microRNA changes in liver tissue associated with fibrosis progression in patients with hepatitis C.

BACKGROUND & AIMS: Accumulating evidence indicates that microRNAs play a role in a number of disease processes including the pathogenesis of liver fibrosis in hepatitis C infection. Our goal is to add to the accruing information regarding microRNA deregulation in liver fibrosis to increase our understanding of the underlying mechanisms of pathology and progression. METHODS: We used next generation sequencing to profile all detectable microRNAs in liver tissue and serum from patients with hepatitis C, stages F1-F4 of fibrosis. RESULTS: We found altered expression of several microRNAs, in particular, miR-182, miR199a-5p, miR-200a-5p and miR-183 were found to be significantly upregulated in tissue from liver biopsies of hepatitis C patients with advanced fibrosis, stage F3 and F4, when compared with liver biopsies from patients with early fibrosis, stages F1 and F2. We also found miR-148-5p, miR-1260b, miR-122-3p and miR-378i among the microRNAs most significantly down-regulated from early to advanced fibrosis of the liver. We also sequenced the serum microRNAs; however, we were not able to detect significant changes in circulating microRNAs associated with fibrosis stage after adjusting for multiple tests. CONCLUSIONS: Adding measurements of tissue microRNAs acquired during routine biopsies will continue to increase our knowledge of underlying mechanisms of fibrosis. Our goal is that these data, in combination with studies from other researchers and future long-term studies, could be used to enhance the staging accuracy of liver biopsies and expand the surveillance of patients at increased risk for cancer and progression to advanced fibrosis.

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer.

BACKGROUND: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. RESULTS: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our "gene therapy" approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ~50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ~35% tumor progression in vivo in already established tumors. CONCLUSIONS: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

The CD24 protein inducible expression system is an ideal tool to explore the potential of CD24 as an oncogene and a target for immunotherapy in vitro and in vivo.

CD24 is a cell surface, heavily glycosylated glycosylphosphatidylinositol-anchored mucin-like protein that is overexpressed in various human malignancies. To accurately analyze CD24 function and dissect its biological role in a defined genetic background, it is critical to tightly regulate its expression and be able to turn it on/off in a restricted environment and at a specific time. The tetracycline-induced expression system is most promising as it exhibits such regulation, lack of pleiotropic effects, and high and rapid induction levels. To evaluate the oncogenic and immunotherapeutic potential of CD24 by applying the Tet-On system, the human CD24 gene was cloned downstream to two tetracycline operator sequences, resulting in pCDNA4/TO-CD24, which was then transfected into tetracycline (Tet) repressor-expressing cells (293T-REx), allowing tight on/off regulation, thereby resulting in a very low background or leaky CD24 expression. Selected clones were chosen for further studies and characterized in vitro and in vivo, and several treatment modalities were examined. In addition, the role of CD24 in promoting cell proliferation and tumor growth was studied. The tetracycline-dependent system was successfully implemented. Tetracycline treatment induced CD24 expression in a dose- and time-dependent fashion, which was abrogated following treatment with anti-CD24 monoclonal antibodies (mAbs). CD24-induced expression led to an increased proliferation rate that was inhibited by mAb treatment. In vivo, significantly larger tumors were developed in tetracycline-fed mice. The CD24 Tet-On system is a good model to unravel the role and underlying CD24 pathogenesis in vivo. This valuable tool allows the successful study of novel treatment options, whose effectiveness depends on the CD24 expression level. This set of experiments supports CD24 oncogenic properties.

An immunoconjugate of anti-CD24 and Pseudomonas exotoxin selectively kills human colorectal tumors in mice.

BACKGROUND & AIMS: Effective and selective treatment options are needed for patients with colorectal cancer (CRC). The CD24 mucin-like glycoprotein is overexpressed in CRCs; monoclonal antibodies (mAbs) against CD24 inhibit tumor cell growth in vitro and in vivo. Based on the tumor-specific expression of CD24, we investigated the potential of anti-CD24 SWA11 mAb, to deliver a cytotoxic agent into CRC cells. METHODS: We conjugated SWA11 to a Pseudomonas exotoxin derivative (PE38) via an Fc-binding ZZ domain from Staphylococcal protein A (which binds the Fc domain of mouse IgG2a immunoglobulins) to generate the immunotoxin SWA11-ZZ-PE38; IgG-ZZ-PE38 was used as control. Human HT-29 and COLO320 (CD24-positive) and HCT116 (CD24-negative) CRC cell lines were assayed for immunotoxin binding, cytotoxicity, viability, and apoptosis. Toxicity and antitumor efficacy were tested in mice. RESULTS: The immunotoxin preserved the affinity and specificity of SWA11, bound and selectively killed CD24-expressing CRC cells via apoptosis. IC(50) values ranged from 20 to 50 ng/mL-several orders of magnitude lower than that of the mAb alone. The immunotoxins were not toxic to mice at the maximum dose of 0.75 mg/kg. Growth of HT-29 xenograft tumors was significantly reduced in mice given SWA11-ZZ-PE38 (by 78%) compared to untreated mice. CONCLUSIONS: Anti-CD24 SWA11 mAb can deliver a PE exotoxin derivative to CRC cells and cause them to undergo apoptosis, without toxicity to normal tissues. This immunotoxin might be developed as a therapeutic treatment for patients with CRC.

Inhibition of pancreatic and lung adenocarcinoma cell survival by curcumin is associated with increased apoptosis, down-regulation of COX-2 and EGFR and inhibition of Erk1/2 activity.

BACKGROUND: Several studies suggested that curcumin inhibits growth of malignant cells via inhibition of cyclooxygenase-2 (COX-2) activity. Other studies indicated that epidermal growth factor receptor (EGFR) is also inhibited by curcumin in vitro and in vivo. Moreover, recent investigations revealed an intracellular cross-talk between EGFR signaling and the COX-2 pathway. Our aim was to evaluate whether the curcumin inhibitory effect on the survival of cancer cells is associated with simultaneous down-regulation of COX-2 and EGFR and inhibition of Erk1/2 (extra-cellular signal regulated kinase) signaling pathway. MATERIALS AND METHODS: Lung and pancreas adenocarcinoma cell lines co-expressing COX-2 and EGFR (PC-14 and p34, respectively) and those expressing EGFR but deficient in COX-2 (H1299 and Panc-1, respectively) were exposed for 72 h to curcumin (0-50 microM). Cell viability was assessed by the XTT assay. Apoptosis was determined by FACS analysis. COX-2, EGFR, ErbB-2 and p-Erk1/2 expressions were measured by Western blot analysis. RESULTS: Curcumin's inhibitory effect on survival and apoptosis of lung and pancreatic adenocarcinoma cell lines was significantly higher in the COX-2-expressing cells than in the COX-2-deficient cells. In the p34 and PC-14 cells, curcumin decreased COX-2, EGFR and p-Erk1/2 expressions in a dose-dependent manner. However, in the Panc-1 and H1299 cell lines, which did not express COX-2, curcumin did not affect EGFR levels. CONCLUSION: Curcumin co-inhibited COX-2 and EGFR expression and decreased Erk1/2 activity. This inhibition was associated with decreased survival and enhanced induction of apoptosis in lung and pancreatic adenocarcinoma cells.

Curcumin induces apoptosis and inhibits growth of orthotopic human non-small cell lung cancer xenografts.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality. Curcumin is involved in various biological pathways leading to inhibition of NSCLC growth. The purpose of this study was to evaluate the effect of curcumin on expression of nuclear factor κB-related proteins in vitro and in vivo and on growth and metastasis in an intralung tumor mouse model. H1975 NSCLC cells were treated with curcumin (0-50 µM) alone, or combined with gemcitabine or cisplatin. The effects of curcumin were evaluated in cell cultures and in vivo, using ectopic and orthotopic lung tumor mouse models. Twenty mice were randomly selected into two equal groups, one that received AIN-076 control diet and one that received the same food but with the addition of 0.6% curcumin 14 days prior to cell implantation and until the end of the experiment. To generate orthotopic tumor, lung cancer cells in Matrigel were injected percutaneously into the left lung of CD-1 nude mice. Western blot analysis showed that the expressions of IkB, nuclear p65, cyclooxygenase 2 (COX-2) and p-ERK1/2 were down-regulated by curcumin in vitro. Curcumin potentiated the gemcitabine- or cisplatin-mediated antitumor effects. Curcumin reduced COX-2 expression in subcutaneous tumors in vivo and caused a 36% decrease in weight of intralung tumors (P=.048) accompanied by a significant survival rate increase (hazard ratio=2.728, P=.036). Curcumin inhibition of COX-2, p65 expression and ERK1/2 activity in NSCLC cells was associated with decreased survival and increased induction of apoptosis. Curcumin significantly reduced tumor growth of orthotopic human NSCLC xenografts and increased survival of treated athymic mice. To evaluate the role of curcumin in chemoprevention and treatment of NSCLC, further clinical trials are required.

Curcumin: a potential radio-enhancer in head and neck cancer.

OBJECTIVES/HYPOTHESIS: To investigate whether curcumin enhances the cytotoxic effect of radiotherapy in head and neck squamous cell carcinoma (HNSCC). METHODS: HNSCC cell lines SCC-1, SCC-9, KB, as well as A431 cell line were treated with curcumin, irradiation, or their combination. Cell viability was evaluated by XTT assay. Cyclooxygenase-2 (COX-2), epithelial growth factor receptor (EGFR), and p-Erk1/2 were measured by Western blot analysis. CD-1 athymic nude mice with orthotopic implanted SCC-1 cells, were treated with control diet, curcumin containing diet, local single-dose radiation, or combination. RESULTS: Curcumin (IC50 range, 15-22 microM) and radiation inhibited cell viability in all cell lines were tested. The combination of curcumin and radiation resulted in additive effect. Curcumin decreased COX-2 expression and inhibited phosphorylation of EGFR in SCC-1 cells. In tumor-bearing mice the combination regimen showed a decrease in both tumor weight (25%, P = .09) and tumor size (15%, P = .23) compared to the nontreated mice. CONCLUSIONS: : Curcumin inhibited HNSCC cell growth and augmented the effect of radiation in vitro and in vivo. A possible mechanism is inhibition of COX-2 expression and EGFR phosphorylation.

Targeting CD24 for treatment of colorectal and pancreatic cancer by monoclonal antibodies or small interfering RNA.

CD24 is a potential oncogene reported to be overexpressed in a large variety of human malignancies. We have shown that CD24 is overexpressed in 90% of colorectal tumors at a fairly early stage in the multistep process of carcinogenesis. Anti-CD24 monoclonal antibodies (mAb) induce a significant growth inhibition in colorectal and pancreatic cancer cell lines that express the protein. This study is designed to investigate further the effects of CD24 down-regulation using mAb or small interfering RNA in vitro and in vivo. Western blot analysis showed that anti-CD24 mAb induced CD24 protein down-regulation through lysosomal degradation. mAb augmented growth inhibition in combination with five classic chemotherapies. Xenograft models in vivo showed that tumor growth was significantly reduced in mAb-treated mice. Similarly, stable growth inhibition of cancer cell lines was achieved by down-regulation of CD24 expression using short hairpin RNA (shRNA). The produced clones proliferated more slowly, reached lower saturation densities, and showed impaired motility. Most importantly, down-regulation of CD24 retarded tumorigenicity of human cancer cell lines in nude mice. Microarray analysis revealed a similar pattern of gene expression alterations when cells were subjected to anti-CD24 mAb or shRNA. Genes in the Ras pathway, mitogen-activated protein kinase, or BCL-2 family and others of oncogenic association were frequently down-regulated. As a putative new oncogene that is overexpressed in gastrointestinal malignancies early in the carcinogenesis process, CD24 is a potential target for early intervention in the prevention and treatment of cancer.

Curcumin augments gemcitabine cytotoxic effect on pancreatic adenocarcinoma cell lines.

BACKGROUND AND AIM: Gemcitabine, the first-line agent in pancreatic adenocarcinoma, has shown limited clinical benefit. Cyclooxygenase-2 (COX-2) represent one of the most promising targets for cancer prevention and treatment. In this study, we investigated whether the phytochemical curcumin, a natural COX-2 inhibitor, can potentiate gemcitabine effect on survival of human pancreatic cancer cells. METHODS: P34 (high COX-2 expression) and Panc-1 (low COX-2 expression) pancreatic cancer cell lines were exposed to different concentrations of gemcitabine (0.1-10 microM), curcumin (0-50 microM), and their combination. Cell viability was evaluated by XTT assay. Cell cycle and apoptosis were assessed by flow cytometry. COX-2, EGFR, and p-ERK1/2 expression was measured by Western blot analysis. RESULTS: Curcumin increased the inhibitory effect of gemcitabine on cell viability as well as its pro-apoptotic effect in COX-2 positive, p34 cells, but not in COX-2 negative, Panc-1 cells. In p34 cells, combination of curcumin and gemcitabine downregulated both COX-2 and p-ERK1/2 in a dose-dependent manner. CONCLUSION: The increased cytotoxic effect of the combination on cell survival and on the induction of apoptosis in COX-2 expressing pancreatic cancer cells is probably associated with downregulation of COX-2 and p-ERK1/2 levels. This finding may contribute to the development of an effective treatment of pancreatic adenocarcinoma.

ErbB-4 may control behavior of prostate cancer cells and serve as a target for molecular therapy.

PURPOSE: To assess ErbB-4 expression in advanced human prostate cancer (PC) cell lines, the role of ErbB-4 in motility, migration, and proliferative/tumorigenic potential of PC cells, and efficacy of anti-ErbB-4 monoclonal antibody (Mab) treatment on PC cells in vitro and tumor growth in vivo. MATERIALS AND METHODS: Established advanced human PC cell lines (PC-3, Cl-1, and Du-145) were evaluated for ErbB-4 expression. Several Cl-1 cell line clones expressing various levels of ErbB-4 were isolated, their motility, migration capacity, and in vitro proliferation as well as survival following Mab treatment were evaluated. Tumorigenicity and proliferation capacity of these clones in vivo and efficacy of Mab treatment on tumor growth were estimated by measurements of subcutaneous tumors developed in nude mice. RESULTS: PC cell lines studied express ErbB-4. Both PC-3 and Du-145 cell lines express high ErbB-4 levels; only 50% of Cl-1 cells express ErbB-4 with large heterogeneity. Cl-1 sub-clones highly expressing ErbB-4 showed increased cell motility, migration, and proliferation rate in vitro and enhanced growth in vivo, compared to clones with low ErbB-4 expression. Mab treatment inhibited the growth of cells expressing high but not low ErbB-4 levels in vitro and decreased the growth of subcutaneous tumors in nude mice generated by ErbB-4 highly expressing cells. CONCLUSIONS: High expression of ErbB-4 in prostate cancer Cl-1 cell clones correlated with high proliferative and migration capacity and high tumorigenic potential. The inhibitory effect of Mab on cell proliferation and on subcutaneous tumor growth suggests ErbB-4's potential as a target for molecular anticancer therapy.

ErbB4 increases the proliferation potential of human lung cancer cells and its blockage can be used as a target for anti-cancer therapy.

Clinical and experimental data suggest that ErbB-4, a member of the epidermal growth factor receptor family, may have a role in cancer progression and response to treatment. We found recently, using a retrospective clinical analysis, that expression of ErbB-4 receptor is correlated with metastatic potential and patient survival in non-small-cell lung cancer (NSCLC). The purpose of this work was to correlate the expression of the ErbB-4 and lung cancer cells growth in vitro and in vivo and to determine the therapeutic potential of a monoclonal antibody to ErbB-4 against lung cancer. For this aim, we ectopically expressed ErbB-4 in a human NSCLC cell line that did not express the ErbB-4 protein. Overexpression of ErbB-4 produced a constitutively activated ErbB-4 receptor. The transfected ErbB-4 positive clones showed an increased cell proliferation in vitro and in vivo in comparison with parental ErbB-4 negative cells and with the cells transfected by neomycin-resistant gene. A monoclonal antibody to ErbB-4 showed both an inhibitory effect on growth rate and an increasing apoptotic rate in the cells expressing ErbB-4. The results of the current study provide evidence that ErbB-4 plays a significant role in human lung cancer and may serve as a molecular target for anticancer therapy.