Loss of human β-defensin 1, 2, and 3 expression in oral squamous cell carcinoma

Introduction:  Human β-defensins (HBDs) are cationic, antimicrobial peptides produced by epithelial cells and involved in various aspects of the innate and acquired immune responses. They are expressed by oral tissues as constitutive and inducible genes. Recently, single nucleotide polymorphisms (SNPs) of β-defensins have been correlated with increased susceptibility to certain diseases. Studies have reported altered expression of β-defensins in cancers suggesting their involvement in carcinogenesis. The purpose of this study was to evaluate the regulation of HBD-1 (also published as DEFB1), HBD-2 (DEFB4) and HBD-3 (DEFB103A) ( http://www.genenames.org/index.html ) and HBD-1 SNPs in oral squamous cell carcinoma cell lines (OSCC) and healthy gingival keratinocytes.
Methods:  β-defensin expression was quantitatively assessed using real-time polymerase chain reactions in OSCC and control cell lines after exposure to interleukin-1β, tumor necrosis factor-α, and interferon-γ. Control data were obtained in a previous study. DNA from 19 OSCC cell lines and 44 control subjects were extracted and the HBD-1 region spanning the 5' untranslated region to the first intron was sequenced and analysed for SNP identification and distribution.
Results:  HBD-1 and HBD-2 basal messenger RNA expression were significantly lower in OSCC. In addition, the ability to be induced was significantly reduced in OSCC for all three β-defensins. Four HBD-1 SNPs were differentially distributed between cancer and control populations. Genotype distribution at the HBD-1 locus also suggested loss of heterozygosity in OSCC.
Conclusions:  The genetic variation observed in OSCC compared with that in control cell lines may account for differences in β-defensin expression. These results suggest a putative role for β-defensins in carcinogenesis and indicate that β-defensins may be useful markers of OSCC.     

Multiplex analysis of serum cytokines in melanoma patients treated with interferon-alpha2b.

PURPOSE: Interferon (IFN)-alpha2b is the only Food and Drug Administration-approved treatment for operable high-risk melanoma that has been shown to significantly and durably prolong relapse-free survival (RFS) of patients with stage IIB-III melanoma. Development of reliable serum assays may contribute to the development of methods for earlier detection of melanoma and the selection of patients who may be most susceptible to current available interventions with IFNalpha. EXPERIMENTAL DESIGN: A powerful high-throughput xMAP multiplex immunobead assay technology (Luminex Corp., Austin, TX) was used to simultaneously test 29 cytokines, chemokines, angiogenic as well as growth factors, and soluble receptors in the sera of 179 patients with high-risk melanoma and 378 healthy individuals. RESULTS: Serum concentrations of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, IL-12p40, IL-13, granulocyte colony-stimulating factor, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, IFNalpha, tumor necrosis factor (TNF)-alpha, epidermal growth factor, vascular endothelial growth factor (VEGF), and TNF receptor II were found to be significantly higher in patients with resected high-risk melanoma compared with healthy controls. Bayesian Network algorithm classification of the data offered 90% sensitivity at 98% specificity with 96.5% of melanoma patients distinguished from healthy individuals. IFN-alpha2b therapy resulted in a significant decrease of serum levels of immunosuppressive and tumor angiogenic/growth stimulatory factors (VEGF, epidermal growth factor, and hepatocyte growth factor) and increased levels of antiangiogenic IFN-gamma inducible protein 10 (IP-10) and IFN-alpha. Pretreatment levels of proinflammatory cytokines IL-1beta, IL-1alpha, IL-6, TNF-alpha, and chemokines MIP-1alpha and MIP-1beta were found to be significantly higher in the serum of patients with longer RFS values of 1 to 5 and >5 years when compared with patients with shorter RFS of <1 year. CONCLUSION: These data show that multiplexed analysis of serum biomarkers is useful for the evaluation of prognostic markers of clinical outcome and potential predictive markers of response to IFN-alpha2b in patients with high-risk operable melanoma.     

Induction of CYP3A by ethanol in multiple in vitro and in vivo models

BACKGROUND: Cytochrome P-450 3A (CYP3A) is responsible for the metabolism of numerous therapeutic agents. The content of CYP3A seems to be affected by ethanol ingestion. Because ethanol is used widely, its potential interaction with CYP3A is of great interest. The effects of ethanol on CYP3A content and activity were assessed in different in vivo and in vitro models. METHODS: Rats fed either the Lieber-DeCarli ethanol-containing diet or an ethanol and liquid diet via the intragastric tube feeding method were used. Additionally, HepG2 cell lines that constitutively and stably express human CYP3A4 were constructed to study ethanol interactions with CYP3A4. RESULTS: In all models tested, ethanol induced CYP3A activity and content, as assessed by the metabolism of fentanyl, a sensitive and specific CYP3A substrate, and Western blot analysis, respectively. In the CYP3A4-expressing HepG2 cell line, incubation with ethanol caused a dose-dependent increase in CYP3A4 activity. Ethanol also increased messenger RNA levels of CYP3A4. In the HepG2-CYP3A4 line, incubation with cycloheximide caused a decrease in fentanyl metabolism secondary to a decrease in CYP3A4 levels; this decrease was prevented by coincubation of cycloheximide with ethanol. CONCLUSIONS: Ethanol induced CYP3A activity and content both in vitro and in vivo. There may be multiple mechanisms of induction of CYP3A4 by ethanol, including stabilization of messenger RNA and protein. Ethanol-induced increases in both the protein level and activity of CYP3A4 may play a role that might be of pathophysiological or clinical significance.     

Chemoimmunohormonal therapy with carmustine,dacarbazine, cisplatin,tamoxifen, and interferon for metastatic melanoma: a prospective phase II study

The objective of this study was to investigate the efficacy of our treatment regimen in metastatic melanoma. Thirty patients entered the study after undergoing a thorough metastatic workup. Treatment protocol included carmustine (BCNU) (150 mg/m(2) IV, day 1) every 6 weeks, dacarbazine (DTIC) (220 mg/m(2) IV, days 1-3), and cisplatin (25 mg/m(2) IV, days 1-3) every 3 weeks, interferon A-2B (6 x 10(6) U/m daily s.c. on days 4-8 and 16-20) and tamoxifen 20 mg/day for 6 weeks. Among 29 evaluable patients, overall response was seen in 15 (52%) and complete response in 5 (17%) patients. Median duration of partial response was 4 months (range, 1-12 months); of complete response was 8 months (range, 2-14 months). Complete response continues in two patients with lung metastases. Median survival time was 8.7 months. Side effects were tolerable. Four (13%) patients developed neutropenic fever, and platelet transfusions were required in five (17%) patients. One patient died of neutropenic sepsis. Thrombocytopenia caused prolongation of the median interval between the first and second courses, and drug doses were reduced in the second course in 8 of 26 (31%) patients. Our chemoimmunohormonal regimen is efficient in metastatic malignant melanoma and can induce durable remission. Severe thrombocytopenia leads to a reduction of carmustine dose in a new protocol.     

Local administration of IL-12-transfected dendritic cells induces antitumor immune responses to colon adenocarcinoma in the liver in mice

Colorectal cancer is one of the most common fatal malignancies in the United States, with an incidence second only to lung cancer. The liver is the most common site of colorectal metastases and frequently the only affected organ once the primary tumor has been surgically removed. The only potentially curative treatment for metastatic colorectal cancer in the liver is surgery, although most patients are not eligible for resection. We have therefore, evaluated the therapeutic efficacy of dendritic cells (DCs) engineered to express IL-12 in a liver metastasis model. Direct administration of DCs into the portal vein significantly inhibited the growth of established MC38 colon carcinoma in the liver in C57BL/6 mice. This effect was accompanied by an intratumoral accumulation of CD4+, CD8+, and NLDC-145+ immune effector cells, and also resulted in a systemic immune response as determined by enhanced production of IFN-gamma by T lymphocytes isolated from both spleen and draining lymph nodes. Evaluation of homing of Cy3-labeled DCs following the portal vein injection confirmed their distribution in the liver and lymphoid tissue. Thus, a local delivery of DCs transduced with the IL-12 gene can not only inhibit colorectal tumor growth in vivo but also mount systemic antitumor immune responses. This approach is likely to improve the outcome of immunotherapy for metastatic colorectal cancer since high numbers of tumor-associated DCs positively correlate with a more favorable prognosis. Simultaneous local gene therapy with IL-12 will further improve clinical efficacy without placing the patient at risk for systemic toxicity.     

Kinase-addiction and bi-phasic sensitivity-resistance of Bcr-Abl- and Raf-1-expressing cells to imatinib and geldanamycin

By activating anti-apoptotic factors (e.g., Hsp70, Raf-1, Bcl-xL), Bcr-Abl blocks apoptotic pathways at multiple levels, thus rendering leukemia cells resistant to chemotherapeutic agents such as doxorubicin (DOX). In Bcr-Abl-transfected HL60 (HL/Bcr-Abl) cells, procaspase-9 was increased and partially processed. The Bcr-Abl inhibitor imatinib (Gleevec, STI-571) released the apoptotic stream. Also, HL/Bcr-Abl cells were hyper-sensitive to geldanamycin (GA), which depletes Bcr-Abl and Raf-1. Raf-1 and Bcr-Abl-transfected FDC-P1 hematopoietic cells were selectively sensitive to GA and imatinib, respectively. Remarkably, cell clones with high levels of Bcr-Abl that could not be depleted by GA were relatively resistant to both GA and imatinib. GA and flavopiridol sensitized such resistant cells to imatinib. These data suggest bi-phasic sensitivity to mechanism-based therapeutic agents. Although Bcr-Abl renders cells hyper-sensitive, an excess of Bcr-Abl results in resistance (due to the remaining activity). We discuss therapeutic approaches to overcome bi-phasic resistance to mechanisms-based agents.     

Function of neuropeptide Y and agouti-related protein at weaning: relation to corticosterone, dietary carbohydrate and body weight

Neuropeptide Y (NPY) and agouti-related protein (AgRP), potent stimulants of feeding, have been linked in adult rats to both corticosterone (CORT) and dietary carbohydrate. To understand the significance of this relationship early in life, measurements were taken of these parameters at different ages around weaning, in rats given a choice of macronutrient diets or maintained on a carbohydrate-rich diet. The results demonstrate that, in both male and female rat pups, the expression and production of NPY and AgRP in the arcuate nucleus (ARC) peak on postnatal day 21 (P21), compared to P15 before weaning and P27 after weaning. These elevated levels of peptide were associated with peak levels of CORT and glucose and also a strong, natural preference for carbohydrate at weaning, which accounted for 55-65% of the pups' total diet. In subgroups defined by their body weight at these stages, rats with as little as 4% lower body weight (compared to higher weight pups) had 30-60% greater expression of NPY and AgRP in the ARC and elevated levels of CORT, with no difference in leptin or insulin. This response was significantly more pronounced at P21 than at P15 or P27. The importance of carbohydrate during this stage was suggested by additional results showing elevated NPY expression, CORT levels, body weight and inguinal fat pad weights in P27 pups raised on a 65% carbohydrate diet vs. 45% carbohydrate. These results suggest that hypothalamic NPY and AgRP, together with CORT, have glucoregulatory as well as feeding stimulatory functions that help mediate the transition from suckling of a fat-rich diet to independent feeding of a carbohydrate-rich diet. During this critical period, the carbohydrate together with the peptides and CORT provide the important signals, including elevated glucose, that promote de novo lipogenesis and enable weanling animals to survive periods of food deprivation.     

Characterization of the acetaminophen-induced degradation of cytochrome P450-3A4 and the proteolytic pathway

This study compared the in vitro responses of malignant and normal cells from the human oral cavity to tea extracts and to its main polyphenolic component, (-)-epigallocatechin gallate (EGCG). The antiproliferative effects of tea polyphenolic extracts and EGCG were more pronounced towards immortalized, tumourigenic (CAL27, HSC-2, and HSG(1)) and non-tumourigenic (S-G) cells than towards normal (GN56 and HGF-1) fibroblasts and green tea was more toxic than black tea. As the addition of tea extract or EGCG to cell culture medium led to the formation of hydrogen peroxide (H(2)O(2)), the research then focused on EGCG as an inducer of oxidative stress, using CAL27, the cancerous cells most sensitive to EGCG, HSG(1), the cancerous cells least sensitive to EGCG, and GN56 cells. The toxicity of EGCG was decreased in the presence of catalase, an enzyme that degrades H(2)O(2), or of deferoxamine, a chelator of Fe(3+). Conversely, pretreatment of the cells with the glutathione depleters, 1-chloro-2,4-dinitrobenzene and 1,3-bis(2-chloroethyl)-N-nitrosourea, potentiated the toxicity of EGCG. A 4-hr exposure to EGCG lessened the intracellular level of reduced glutathione in the CAL27 and HSG(1) cells, but not in the GN56 fibroblasts. Whereas EGCG itself did not induce lipid peroxidation, Fe(2+)-induced lipid peroxidation was potentiated by EGCG. A 72-hr exposure to cytotoxic concentrations of EGCG induced significant cytoplasmic vacuolization in all cell types. The results presented herein are consistent with EGCG acting as a prooxidant, with the cancerous cells more sensitive to oxidative stress than the normal cells.     

Chemotherapy of glioblastoma in rats using doxorubicin-loaded nanoparticles

Glioblastomas belong to the most aggressive human cancers with short survival times. Due to the blood-brain barrier, they are mostly inaccessible to traditional chemotherapy. We have recently shown that doxorubicin bound to polysorbate-coated nanoparticles crossed the intact blood-brain barrier, thus reaching therapeutic concentrations in the brain. Here, we investigated the therapeutic potential of this formulation of doxorubicin in vivo using an animal model created by implantation of 101/8 glioblastoma tumor in rat brains. Groups of 5-8 glioblastoma-bearing rats (total n = 151) were subjected to 3 cycles of 1.5-2.5 mg/kg body weight of doxorubicin in different formulations, including doxorubicin bound to polysorbate-coated nanoparticles. The animals were analyzed for survival (% median increase of survival time, Kaplan-Meier). Preliminary histology including immunocytochemistry (glial fibrillary acidic protein, ezrin, proliferation and apoptosis) was also performed. Rats treated with doxorubicin bound to polysorbate-coated nanoparticles had significantly higher survival times compared with all other groups. Over 20% of the animals in this group showed a long-term remission. Preliminary histology confirmed lower tumor sizes and lower values for proliferation and apoptosis in this group. All groups of animals treated with polysorbate-containing formulations also had a slight inflammatory reaction to the tumor. There was no indication of neurotoxicity. Additionally, binding to nanoparticles may reduce the systemic toxicity of doxorubicin. This study showed that therapy with doxorubicin bound to nanoparticles offers a therapeutic potential for the treatment of human glioblastoma.