TIMP-1 overexpression promotes tumorigenesis of MDA-MB-231 breast cancer cells and alters expression of a subset of cancer promoting genes in vivo distinct from those observed in vitro

TIMP-1 (Tissue inhibitor of matrix metalloproteinase-1) is typically associated with inhibition of matrix metalloproteinases (MMP) induced invasion. However, TIMP-1 is overexpressed in many malignancies and is associated with poor prognosis in breast cancer. The mechanisms by which TIMP-1 promotes tumorigenesis are unclear. Reduced levels of TIMP-1 mediated by shRNA in MDA-MB-231 breast cancer cells had no effect on cellular physiology in vitro or tumor growth in SCID mice compared to vector control MDA-MB-231 cells. However, overexpression of TIMP-1 in MDA-MB-231 cells resulted in inhibition of cell invasion and enhanced phosphorylation of p38 MAPK and AKT in vitro. Additionally, treatment of parental MDA-MB-231 cells with purified TIMP-1 protein led to activation of p38 MAPK and MKK 3/6. cDNA array analysis demonstrated that high expression of TIMP-1 in MDA-MB-231 cells resulted in alterations in expression of approximately 200 genes, 1.5 fold or greater compared to vector control cells (P < 0.1). Real-time RT-PCR confirmed changes in expression of several genes associated with cancer progression including DAPK1, FGFR4 and MAPK13. In vivo, high TIMP-1 expression induced tumor growth in SCID mice compared to vector control cells and increased tumor vessel density. Affymetrix array analysis of vector control and TIMP-1 MDA-MB-231 xenograft tumors revealed that TIMP-1 altered expression of approximately 600 genes in vivo, including MMP1, MMP13, S100A14, S100P, Rab25 and ID4. These combined observations suggest that the effects of TIMP-1 differ significantly in a 2-D environment compared to the 3-D environment and that TIMP-1 stimulates tumor growth. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tanshinone IIA induces growth inhibition and apoptosis in gastric cancer in vitro and in vivo

As a phytochemical derived from the roots of Salvia miltiorrhiza Bunge, Tanshinone?IIA has been reported to possess anti-inflammatory and antioxidant activity. Studies in breast, colon, prostate and lung cancer indicate that Tanshinone?IIA may exhibit a promising antitumor activity. However, systemic studies of the cytotoxic effects of Tanshinone?IIA on gastric cancer have not been described. The present study offers a comprehensive evaluation of the antitumor effects of Tanshinone?IIA in gastric cancer cells in?vitro and in a mouse xenograft model. Cell viability and apoptosis in?vitro were evaluated through the MTT assay and flow cytometry analysis. The results indicate that Tanshinone?IIA can induce gastric cancer cell growth inhibition and apoptosis in a time- and concentration-dependent manner. Furthermore, we investigated the mechanism of the apoptotic effects induced by Tanshinone?IIA. We found that Tanshinone?IIA can not only cause cell cycle arrest in the G2/M phase, but also trigger the intrinsic apoptotic signaling pathway. The results suggest that Tanshinone?IIA may serve as an effective adjunctive reagent in the treatment of gastric cancer.     

An integrated in vitro and in vivo high-throughput screen identifies treatment leads for ependymoma

Using a mouse model of ependymoma-a chemoresistant brain tumor-we combined multicell high-throughput screening (HTS), kinome-wide binding assays, and in?vivo efficacy studies, to identify potential treatments with predicted toxicity against neural stem cells (NSC). We identified kinases within the insulin signaling pathway and centrosome cycle as regulators of ependymoma cell proliferation, and their corresponding inhibitors as potential therapies. FDA approved drugs not currently used to treat ependymoma were also identified that posses selective toxicity against ependymoma cells relative to normal NSCs both in?vitro and in?vivo, e.g., 5-fluorouracil. Our comprehensive approach advances understanding of the biology and treatment of ependymoma including the discovery of several treatment leads for immediate clinical translation.     

Effects of polyamine depletion by α-difluoromethylornithine on in vitro and in vivo biological properties of 4T1 murine mammary cancer cells

Increased polyamine synthesis has been associated with proliferation and progression of breast cancer, and thus, is a potential target for anti-cancer therapy. Polyamine depletion by DFMO has been shown to decrease pulmonary and bone metastasis from human breast cancer cell xenografts. Following these observations, this study was designed to test the effects of DFMO on in vitro and in vivo features of the highly invasive and metastatic 4T1 murine mammary cancer cells. DFMO inhibited proliferation, caused G1-S arrest, and suppressed in vitro invasiveness of 4T1 cells. In contrast to our previous findings with MDA-MB-435 cells, DFMO did not affect the activation of STAT3, JNK, and ERK, but decreased phosphorylation of p38. DFMO did not alter the expression of Twist. DFMO delayed the orthotopic growth of 4T1 xenografts in association with suppressed putrescine and spermidine levels but increased levels of spermine. DFMO did not affect pulmonary metastasis when primary tumors of control and DFMO-treated mice were matched for size. Interestingly, DFMO reduced Ki-67 expression only in the primary tumors but did not affect its expression in the metastatic tumors in the lung. Cleaved caspase-3 expression was not affected by DFMO in either the primary tumors or pulmonary metastasis. In summary, DFMO treatment markedly inhibited in vitro proliferation and invasiveness of 4T1 cells and retarded the growth of orthotopic xenografts in mice. The failure of DFMO to inhibit pulmonary metastasis in this system appears to be due, at least in part, to its lack of anti-proliferative effect at the metastatic sites.     

Influence of stromal–epithelial interactions on breast cancer in vitro and in vivo

Stromal cell-secreted chemokines including CCL2 have been implicated in the primary tumor microenvironment, as mediators of tumor cell migration, proliferation, and angiogenesis. Expression of CCL2 and its principal receptor CCR2 was analyzed by RQ-PCR in primary tumor cells and breast cancer cell lines. Breast cancer cell lines (MDA-MB-231, T47D) were co-cultured directly on a monolayer of primary breast tumor and normal stromal cells, retrieved using EpCAM+ magnetic beads, and changes in expression of CCL2, CCR2, MMP11, ELK1, VIL2, and Ki67 detected by RQ-PCR. Epithelial cell migration and proliferation in response to stromal cell-secreted factors was also analyzed. In vivo, tumor xenografts were formed by co-injecting T47D cells with primary tumor stromal cells. Following establishment, tumors were harvested and digested, epithelial cells retrieved and analyzed by RQ-PCR. Whole tumor tissue was also analyzed by immunohistochemistry for CD31 and the VIL2 encoded protein Ezrin. Tumor stromal cells expressed significantly higher levels of CCL2 than normal cells, with no CCR2 expression detected. Primary epithelial cells and breast cancer cell lines expressed elevated CCL2, with relative expression of CCR2 found to be higher than the ligand. Interaction of breast cancer epithelial cells with primary tumor, but not normal stromal cells, stimulated increased expression of CCL2 (8-fold), ELK1 (6-fold), VIL2 (6-fold), and MMP11 (17-fold). Factors secreted by stromal cells, including CCL2, stimulated a significant increase in epithelial cell migration, with no effect on cell proliferation in vitro observed. In vivo, the presence of stromal cells resulted in tumors of increased volume, mediated at least in part through neoangiogenesis demonstrated by immunohistochemistry (CD31). Admixed tumor xenografts exhibited increased expression of Ki67, MMP11, VIL2, and ELK1. Elevated Ezrin protein was also detected, with increased cytoplasmic localization. The results presented highlight mechanisms through which breast cancer epithelial cells can harness stromal cell biology to support tumor progression.     

Berberine enhances radiosensitivity of esophageal squamous cancer by targeting HIF-1α in vitro and in vivo

Radiation therapy is an important treatment approach for esophageal squamous cell carcinoma (ESCC). However, how to promote radiation sensitivity in ESCC remains a challenge. This study aimed to evaluate the effects of berberine, a common used Chinese medicine, on the radiosensitivity of ESCC. ECSS cell line ECA109 and TE13 were subjected to hypoxia and/or ionizing radiation (IR), in the presence or absence of berberine treatment. Cell growth and survival, and apoptosis were evaluated. In addition, ECA109 cells were xenografted into nude mice and subjected to IR and/or berberine treatment. The expression of HIF-1α and VEGF was detected by western blot and immunohistochemical analysis. Our results showed that berberine increased radiosensitivity of ESCC cells and xenografts, and this was associated with the inhibition of HIF-1α and VEGF expression. These data suggest that berberine may be a potential radiotherapy sensitization drugs due to its significant anti-hypoxia activity.     

Silencing of IKKε using siRNA inhibits proliferation and invasion of glioma cells in vitro and in vivo

Recent studies implicated IKKε in the pathogenesis of many human cancers by promoting cell proliferation, increasing tumor angiogenesis and metastasis, and generating resistance to cell apoptosis. However, whether IKKε can influence the invasive ability and proliferation of glioma cells remains largely unknown. In this study, we showed that overexpression of IKKε is positively correlated to glioma pathological grade, suggesting that IKKε plays a role in tumor progression, rather than tumor initiation. Targeted knockdown of IKKε in human glioma cells using siRNA, was associated with inhibition of cell growth, cell cycle arrest and decreased cell invasion; however, notable apoptosis was not observed. Furthermore, we demonstrated that transposition of NF-κB p65 resulted in the alteration of these phenotypes. Tumor growth was attenuated in established subcutaneous gliomas in nude mice treated with IKKε siRNA in vivo. Collectively, our results suggest that deregulation of IKKε plays a pivotal role in the uncontrolled proliferation and malignant invasion of glioma cells in vitro and in vivo by targeting NF-κB. Silencing of IKKε using synthetic siRNAs may offer a novel therapeutic strategy for the treatment of glioma.     

Phase II trial of sequential paclitaxel and 1 h infusion of bryostatin-1 in patients with advanced esophageal cancer

BACKGROUND: We sought to determine the response rate and toxicity profile of sequential paclitaxel and bryostatin-1, a novel, selective inhibitor of protein kinase C, in patients with advanced esophageal cancer. PATIENTS AND METHODS: Patients with advanced esophageal and gastroesophageal junction cancer were enrolled. All gave informed consent. They were initially treated with paclitaxel 90 mg/m(2) intravenously on Day 1 and bryostatin-1 50 mug/m(2) on Day 2 weekly for three consecutive weeks out of four. Because of severe myalgias, dosing was reduced to paclitaxel 80 mg/m(2) with bryostatin-1 40 mug/m(2) and then to paclitaxel 80 mg/m(2) with bryostatin-1 25 mug/m(2). RESULTS: Twenty-four patients were enrolled, with 22 assessable for response. The partial response rate was 27%. 10 patients treated with bryostatin-1 40-50 mug/m(2) had a response rate of 40 versus 17% at bryostatin-1 25 mug/m(2) (p-value = 0.3). Median time-to-progression was 3.7 months and median survival was 8.3 months. Grade 3/4 myalgias were seen in 50% of patients. Myalgias appeared to be related to bryostatin-1 dose. Because of toxicity, the trial was closed prior to full accrual. CONCLUSIONS: Despite potential anti-tumor activity of this combination in patients with advanced esophageal cancer, further development is not warranted, given the severe toxicity, especially myalgias, that were seen.     

Evaluating near-infrared photoimmunotherapy for targeting fibroblast activation protein-α expressing cells in vitro and in vivo

Photoimmunotherapy (PIT), carried out using an Ab conjugated to the near infrared dye IRDye700DX, is achieving significant success in target-specific elimination of cells. Fibroblast activation protein alpha (FAP-α) is an important target in cancer because of its expression by cancer-associated fibroblasts (CAFs) as well as by some cancer cells. Cancer-associated fibroblasts that express FAP-α have protumorigenic and immune suppressive functions. Using immunohistochemistry of human breast cancer tissue microarrays, we identified an increase of FAP-α⁺ CAFs in invasive breast cancer tissue compared to adjacent normal tissue. We found FAP-α expression increased in fibroblasts cocultured with cancer cells. In proof-of-principle studies, we engineered human FAP-α overexpressing MDA-MB-231 and HT-1080 cancer cells and murine FAP-α overexpressing NIH-3T3 fibroblasts to evaluate several anti-FAP-α Abs and selected AF3715 based on its high binding affinity with both human and mouse FAP-α. After conjugation of AF3715 with the phthalocyanine dye IR700, the resultant Ab conjugate, FAP-α-IR700, was evaluated in cells and tumors for its specificity and effectiveness in eliminating FAP-α expressing cell populations with PIT. Fibroblast activation protein-α-IR700-PIT resulted in effective FAP-α-specific cell killing in the engineered cancer cells and in two patient-derived CAFs in a dose-dependent manner. Following an intravenous injection, FAP-α-IR700 retention was three-fold higher than IgG-IR700 in FAP-α overexpressing tumors, and two-fold higher compared to WT tumors. Fibroblast activation protein-α-IR700-PIT resulted in significant growth inhibition of tumors derived from FAP-α overexpressing human cancer cells. A reduction of endogenous FAP-α⁺ murine CAFs was identified at 7 days after FAP-α-IR700-PIT. Fibroblast activation protein-α-targeted near infrared PIT presents a promising strategy to eliminate FAP-α⁺ CAFs.     

Liposome encapsulated Disulfiram inhibits NFκB pathway and targets breast cancer stem cells in vitro and in vivo

Breast cancer stem cells (BCSCs) are pan-resistant to different anticancer agents and responsible for cancer relapse. Disulfiram (DS), an antialcoholism drug, targets CSCs and reverses pan-chemoresistance. The anticancer application of DS is limited by its very short half-life in the bloodstream. This prompted us to develop a liposome-encapsulated DS (Lipo-DS) and examine its anticancer effect and mechanisms in vitro and in vivo.The relationship between hypoxia and CSCs was examined by in vitro comparison of BC cells cultured in spheroid and hypoxic conditions. To determine the importance of NFκB activation in bridging hypoxia and CSC-related pan-resistance, the CSC characters and drug sensitivity in BC cell lines were observed in NFκB p65 transfected cell lines. The effect of Lipo-DS on the NFκB pathway, CSCs and chemosensitivity was investigated in vitro and in vivo.The spheroid cultured BC cells manifested CSC characteristics and pan-resistance to anticancer drugs. This was related to the hypoxic condition in the spheres. Hypoxia induced activation of NFκB and chemoresistance. Transfection of BC cells with NFκB p65 also induced CSC characters and pan-resistance. Lipo-DS blocked NFκB activation and specifically targeted CSCs in vitro. Lipo-DS also targeted the CSC population in vivo and showed very strong anticancer efficacy. Mice tolerated the treatment very well and no significant in vivo nonspecific toxicity was observed.Hypoxia induced NFκB activation is responsible for stemness and chemoresistance in BCSCs. Lipo-DS targets NFκB pathway and CSCs. Further study may translate DS into cancer therapeutics.     

Anti-cancer activity of DHA on gastric cancer—an in vitro and in vivo study

Treatment of gastric cancer remains a major challenge, and new anticancer drugs are urgently required. This study investigated whether dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, could inhibit the growth of gastric cancer both in vitro and in vivo. A series of in vitro experiments including MTT, colony-forming, wound healing, invasion, cell cycle, cellular senescence, and apoptosis assays were performed to examine the antiproliferative and antimetastatic effects of DHA on three gastric cancer cell lines, SGC-7901, BGC823, and MGC803. The result showed that the proliferation rate and colony-forming abilities of gastric cancer cells were significantly suppressed by DHA together with significant suppression of the expressions of proliferation markers (PCNA, cyclin E, and cyclin D1), and upregulation of p21 and p27. Moreover, DHA induced cellular senescence, G1 phase cell cycle arrest and hindered the migration and invasion of gastric cancer cells corresponding with downregulation of MMP-9 and MMP-2. Furthermore, DHA significantly induced apoptosis through suppressing Bcl-2 as well as activating caspase-9 and PARP. Treatment of gastric cancer cells with DHA increased miR-15b and miR-16 expression, caused a downregulation of Bcl-2, resulting in apoptosis of gastric cancer cells. In vivo, our data showed that DHA significantly inhibited the growth of SGC7901 cell-transplanted tumors. In summary, we have shown that DHA is able to inhibit the growth and metastasis of human gastric cancer. The modulation of miR-15b and miR-16 mediated the apoptosis effects of DHA in gastric cancer cells. Our work suggested that DHA has significant anticancer effects against gastric cancer both in vivo and in vitro, indicating that it is a promising therapy for human gastric cancer.     

Antitumor activities of recombinant human interferon (IFN)-λ1 in vitro and in xenograft models in vivo for colon cancer

Interferon (IFN)-λ1, as a newly identified IFN, interacts with the structurally unique IFN-λ1 receptor complex and exhibits antiviral and antiproliferation effects. The major focus of our work was to study the antitumor activities of recombinant human IFN-λ1 (rhIFN-λ1) in vivo and in vitro. The MTT analysis showed that rhIFN-λ1 inhibited the proliferation of HCT116 cells in a dose-dependent manner, but with a less efficacy in HT29 cells than HCT116 cells. IFN-λ1 also activated the STATs and induced apoptosis in both types of cells. In the in vivo study, we found that rhIFN-λ1 suppressed tumor growth in a dose-dependent fashion, with an inhibition rate of 52% of HCT116 (P < 0.01) and 56% of HT29 (P < 0.01). These results indicate that rhIFN-λ1 could be a new potential IFN reagent for cancer therapy.     

Bullatacin — in vivo and in vitro experience in an ovarian cancer model

The cytotoxicity and antitumor effects of the acetogenin Bullatacin were evaluated in vitro in multiple ovarian cancer cell lines and in vivo in a murine ovarian teratocarcinoma (MOT) model in C3HeB/FeJ mice. The in vitro cytotoxicity of Bullatacin against four human ovarian epithelial tumor cell lines (OC-194, OC-222, OVCAR-3, and A-2780) was assessed in 48- and 72-h tetrazolium-dye (MTT) cytotoxicity assays. The percentage of cytotoxicity was determined on the basis of the mean optical density of the respective untreated cells and the dose effective against 50% of the cells (ED₅₀) was calculated for each cell line. In vivo experiments were performed on adult female C3HeB/FeJ mice, which were injected i.p. with 10⁵ MOT cells and varying amounts of Bullatacin given either in a single dose or in 5 subsequent doses over 72 h. All mice were observed for survival relative to that of the control groups, which were injected either with 10⁵ MOT cells with or without serial injections of vehicle or with vehicle only. All four epithelial ovarian cancer cell lines displayed sensitivity to Bullatacin. The relative cytotoxic effects were very heterogeneous, with the ED₅₀ value ranging between 10^–7 g/ml for OC-194 and 4 g/ml for the cisplatin-resistant cell line OVCAR-3 in a 72-h MTT cytotoxicity assay. All mice that had been injected i.p. with 10⁵ MOT cells and 1.4 mg/kg or more of Bullatacin died within the first 24 h after injection, whereas all mice that had received 600 g/kg of Bullatacin or less survived equally as long as the controls that had been injected with MOT only (21.1±0.9 days). Mice that had received Bullatacin at a dose ranging from 600 g/kg to 1.4 mg/kg either died during the 1st day postinjection or survived, but not longer than the MOT control group. Serial i.p. injections of Bullatacin again either led to death of the mice within 24–48 h of the last dose of Bullatacin or did not have any effect on the survival of the mice as compared with the respective control groups, which had been injected with the tumor and serial injections of vehicle (22.5±2.2 days). In summary, Bullatacin showed no effect on MOT-caused animal death in C3HeB/FeJ mice at nonlethal dose ranges, whether it was given as a single i.p. dose or serially over 72 h. In vitro, however, it proved to be a very potent cytotoxic agent in a variety of ovarian cancer cell lines. As compared with other chemotherapeutic agents, which we accept as having clinically important antitumor efficacy against ovarian cancer, such as cisplatin or carboplatin, Bullatacin demonstrated a very favorable ED_{50 in vitro}/LD_{50 in vivo} (the dose lethal to 50% of the mice) ratio.This work was supported by the Bertha Warshaver Rubin Research Fund and was presented at the 21st annual meeting of the Western Association of Gynecologic Oncologists, May 19–23, 1993, Santa Monica, California     

The combined effects of oncolytic reovirus plus Newcastle disease virus and reovirus plus parvovirus on U87 and U373 cells in vitro and in vivo

Previous results had documented oncolytic capacity of reovirus, parvovirus and Newcastle disease virus (NDV) on several tumor cell types. To test whether combinations of these viruses may increase this capacity, human U87- and U373-glioblastoma cells, in vitro or xenografted into immuno-compromised mice, were subjected to simultaneous double infections and analyzed. Our results show that reovirus (serotype-3) plus NDV (Hitcher-B1) and reovirus plus parvovirus-H1 lead to a significant increase in tumor cell killing in vitro in both cell lines (Kruskal–Wallis test, P < 0.01) and in vivo. Immunofluorescence and flow cytometry analyses demonstrated the simultaneous replication of the viruses in nearly all cells (>95%) after combined infection. These data thus indicate that a synergistic anti-tumor effect can be achieved by the combined infection with oncolytic viruses.     

Primitive origins of prostate cancer: In vivo evidence for prostate‐regenerating cells and prostate cancer‐initiating cells

Tissue stem cells have been linked to cancers of epithelial origin including the prostate. There are three relevant issues concerning stem cells and cancer that rely solely on functional studies: 1. Are there tissue-regenerating stem cells in the adult organ? 2. Can tissue-regenerating cells serve as targets for transformation? 3. Do primary tumors contain tumor-propagating (cancer stem) cells? We will review the recent literature with respect to these critical issues to provide a direct link between primitive cells and prostate cancer.     

Phosphoprotein associated with glycosphingolipid microdomains 1 inhibits the proliferation and invasion of human prostate cancer cells in vitro through suppression of Ras activation

Phosphoprotein associated with glycosphingolipid microdomains 1 (PAG) is an important negative regulator of immune signaling in T lymphocytes. However, newly emerging evidence has indicated that PAG may play important roles in tumor cells. Our previously reported cDNA microarray experiments identified PAG as a gene down-regulated in the high metastatic potential prostate cancer cell line PC-3M-1E8. In this study, we investigated the role of PAG in the proliferation, invasion and metastasis of prostate cancer cells and the underlying mechanisms. We confirmed that the expression of PAG at both the mRNA and protein levels was low in PC-3M-1E8 and DU145 cells compared to low metastatic potential prostate cancer cells PC-3M-2B4. In addition, we demonstrated that the reintroduction of PAG to PC-3M-1E8 and DU145 cells led to reduced proliferation through cell cycle arrest, decreased anchorage-independent growth and reduced invasion ability of tumor cells in vitro. This is the first report demonstrating that PAG inhibits the proliferation and invasion potential of prostate cancer cells via the interaction with RasGAP to recruit RasGAP to the cell membrane, where RasGAP hydrolyzes GTP to GDP, reduces the level of activated Ras, and ultimately suppresses the activation of ERK1/2, cyclin D1 and other effectors of the Ras signaling pathway. Morphologically, we observed that PAG could diminish the formation of pseudopodia on the cell surface and redistribute the intracellular F-actin in PC-3M-1E8 cells, which directly leads to the decreased invasion and metastasis potential of tumor cells. Taken together, these results suggest that PAG acts to inhibit the development and metastasis of prostate cancers and represents a novel therapeutic target for prostate cancer.     

Nitric oxide (NO) enhances pemetrexed cytotoxicity via NO‑cGMP signaling in lung adenocarcinoma cells in vitro and in vivo

Pemetrexed (PEM) is a novel, multitargeted, antifolate, antineoplastic agent for the treatment of non-small cell lung cancer and malignant pleural mesothelioma. Additional effects of nitric oxide (NO) donors on the chemosensitivity of cancers have been reported. However, the effects of an NO donor on PEM-induced cytotoxicity remain unknown. In this study, we investigated the effects of the NO donors, NOC-18 on the cytotoxicity in A549 cells in vitro and of nitroglycerin (GTN), on the tumor growth of Lewis lung carcinoma cells in a murine syngraft model treated with PEM. The effects of NO donors on the expression of proteins associated with PEM metabolism, including thymidylate synthase (TS), reduced folate carrier 1 (RFC1), folylpolyglutamate synthase (FPGS), γ-glutamyl hydrolase (GGH) and multidrug resistance-related protein (MRP)5, and the effects of cyclic guanosine mono-phosphate (cGMP) signaling on these proteins were examined in A549 cells. Treatment with 100 nM NOC-18 for 3 days significantly enhanced PEM-induced cytotoxicity and increased the expression of RFC1 and FPGS in A549 cells. Treatment with 10 nM 8-bromo-cGMP (8-Br-cGMP) for 3 days also increased the expression of RFC1 and FPGS in A549 cells. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 μm) significantly reversed the increase in RFC1 and FPGS expression induced by 100 nM NOC-18 in A549 cells. Combination therapy with GTN and PEM significantly reduced tumor growth compared with PEM alone in the syngraft model. The enhanced antitumor effect of GTN plus PEM was significantly reversed by the concomitant addition of ODQ. These findings suggest that NO donors, such as NOC-18 and GTN, enhance the anticancer effects of PEM by increasing the RFC1 and FPGS expression and stimulating cGMP signaling pathways in cancer cells.     

Xanthine oxidoreductase – Clinical significance in colorectal cancer and in vitro expression of the protein in human colon cancer cells

Xanthine oxidoreductase (XOR) is a key enzyme in degradation of DNA and RNA, and has previously been shown to be decreased in aggressive breast and gastric cancer. In this study, XOR expression was assessed in tissue microarray specimens of 478 patients with colorectal cancer and related to clinical parameters. In addition, we performed in vitro studies of XOR activity, protein and mRNA in colon cancer cells (Caco-2). Results from the tissue expression analyses show that XOR was decreased in 62% and undetectable in 22% of the tumours as compared to normal tissue. Loss of XOR was associated with poor grade of differentiation (p = 0.006) and advanced Dukes stage (p = 0.03). In multivariate survival analysis, XOR was a prognostic factor (p = 0.008), independent of Dukes stage, histological grade, age and tumour location. The in vitro analyses show that XOR is not measurable in undifferentiated Caco-2 cells, but appears and increases with differentiation. We conclude that XOR expression is associated with histological grade of differentiation and extent of disease in colorectal cancer, and it provides significant prognostic information independently of established factors.