or cycloheximide (CHX; 10 μg/ml) decreased FLIP expression levels in all three cell lines. The decrease in cellular levels of FLIP was associated with sensitization to TRAIL-mediated apoptosis, as demonstrated by enhanced cell death and caspase-3 activity compared with either Act D or CHX alone. Our findings suggest that reduction of FLIP levels by Act D or CHX renders TRAIL-resistant human colon cancer cells sensitive to TRAIL-mediated apoptosis. The combination of TRAIL along with agents such as Act D or CHX, which target proteins that prevent cell death, may provide a more effective and less toxic regimen for treatment of resistant colon cancers.

Molecular Mechanisms Contributing to Necrotizing Enterocolitis

ObjectiveTo examine the cellular mechanisms involved in the pathogenesis of necrotizing enterocolitis (NEC). Summary Background DataNecrotizing enterocolitis is a major cause of death and complications in neonates; the cellular mechanisms responsible for NEC are unknown. The inducible form of cyclooxygenase (i.e., COX-2) is activated by the transcription factor nuclear factor (NF)-&kgr;B and is thought to play a role in inflammation. MethodsSegments of perforated and adjacent uninvolved small intestine from neonates with NEC were analyzed for COX-2 expression by immunohistochemistry. NEC was induced in weanling (18 days old) rats by occlusion of superior mesenteric vessels for 1 hour and intraluminal injection of platelet activating factor (50 &mgr;g/kg). Small intestine was harvested for protein extraction. Western immunoblot was performed to determine expression of COX-2. Gel shift assays were performed to assess NF-&kgr;B binding activity. ResultsImmunohistochemical analysis showed increased COX-2 protein expression in the perforated intestinal sections of all 36 neonates but not in adjacent normal intestine. Increased expression of COX-2 protein and NF-&kgr;B binding activity was noted in the small intestine of weanling rats at 0 and 3 hours after induction of NEC. ConclusionsIncreased COX-2 expression was identified in all neonatal intestinal segments resected for perforated NEC. In addition, a coordinate induction of COX-2 expression and NF-&kgr;B binding was noted in a rodent model of NEC. These findings suggest that the COX-2/NF-&kgr;B pathway may play a role in the pathogenesis of NEC. Therapeutic agents that target this pathway may prove useful in the treatment or possible prevention of NEC.

Mitochondrial DNA damage and altered membrane potential (ΔΨ) in pancreatic acinar cells induced by reactive oxygen species

Background: Reactive oxygen species (ROS) have been implicated in the induction of acute pancreatitis. Mitochondria possess a distinct genome (mtDNA) that is more susceptible to ROS-induced damage than nuclear DNA (nDNA). The purpose of our study was to determine the effect of ROS on mitochondrial function and membrane potential (ΔΨmt), to identify signal transduction mechanisms activated by ROS, and to quantify damage to mtDNA in an in vitro pancreatitis model. Methods: Pancreatic acinar cells, AR4-2J, were treated with saline solution (control) or hydrogen peroxide (H2O2), a representative ROS. Mitochondrial function was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; to determine ΔΨmt, rhodamine-123 uptake was measured. Intracellular calcium levels and c-Jun N-terminal kinase activity was determined; gel mobility shift assays were performed to assess induction of the transcription factor NF-κB. To quantitate DNA damage, a novel polymerase chain reaction–based procedure was performed. Results: Mitochondrial function and ΔΨmt were significantly decreased with oxidative damage. H2O2 treatment resulted in increased intracellular calcium levels, activation of c-Jun N-terminal kinase, and induction of NF-κB DNA binding. Treatment of AR4-2J cells with H2O2 resulted in selective mtDNA damage; nDNA was not affected. Conclusions: Our data demonstrate that pancreatic mtDNA is more susceptible to oxidative damage than nDNA; this damage is associated with decreases in mitochondrial function and ΔΨmt and activation of downstream signal transduction pathways. Mitochondrial damage mediated by ROS may play a central role in pancreatic cell injury associated with acute pancreatitis. (Surgery 1999:126:148-55.)

Assessment of differential gene expression patterns in human colon cancers.

ObjectiveTo use a novel genomic approach to determine differential gene expression patterns in colon cancers of different metastatic potential. Summary Background DataColorectal cancer is the third leading cause of cancer deaths in the United States; despite aggressive treatment strategies, the 5-year survival rate for metastatic cancer has not changed in 50 years. The analysis of changes in gene expression patterns associated with metastasis may provide new treatment strategies. MethodsHuman colon cancer cells KM12C (derived from a Dukes B colon cancer), KML4A (a metastatic variant derived from KM12C), and KM20 (derived from a Dukes D colon cancer) were extracted for RNA. In addition, RNA was extracted from normal colon, primary cancer, and liver metastasis in a patient with metastatic colon cancer. Gene expression patterns for approximately 1,200 human genes were analyzed and compared by cDNA array techniques. ResultsOf the roughly 1,200 genes assessed in the KM cell lines, 9 genes were noted to have a more than threefold change in expression (either increased or decreased) in the more metastatic KML4A and KM20 cells compared with KM12C. Assessment of tissues from a patient with metastatic colon cancer demonstrated a more than threefold change in the expression of 14 genes in the primary cancer and liver metastasis compared with normal mucosa. ConclusionsUsing cDNA expression array technology, the authors identified genes with expression levels that are altered with metastasis. The ability to analyze and compare the expression patterns of multiple genes simultaneously provides a powerful technique to identify potential molecular targets for novel therapeutic strategies.

Differential gene expression in primary and recurrent carotid stenosis

Apoptosis of the cellular components of complex atherosclerotic plaque may lead to plaque instability and rupture. In this study, five primary plaques and one recurrent fibrointimal lesion obtained from patients undergoing carotid endarterectomy for symptomatic carotid stenosis > or = 70% were analyzed by immunohistochemistry and cDNA microarray to identify gene expression patterns that may determine plaque susceptibility or resistance to apoptosis. Immunohistochemistry showed expression of active caspase 3, an effector of apoptosis, in macrophages and lymphocytes surrounding the lipid core, in smooth muscle cells in the fibrous cap, and media of primary plaques as well as in occasional smooth muscle cells in the recurrent lesion. Among the genes demonstrating increased expression in primary plaques were IGFR2, DR4, DAPK1, Bak, and ERK 1 and 2 and those showing decreased expression included the TNF receptors 1 and 2, akt1, and IGFBP3. When comparing the recurrent lesion to the normal tissue, the expression of 13 genes was decreased by 3-fold, including IGFBP2 and IGFBP3, and none were increased by more than 1.5-fold. The analysis of gene expression patterns in primary and recurrent stenotic lesions provides a powerful approach to identify the signaling pathways that alter cellular apoptotic patterns in such lesions.

TRAIL Mediated Apoptosis Increases Carotid Plaque Vulnerability

P116 Background: TRAIL, a TNF family protein, and its receptors DR 4 and 5 (TNF-R) are known to induce apoptosis in tumor cells via caspase activation. In this study, we examined the relationship between TRAIL, its effector caspases and apoptosis in carotid plaque (CP). Materials and Methods: Forty CPs were examined by immunohistochemistry, Western blotting and RNA protection assay for evidence of apoptosis (Tunel), TRAIL, caspase 3, 8 and PARP. We compared the expression of these pro-apoptotic proteins within plaque (P), and in areas of intimal thickening (IT) adjacent to the plaque to that of normal intima (N). Results: Apoptosis was detected in 10–25% of ECs, SMCs, macrophages and T lymphocytes in complex CP, but only in 1–3% SMCs in fibrous plaques. TRAIL and TNF-R, as well as caspases 3, 8, and PARP were present in all complex lesions. In N, there were focal areas of caspase 3 expression localized to ECs, whereas both caspase 3 and TRAIL were detected in all 3 regions of complex CP. There was a significant increase in the expression of TRAIL, TNF-R and caspase 8 in areas of IT compared to N (Fig. 1). Conclusions: The increased expression of TRAIL, TNF-R and caspases 3 and 8 in areas of IT and caspase 3 in ECs from N suggest that the mechanisms for ongoing EC injury and TRAIL-mediated cell death are present in complex CP. Modulation of these pro-apoptotic proteins and/or their inhibitors could potentially result in the introduction of therapeutic agents to attenuate plaque vulnerability.