Food intake, tumor growth, and weight loss in EP2 receptor subtype knockout mice bearing PGE2-producing tumors.

Previous studies in our laboratory have demonstrated that prostaglandin (PG) E2 is involved in anorexia/cachexia development in MCG 101 tumor-bearing mice. In the present study, we investigate the role of PGE receptor subtype EP2 in the development of anorexia after MCG 101 implantation in wild-type (EP2 (+/+)) or EP2-receptor knockout (EP2(-/-)) mice. Our results showed that host absence of EP2 receptors attenuated tumor growth and development of anorexia in tumor-bearing EP2 knockout mice compared to tumor-bearing wild-type animals. Microarray profiling of the hypothalamus revealed a relative twofold change in expression of around 35 genes including mRNA transcripts coding for Phospholipase A2 and Prostaglandin D2 synthase (Ptgds) in EP2 receptor knockout mice compared to wild-type mice. Prostaglandin D2 synthase levels were increased significantly in EP2 receptor knockouts, suggesting that improved food intake may depend on altered balance of prostaglandin production in hypothalamus since PGE2 and PGD2 display opposing effects in feeding control.

Preoperative low dose NSAID treatment influences the genes for stemness, growth, invasion and metastasis in colorectal cancer.

Preclinical data, and an increasing list of clinical investigations, show anti-inflammatory agents to favourably influence the biology of colorectal tumor. We have earlier reported on re-expression of activated immune cells after three days preoperative treatment of patients with colorectal carcinoma, randomized to receive oral NSAID (indomethacin or celebrex). Antisecretory prophylaxis (esomeprasol) was provided to all patients and served as sham treatment. Concomittant to MHC locus activation, Prominin1/CD133, a marker associated with stemness and poor prognosis in several solid tumors, was downregulated. The aim of the present study was to evaluate expression of additional regulators belonging to the stem cell niche, OCT4, SOX2 and BMP7, as well as some microRNAs, reported to act as tumor suppressors or oncomiRs. Peroperative tumor biopsies were analyzed by microarrays, quantitative real-time PCR and immunohistochemistry (IHC). The stem cell master regulator SOX2 was increased by NSAIDs (p<0.01), as well as the tumor suppressor miR-630 (p<0.01), while BMP7, a marker for poor prognosis in CRC, was downregulated by NSAID (indomethacin, p<0.02). The upregulation of SOX2, but not of its heterodimer binding partner OCT4, could imply a negative feed-back loop, with a switch­off for stemness preservation of tumor cells. This is supported by the overall evaluation of gene expression profiles with subsequent events, indicating less aggressive tumors following NSAID treatment.

Downregulation of Prominin 1/CD133 expression in colorectal cancer by NSAIDs following short-term preoperative treatment.

Expression of Prominin 1/CD133 is associated with poor prognosis and chemoresistance in several types of solid tumors. The aim of the present study was, therefore, to evaluate Prominin 1/CD133 expression in colorectal carcinoma after short-term preoperative treatment by non-steroidal anti-inflammatory drugs (NSAIDs). Patients aimed at curative operation for colon cancer were randomized to receive NSAIDs (indomethacin 50 mg x2 or celecoxib 100 mg x2) three days preoperatively. Antisecretory prophylaxis (esomeprasol 40 mg x1) was provided to all patients and served as sham intake. CD133 expression in tumor tissue was also assessed in tumors from Dukes' B patients selected for either long or short postoperative survival. No patients received perioperative chemoradiotherapy. Tumor tissue was collected at surgery for quantification of mRNAs (Prom1 and Wnt4) by qPCR. Immunohistochemistry stained for CD133, Ep-CAM, CD34 and CD45. PGE2 content in tumor tissue was determined. Transcript of CD133 in tumor tissue was lower in patients treated with NSAIDs (0.28 ± 0.07 vs. 0.51 ± 0.08; p<0.03) as well as some other stem cell-related transcripts. In treated patients 36% of all tumors stained positive for CD133 compared to 71% in sham-treated control patients (p<0.05). Short survivors with Dukes' B tumors displayed 78% CD133 expression as compared to 33% of tumors in long-term survivors (p<0.002). Tumor tissue PGE(2) content was negatively related to patient survival. Our results show that short-term preoperative NSAID treatment downregulates colon cancer tissue expression of Prominin 1/CD133, a stem cell marker indicative of survival prognosis as confirmed.

COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status.

BACKGROUND: Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. METHOD: Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. RESULTS: Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1ß, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4) showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3) were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. CONCLUSIONS: Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue.

Mechanisms behind COX-1 and COX-2 inhibition of tumor growth in vivo.

Non-steroidal anti-inflammatory drugs (NSAIDs) attenuate tumor net growth in clinical and experimental cancer. Evaluations in cell culture experiments have implied involvement of growth factor and G-protein related signaling pathways to explain decreased proliferation, angiogenesis, increased cell adhesion and apoptosis. Sparse information is however available from studies on growing tumors in vivo. The aim of the present study was to map alterations in selected signal proteins in relation to heterogeneous tissue expression of COX-2 in tumors during COX inhibition. MCG 101 cells were exposed to indomethacin treatment both in vivo and in vitro to reduce PGE2 production. Tumor tissue specimens were taken for immunohistochemical analyses and qPCR determinations. Protein markers were selected to reflect cell proliferation and cell cycling, angiogenesis and metastasis in relationship to COX-2 staining in tumor tissue. Indomethacin did not change overall COX-2 staining in tumor tissue, but altered its distribution towards increased staining in cell nuclei/nucleoli and decreased COX-2 staining heterogeneity in tumor tissue. P53 staining was decreased, while PCNA and TGFß3 staining were increased by indomethacin in tumor areas with high presence of COX-2, which correlated to staining of BAX, TUNEL, Bcl-2, c-jun, p21, p27, p53 and NM23. Net tumor growth was predicted by EGF-R, p21 and p27 proteins in tumor tissue during indomethacin treatment (multivariate analysis). RNA transcript analyses showed decreased EGF-R and KRas expression in vivo, following indomethacin treatment, which also included KRas, PI3K, JAK1, STAT3 and c-jun, mRNAs in cultured tumor cells. In conclusion, our results extend earlier studies on cell culture experiments and demonstrate that EGF-R and downstream KRas pathways communicate effects of increased prostaglandin activity in tumor tissue in vivo.

Genes with relevance for early to late progression of colon carcinoma based on combined genomic and transcriptomic information from the same patients.

BACKGROUND: Genetic and epigenetic alterations in colorectal cancer are numerous. However, it is difficult to judge whether such changes are primary or secondary to the appearance and progression of tumors. Therefore, the aim of the present study was to identify altered DNA regions with significant covariation to transcription alterations along colon cancer progression. METHODS: Tumor and normal colon tissue were obtained at primary operations from 24 patients selected by chance. DNA, RNA and microRNAs were extracted from the same biopsy material in all individuals and analyzed by oligo-nucleotide array-based comparative genomic hybridization (CGH), mRNA- and microRNA oligo-arrays. Statistical analyses were performed to assess statistical interactions (correlations, co-variations) between DNA copy number changes and significant alterations in gene and microRNA expression using appropriate parametric and non-parametric statistics. RESULTS: Main DNA alterations were located on chromosome 7, 8, 13 and 20. Tumor DNA copy number gain increased with tumor progression, significantly related to increased gene expression. Copy number loss was not observed in Dukes A tumors. There was no significant relationship between expressed genes and tumor progression across Dukes A-D tumors; and no relationship between tumor stage and the number of microRNAs with significantly altered expression. Interaction analyses identified overall 41 genes, which discriminated early Dukes A plus B tumors from late Dukes C plus D tumor; 28 of these genes remained with correlations between genomic and transcriptomic alterations in Dukes C plus D tumors and 17 in Dukes D. One microRNA (microR-663) showed interactions with DNA alterations in all Dukes A-D tumors. CONCLUSIONS: Our modeling confirms that colon cancer progression is related to genomic instability and altered gene expression. However, early invasive tumor growth seemed rather related to transcriptomic alterations, where changes in microRNA may be an early phenomenon, and less to DNA copy number changes.

Receptor and enzyme expression for prostanoid metabolism in colorectal cancer related to tumor tissue PGE2.

Prostaglandins support progression of colorectal cancer by several mechanisms. This conclusion is based on epidemiological and drug intervention long-term studies or retrieved from animal and cell culture experiments. The aim of the present study was to map receptor and enzyme expression for prostanoid metabolism in the presence of high or low PGE2 content within colon cancer tissue at primary tumor operation and after short-term preoperative provision of non-steroidal anti-inflammatory drug (NSAID). Twenty-three unselected patients with colon cancer were randomly selected to receive indomethacin (NSAID) or sham treatment for 3 days before surgery. Normal colon and tumor tissue were collected at operation for RNA extraction. Tissue PGE2 levels were measured by radioimmunoassay. Gene expression was quantified by microarray and real-time PCR. COX-1 expression increased proportionally to COX-2 expression in colon cancer tissue from untreated patients. Indomethacin reduced PGE2 content in normal and tumor tissue with subsequently decreased IP, HPGD and PPARgamma receptor expression in both tumor and normal colon tissue, while subtype EP1-4 receptors were not significantly influenced by indomethacin treatment. MPGES-1 expression was not related to overall PGE2 content in tumor and colon tissue, but decreased significantly in normal tissue during indomethacin exposure. Reduction of tumor tissue PGE2 was related to significant alteration in expression of several hundred genes indicating decreased cell cycling and increased apoptosis during indomethacin treatment, probably related to upregulation of acute phase reactants in tumor tissue. Increased prostanoid activity in colon cancer tissue is related to cross-talk between tumor and stroma cells.

Preoperative treatment with a non-steroidal anti-inflammatory drug (NSAID) increases tumor tissue infiltration of seemingly activated immune cells in colorectal cancer.

This study evaluates HLA gene expression and tumor infiltration by B-cells, macrophages, dendritic cells, T-helper and cytotoxic T-lymphocytes in response to short-term preoperative treatment with cyclooxygenase inhibitors. Patients with colorectal carcinoma were randomized to receive oral NSAID (indomethacin or celebrex) for three days preoperatively; controls received esomeprazol. Peroperative tumor biopsies and normal colon tissue were analyzed by microarray, quantitative PCR and immunohistochemistry. Efficacy of short-term systemic NSAID treatment was confirmed by measurement of PGE2 production in blood monocytes from healthy volunteers. NSAID treatment upregulated genes at the MHC locus on chromosome 6p21 in tumor tissue, but not in normal colon tissue, from the same patient. 23 of the 100 most upregulated genes belonged to MHC class II. HLA-DM, -DO (peptide loading), HLA-DP, -DQ, -DR (antigen presentation), granzyme B, H, perforin and FCGR3A (CD16) (cytotoxicity) displayed increased expression, as did CD8, a marker of cytotoxic T-lymphocytes, while HLA-A and -C expression were not increased by NSAID treatment. MHC II protein (HLA-DP, -DQ, -DR) levels and infiltration by CD4+ T-helper cells of tumor stroma increased upon NSAID treatment, while CD8+ cytotoxic T-lymphocytes increased in both tumor stroma and epithelium. Molecules associated with immunosuppressive T regulatory cells (FOXP3, IL-10) were significantly decreased in indomethacin-exposed tumors. Standard oral administration of NSAID three days preoperatively was enough to increase tumor infiltration by seemingly activated immune cells. These findings agree with previous information that high prostanoid activities in colorectal cancer increase the risk for reduced disease-specific survival following tumor resection.

Growth associated proteins in tumor cells and stroma related to disease progression of colon cancer accounting for tumor tissue PGE2 content.

Connections among specific proteins (Bax, Bcl-2, bFGF, COX-1, COX-2, E-cad, p15, p53, PCNA, TGFbeta3, TUNEL, vWF) in control of cell proliferation, apoptosis, cell adhesion, tumor vascularity and PGE2 content were evaluated in colon cancer as related to disease progression and survival. Tumor tissue and adjacent normal colon mucosa were obtained at curative resection in 22 patients. PGE2 concentrations were assessed in tumor tissue and tumor derived blood, splanchnic blood, peripheral venous blood and urine. Host inflammation was determined (CRP, ESR) in relationship to tumor differentiation and stage. Patients survived as expected according to Dukes A-D staging. Growth-related proteins correlated between tumor cells and stroma as well as between protein factors within tumor cells and tumor stroma. COX-2 predicted tumor tissue content of PGE2 (p<0.002), without reflection in tumor derived blood. Systemic inflammation was predicted by p15, TGFbeta3 and Bcl-2 in tumor tissue (p<0.001). p15 and vWF predicted reduced survival in ungrouped patients (p<0.02), while p15, PCNA, TGFbeta3 and vWF predicted reduced survival (p<0.0001) when patient grouping accounted for high tumor content of PGE2. Our results connect systemic inflammation and survival to COX-2 staining and increased PGE2 in colon cancer. Thus, it seems important to understand proximal signals behind upregulation of COX-2 and subsequent PGE2 production in certain tumor cells in colon cancer.

Survival and erythropoietin receptor protein in tumours from patients randomly treated with rhEPO for palliative care.

BACKGROUND: Recombinant erythropoietin (rhEPOalpha) corrects anaemia, improves physical functioning and quality of life in cancer patients. However, published reports have suggested risks for tumour stimulation by provision EPO to patients with remaining tumour cells perhaps related to the presence of EPO receptor protein in tumour tissue. Therefore, the aim of the present study was to exclude a possibility that cancer patients who respond favourably to EPO treatment have mainly tumours with low EPO receptor protein expression. METHODS: Tumour tissue was evaluated in 87 patients out of 108 randomly allocated for treatment with rhEPOalpha (n = 50) versus controls (n = 58). Tumour cell proliferation (Ki-67 index) and EPO receptor protein expression were evaluated by immunohistochemistry. RESULTS: EPO treatment varied between 2 and 35 months, in doses between 10,000 and 40,000 Units/week. Ki-67 index did not differ between study and control patients before EPO treatment. Tumour tissue erythropoietin receptor protein was also similar between treated and untreated patients. Around 40% of tumour cells contained EPO receptors. Survival did not differ among EPO treated and control patients analysed as intention to treat, while survival was significantly improved in EPO treated patients per protocol treatment (P < 0.05). Ki-67 index and tumour tissue erythropoietin receptor protein did not predict survival, which systemic inflammation (ESR) did (P < 0.02). CONCLUSIONS: Our results support that reported risk to accelerate disease progression by EPO treatment in palliative care is not justified in patients with solid, gastrointestinal cancer despite tumour presence of EPO receptor protein.

Prostanoid receptor expression in colorectal cancer related to tumor stage, differentiation and progression.

INTRODUCTION: Alterations in eicosanoid metabolism is well established in a variety of malignant tumors, particularly colorectal carcinoma. Recent studies in our laboratory have emphasized a role for EP subtype receptors in progression of colorectal cancer and disease specific mortality. Therefore, the aim of the present study was to extend our knowledge to include additional receptor expression (DP1, DP2, FP, IP, TP) for prostanoids (PGD2, TXA2, PGF2alpha, PGI2) in relationship to tumor stage, differentiation and progression of colorectal cancer. MATERIAL AND METHODS: Total RNA from 62 tumors and adjacent normal colon tissue (n = 48) was extracted. Quantification of receptor expression was performed by realtime PCR and related to the expression of an appropriate housekeeping gene (GAPDH). Tumors were assessed according to Dukes A-D (stage I-IV). RESULTS: DP1, DP2, FP and IP receptor subtypes displayed significantly reduced overall expression in tumor tissue compared to normal colon tissue, while the TP receptor subtype showed significantly higher expression in tumor tissue. Overall expression of the prostanoid receptors in tumor tissue was not related to clinical indexes as tumor stage and tumor cell differentiation evaluated by multivariate analyses. Cultured colorectal cancer cell lines with low (HT-29) and high (HCA-7) intrinsic PGE2 production at confluent state did not express DP1 and IP receptor subtypes, but displayed low expression of DP2, FP and TP receptor subtypes. CONCLUSION: The results in the present study indicate imbalanced expression of prostanoid receptors in colorectal cancer compared to normal colon tissue without clear cut relationship to disease progression. Therefore, future studies should be performed on defined cells within the tumor tissue compartment determining whether any prostanoid receptor(s) is useful as a molecular target in treatment or prevention of colorectal cancer.

EP1-4 subtype, COX and PPAR gamma receptor expression in colorectal cancer in prediction of disease-specific mortality.

The importance of prostaglandins in tumor growth and progression is well recognized, including antineoplastic activities by cyclooxygenase (COX) inhibitors. Variation in treatment response to COX inhibition has questioned differences in expression of cell surface and nuclear membrane receptors among tumors with different disease progression. The purpose of this study was to evaluate whether EP(1-4) subtype, PPAR gamma receptor and COX-1/COX-2 expression in colorectal cancer are related to tumor-specific mortality. Reverse transcription-polymerase chain reaction and immunohistochemistry were used to demonstrate expression and protein appearance in tumor tissue compared with normal colon tissue. EP(1) and EP(2) subtype receptor protein was highly present in tumor cells, EP(3) occurred occasionally and EP(4) was not visible. PPAR gamma, EP(2) and EP(4) mRNA were significantly higher in normal colon tissue compared with tumor tissue, without any distinct relationship to Dukes A-D tumor stage. Multivariate analyses indicated that increased tumor tissue EP(2) and COX-2 expression predicted poor survival (p<0.001). COX-1 expression was significantly higher than COX-2 expression in normal colon tissue. Average COX-2 mRNA was not increased in tumor tissue compared with normal colon. However, most tumor cells stained positive for COX-2 protein, which was low or undetectable in normal mucosa cells. COX-1 protein was preferentially visible in stroma. EP(1-4) subtype receptor mRNAs were generally positively correlated to both COX-1 and COX-2 in tumor tissue, but not in normal colon. Our results imply that both prostaglandin production (COX-2) and signaling via EP(1-4) subtype receptors, particularly EP(2), predict disease-specific mortality in colorectal cancer.

Global tumor RNA expression in early establishment of experimental tumor growth and related angiogenesis following COX-inhibition evaluated by microarray analysis.

Altered expression of COX-2 and overproduction of prostaglandins, particularly prostaglandin E(2), are common in malignant tumors. Consequently, non-steroidal anti-inflammatory drugs (NSAIDs) attenuate tumor net growth, tumor related cachexia, improve appetite and prolong survival. We have also reported that COX-inhibition (indomethacin) interfered with early onset of tumor endothelial cell growth, tumor cell proliferation and apoptosis. It is however still unclear whether such effects are restricted to metabolic alterations closely related to eicosanoid pathways and corresponding regulators, or whether a whole variety of gene products are involved both up- and downstream effects of eicosanoids. Therefore, present experiments were performed by the use of an in vivo, intravital chamber technique, where micro-tumor growth and related angiogenesis were analyzed by microarray to evaluate for changes in global RNA expression caused by indomethacin treatment. Indomethacin up-regulated 351 and down-regulated 1852 genes significantly (p < 0.01); 1066 of these genes had unknown biological function. Genes with altered expression occurred on all chromosomes. Our results demonstrate that indomethacin altered expression of a large number of genes distributed among a variety of processes in the carcinogenic progression involving angiogenesis, apoptosis, cell-cycling, cell adhesion, inflammation as well as fatty acid metabolism and proteolysis. It remains a challenge to distinguish primary key alterations from secondary adaptive changes in transcription of genes altered by cyclooxygenase inhibition.

Tumor genome wide DNA alterations assessed by array CGH in patients with poor and excellent survival following operation for colorectal cancer.

Genome wide DNA alterations were evaluated by array CGH in addition to RNA expression profiling in colorectal cancer from patients with excellent and poor survival following primary operations. DNA was used for CGH in BAC and cDNA arrays. Global RNA expression was determined by 44K arrays. DNA and RNA from tumor and normal colon were used from cancer patients grouped according to death, survival or Dukes A, B, C and D tumor stage. Confirmed DNA alterations in all Dukes A - D were judged relevant for carcinogenesis, while changes in Dukes C and D only were regarded relevant for tumor progression. Copy number gain was more common than loss in tumor tissue (p < 0.01). Major tumor DNA alterations occurred in chromosome 8, 13, 18 and 20, where short survival included gain in 8q and loss in 8p. Copy number gains related to tumor progression were most common on chromosome 7, 8, 19, 20, while corresponding major losses appeared in chromosome 8. Losses at chromosome 18 occurred in all Dukes stages. Normal colon tissue from cancer patients displayed gains in chromosome 19 and 20. Mathematical Vector analysis implied a number of BAC-clones in tumor DNA with genes of potential importance for death or survival. The genomic variation in colorectal cancer cells is tremendous and emphasizes that BAC array CGH is presently more powerful than available statistical models to discriminate DNA sequence information related to outcome. Present results suggest that a majority of DNA alterations observed in colorectal cancer are secondary to tumor progression. Therefore, it would require an immense work to distinguish primary from secondary DNA alterations behind colorectal cancer.

Effects of ghrelin on anorexia in tumor-bearing mice with eicosanoid-related cachexia.

Ghrelin is a novel brain-gut peptide that stimulates food intake and may secondarily increase body weight via a growth hormone secretagogue receptor (GHS-R). Tumor-bearing mice (MCG101), characterized by anorexia, fat loss and muscle wasting due to increased concentration of PGE2 and proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha), were provided ghrelin i.p. at a low (20 microg/day) and high dose (40 microg/day) to examine the ability of ghrelin to counteract tumor-induced anorexia. Immunohistochemical staining and Western blot analyses were used to identify GHS-R expression in the brain as well as its relationship to NPY expression in hypothalamic neurons. GHS-R mRNA in hypothalamus and ghrelin mRNA in gastric fundus were quantified by RT-PCR. Body composition was determined by carcass extractions. GHS-R expression in hypothalamus and plasma ghrelin levels were significantly increased in freely-fed tumor-bearing mice, while gastric fundus expression of ghrelin was unaltered compared to non-tumor-bearing mice (controls). Ghrelin treatment increased food intake, body weight and whole body fat at both low and high doses of ghrelin in normal controls, while tumor-bearing mice showed improved intake and body composition at the high dose of ghrelin only. Exogenous ghrelin normalized the GHS-R expression in hypothalamus from tumor-bearing mice without alterations in the gastric fundus expression of ghrelin. Tumor growth was not altered by exogenous ghrelin. Our results indicate that MCG 101-bearing mice became ghrelin resistant despite upregulation of hypothalamic GHS-R expression, which confirms similar indirect observations in cancer patients. Thus, other factors downstream of the ghrelin-GHS-R system appear to be more important than ghrelin to explain cancer-induced anorexia.

The effects of non-selective, preferential-selective and selective COX-inhibitors on the growth of experimental and human tumors in mice related to prostanoid receptors.

Earlier observations on cyclo-oxygenase inhibitors (NSAIDs) restricting tumor growth were re-evaluated by comparing the effects of non-selective, preferential selective and selective derivatives of COX-inhibitors on tumor growth in mouse models with either prostaglandin-sensitive (MCG-101, human tumors) and -insensitive transplants (K1735-M2). Tumor growth, with and without provision of a classical cyclo-oxygenase inhibitor (indomethacin), was related to tumor content of COX-1/COX-2 protein as well as to EP1-EP4 and prostacyclin receptor expression. Mouse serum amyloid protein (SAP) was measured as an indicator of systemic inflammation, which relates to pro-inflammatory cytokines. Indomethacin inhibited tumor growth and prolonged the survival of mice bearing MCG-101 tumors, which display a high production of PGE2, while K1735-M2 tumors with insignificant amounts of PGE2 did not respond to indomethacin at all. However, the effects of various NSAIDs on tumor growth were highly variable in combination with the fact that most preferential selective and selective COX-2 inhibitors attenuated poorly systemic inflammation evaluated by plasma concentrations of mouse SAP. The ability of NSAIDs to attenuate tumor growth was not related to the tumor content of COX-2 protein as expected. Multivariate analysis suggests that significant COX-inhibition of tumor growth may be related to tumor expression of subtype EP2, EP3 (p<0.005) and perhaps EP4 (p<0.09) in complex interplay. The extent of tumor growth inhibition by COX-inhibitors is not simply related to drug specificity on COX-1 or COX-2 pathways. Such effects may instead be related to tumor expression of prostanoid receptors in tumor tissue.

Prostaglandin E and prostacyclin receptor expression in tumor and host tissues from MCG 101-bearing mice: a model with prostanoid-related cachexia.

Preclinical and clinical studies in our laboratory have suggested that prostaglandin (PG) E2 is involved in anorexia and cachexia development, although the role of COX pathways on the pathogenesis of cancer cachexia remains to be clarified. Expressions of PGE (EP1, EP2, EP3alpha,beta,gamma and EP4) and PGI (IP) receptors in the central nervous system (brain cortex, hypothalamus and brain stem), in peripheral (liver, white adipose tissue and skeletal muscle) and tumor tissue from MCG-101-bearing mice with and without indomethacin treatment were investigated by RT-PCR and immunohistochemistry. Expression of EP1 in the liver and EP4 receptor in white adipose tissue were upregulated and responded to indomethacin treatment, while downregulated expression of EP3 in skeletal muscle from tumor-bearing mice was unresponsive to indomethacin treatment despite improved carcass weight. Expression of EP and IP receptors in brain and tumor tissue from tumor-bearing mice were neither related nor responsive to systemic PGE2 levels including increased IL-1beta, IL-6 and TNF-alpha host activities. The expression IP receptor in CNS, peripheral tissue and tumor tissue was unchanged by cachexia development. Our results suggest that transcription of EP receptors in liver, fat and skeletal muscle tissue may be a control level for host metabolic alterations during tumor progression, while overall EP and IP receptor expression in CNS did not indicate an important control level for appetite regulation in MCG 101-bearing mice despite prostanoid related anorexia.

Cyclooxygenase inhibition in early onset of tumor growth and related angiogenesis evaluated in EP1 and EP3 knockout tumor-bearing mice.

It is well established that prostanoids are essential for local inflammation including cell proliferation and apoptosis. Accordingly, prostaglandin E2 (PGE(2)) is a critical factor in wound healing, tumor invasiveness and progression. Therefore, the aim of the present work was to evaluate effects by PGE(2) on tumor vascular density at early onset of tumor growth where hypoxia is limited. Wild-type mice (C57Bl, C3H/HeN) bearing either MCG-101 tumors or a malignant melanoma (K1735-M2) with either high or insignificant PGE(2) production and subsequently different in sensitivity to cyclooxygenase (COX) inhibition were used. Tumor angiogenesis was estimated by intravital microscopy and immune histochemical analysis in wild type and EP(1) or EP(3) subtype receptor knockout mice (C57Bl). Both MCG-101 and K1735-M2 tumor cells stimulated early outgrowth of tumor vessels in proportion to intrinsic growth rate of tumor cells. Indomethacin had no effects on tumor growth or tumor related vascular area in K1735-M2 bearing mice. By contrast, indomethacin decreased tumor cell proliferation and increased apoptosis in MCG-101 tumors with subsequent adaptation in tumor vascular density. Effects of indomethacin on early growth of MCG-101 tumors were not related to tumor content of bFGF protein, while our earlier studies on long-term tumor growth have shown decreased mRNA levels of bFGF during indomethacin treatment. Early onset of tumor growth was significantly promoted in EP(3)- but not in EP(1)-knockouts, although long-term tumor growth is attenuated in EP(1)-knockouts as reported elsewhere. Our results demonstrate that tumor production of PGE(2) promotes primarily net growth of tumor cells with subsequent adaptations in development of the tumor vasculature. Therefore, it is likely that angiogenesis is not a limiting step at the early onset of tumor growth.

P53 mutations in colorectal cancer assessed in both genomic DNA and cDNA as compared to the presence of p53 LOH.

Mutations in p53 is a most common genetic alteration in human cancer and has been related to outcome, also in colorectal cancer. However, published results are not unanimous on this point without clear-cut explanations. Different analytical methods have been applied which could explain some discrepancies. Another factor of importance may be the source of nucleic acids used for identification of sequence alterations. Therefore we compared mutations in exons 7 and 8 of p53 found in genomic DNA, with mutations in cDNA from the same patients and evaluated to what extent LOH and mutations in p53 occurred simultaneously, which are prerequisites for a complete loss of p53 function according to the classic concept. cDNA and genomic DNA from tumor tissue from 123 patients were used. Thirty-four mutations were found in both tumor cDNA and genomic DNA. Twenty missense mutations were the same in both cDNA and genomic DNA. Two missense mutations, one 1 bp deletion and one nonsense mutation were only found in genomic DNA, while nine missense mutations and one 9 bp deletion were found in cDNA only. Statistical analysis showed no significant difference among mutations in cDNA and genomic DNA (p=1.00). Only 2 patients (5%) had both LOH and mutations in exons 2-11 of p53. One patient had LOH without p53 mutation, while 18 patients (44%) showed p53 mutation without LOH and 20 patients (49%) had wt p53 without LOH. Forty-four patients (52%) were non-informative for LOH of the entire cohort consisting of 85 patients. Our findings suggest that different results reported on the role mutated p53 may have in colorectal cancer are probably not explained by the source of nucleic acids (RNA vs DNA) for sequence determinations. Rather, unknown information about simultaneous but different presence of p53 LOH in previously published reports is more likely. However, an unexpectedly infrequent occurrence (5-10%) of complete and isolated p53 ablation (LOH + mutation) would demand considerably larger patient populations (1500-2000 patients) than ever published for relating p53 function to survival in patients with colorectal cancer. Therefore, the role of p53 dysfunction in progression of colorectal cancer remains uncertain.

Prediction of postoperative survival by preoperative serum concentrations of anti-p53 compared to CEA, CA 50, CA 242 and conventional blood tests in patients with colorectal carcinoma.

Blood samples were obtained preoperatively from 151 consecutive patients who were operated for colorectal carcinoma; of these 132 patients underwent curatively aimed operations. Patients who lived for more than five years without any evidence of relapse were classified as survivors and those who died during follow-up were classified as non-survivors. Tumors were staged according to Dukes and tumor differentiation was classified as high, intermediate or low. Serum anti-p53 was determined in all patients and compared to CEA, CA 50 and CA 242 in serum as well as to blood hemoglobin concentration (Hb), erythrocyte sedimentation rate (ESR), serum alkaline phosphatases (ALP), in the same preoperative blood samples for comparison of power to predict mortality in colorectal cancer. Tumor differentiation and Dukes classification predicted survival and the risk to die of colorectal carcinoma as expected. CA 242 and anti-p53 titers in serum were not significantly different among groups of patients with Dukes A-D tumors, while Hb, ESR, ALP, CEA and CA 50 were significantly different with increasing levels appearing in Dukes C-D, except for Hb which decreased. Survivors had lower ESR, ALP, CEA, CA 50 and CA 242, while Hb and serum anti-p53 titers were not different among survivors and non-survivors. Survivors among Dukes A-C patients had normal Hb, ESR, ALP and CA 242 before operation, while non-survivors had increased ESR, CEA and CA 50. Levels of anti-p53 were unrelated to concentrations of all other blood and serum variables, and did not predict survival in Dukes A-D patients. Serum levels of CEA and CA 50 displayed a significantly altered distribution among survivors and non-survivors (p<0.01). This altered distribution was independent of Dukes A-C tumor stage for CEA (p<0.05) but not for CA 50. Based on these results, we conclude that patients with CEA levels above 15 ng/ml should be regarded as high risk patients even when their tumors are classified as Dukes B. These patients may benefit from neoadjuvant or adjuvant chemotherapy.