Role of the Cyclooxygenase Pathway in Chemotherapy-Induced Oral Mucositis: A Pilot Study

Goals Oral mucositis can be a significant and dose-limiting complication of high-dose cancer therapy. Mucositis is a particularly severe problem in patients receiving myeloablative chemotherapy prior to bone marrow or hematopoetic stem cell transplant (HSCT). The cyclooxygenase (COX) pathway mediates tissue injury and pain through upregulation of pro-inflammatory prostaglandins, including prostaglandin E2 (PGE2) and prostacyclin (PGI2). The objective of this small (n=3) pilot study was to examine the role of the COX pathway in causing mucosal injury and pain in chemotherapy-induced oral mucositis. Materials and methods We collected blood, saliva, and oral mucosal biopsy specimens from three autologous HSCT patients at the following time-points before and after administration of conditioning chemotherapy: Day −10, +10, +28, and +100, where day 0 is day of transplant. RNA extracted from full-thickness tissue samples was measured by RT-PCR for the following: COX-1, COX-2, microsomal prostaglandin E synthase (mPGES), IL-1β, and TNF-α. Blood and saliva samples were measured by ELISA for PGE2 and PGI2, which are markers of COX activity. Severity of oral mucositis was determined using the Oral Mucositis Index. Severity of pain due to oral mucositis was measured using a Visual Analog Scale. Relationships between the different variables were examined using Spearman rank correlation coefficients. Main results Mean mucositis and pain scores increased significantly after administration of chemotherapy and then gradually declined. The correlation between changes in mucositis and pain scores was strong and statistically significant. The following additional correlations were statistically significant: between tissue COX-1 and pain; between tissue mPGES and pain; between salivary PGE1 and pain; between salivary PGI2 and pain. Other relationships were not statistically significant. Conclusions Our finding of significant associations of pain scores with tissue COX-1 and mPGES, as well as salivary prostaglandins, is suggestive of a role for the cyclooxygenase pathway in mucositis, possibly via upregulation of pro-inflammatory prostaglandins. However, our small sample size may have contributed to the lack of significant associations between COX-2 and other inflammatory mediators with mucosal injury and pain. Thus, additional studies with larger numbers of subjects are warranted to confirm the involvement of the cyclooxygenase pathway in chemotherapy-induced mucositis

Null mutation for Macrophage Migration Inhibitory Factor (MIF) is associated with less aggressive bladder cancer in mice

<p>Abstract</p> <p>Background</p> <p>Inflammatory cytokines may promote tumorigenesis. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with regulatory properties over tumor suppressor proteins involved in bladder cancer. We studied the development of bladder cancer in wild type (WT) and MIF knockout (KO) mice given N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), a known carcinogen, to determine the role of MIF in bladder cancer initiation and progression.</p> <p>Methods</p> <p>5-month old male C57Bl/6 MIF WT and KO mice were treated with and without BBN. Animals were sacrificed at intervals up to 23 weeks of treatment. Bladder tumor stage and grade were evaluated by H&E. Immunohistochemical (IHC) analysis was performed for MIF and platelet/endothelial cell adhesion molecule 1 (PECAM-1), a measure of vascularization. MIF mRNA was analyzed by quantitative real-time polymerase chain reaction.</p> <p>Results</p> <p>Poorly differentiated carcinoma developed in all BBN treated mice by week 20. MIF WT animals developed T2 disease, while KO animals developed only T1 disease. MIF IHC revealed predominantly urothelial cytoplasmic staining in the WT control animals and a shift toward nuclear staining in WT BBN treated animals. MIF mRNA levels were 3-fold higher in BBN treated animals relative to controls when invasive cancer was present. PECAM-1 staining revealed significantly more stromal vessels in the tumors in WT animals when compared to KOs.</p> <p>Conclusion</p> <p>Muscle invasive bladder cancer with increased stromal vascularity was associated with increased MIF mRNA levels and nuclear redistribution. Consistently lower stage tumors were seen in MIF KO compared to WT mice. These data suggest that MIF may play a role in the progression to invasive bladder cancer.</p

Cyclooxygenase-2 suppresses the anabolic response to PTH infusion in mice.

We previously reported that the ability of continuously elevated PTH to stimulate osteoblastic differentiation in bone marrow stromal cell cultures was abrogated by an osteoclastic factor secreted in response to cyclooxygenase-2 (Cox2)-produced prostaglandin E2. We now examine the impact of Cox2 (Ptgs2) knockout (KO) on the anabolic response to continuously elevated PTH in vivo. PTH (40 μg/kg/d) or vehicle was infused for 12 or 21 days in 3-mo-old male wild type (WT) and KO mice in the outbred CD-1 background. Changes in bone phenotype were assessed by bone mineral density (BMD), μCT and histomorphometry. PTH infusion for both 12 and 21 days increased femoral BMD in Cox2 KO mice and decreased BMD in WT mice. Femoral and vertebral trabecular bone volume fractions were increased in KO mice, but not in WT mice, by PTH infusion. In the femoral diaphysis, PTH infusion increased cortical area in Cox2 KO, but not WT, femurs. PTH infusion markedly increased trabecular bone formation rate in the femur, serum markers of bone formation, and expression of bone formation-related genes, growth factors, and Wnt target genes in KO mice relative to WT mice, and decreased gene expression of Wnt antagonists only in KO mice. In contrast to the differential effects of PTH on anabolic factors in WT and KO mice, PTH infusion increased serum markers of resorption, expression of resorption-related genes, and the percent bone surface covered by osteoclasts similarly in both WT and KO mice. We conclude that Cox2 inhibits the anabolic, but not the catabolic, effects of continuous PTH. These data suggest that the bone loss with continuously infused PTH in mice is due largely to suppression of bone formation and that this suppression is mediated by Cox2

Morphometry of femoral trabecular bone and L3 vertebrae in mice infused for 21 days.

<p><b>A.</b> Representative μCT images (top panel) and morphometric analyses (bottom panel) of trabecular bone in the metaphyseal region of distal femur. <b>B.</b> Representative μCT images (top panel) and morphometric analyses (bottom panel) of trabecular bone in L3 vertebrae. Bars are means ± SEM for 6 WT and 6 KO mice infused with vehicle and 7 WT and 7 KO mice infused with PTH. ^aSignificant effect of PTH, p<0.01; ^bp<0.05. ^cSignificant effect of genotype, p<0.01. <b>C</b>. Representative μCT cross-sectional images of midshaft of femurs.</p

List of genes and primers used to analyze gene expression by quantitative real time PCR.

<p>(A) Genes analyzed by TaqMan Gene Expression Assay and the Design Probes used to analyze their expression. (B) Genes analyzed by Syber Green validated primer sequences.</p><p>List of genes and primers used to analyze gene expression by quantitative real time PCR.</p

Static and dynamic histomorphometric analysis of distal femurs in mice infused for 12 days.

<p><b>A.</b> Representative microscopic images of distal femur. (1) Hematoxylin staining at 20x magnification. (2) Tartrate resistant acid phosphatase (TRAP) staining and counter staining with hematoxylin at 400x magnification in PTH-infused WT and KO mice. (3) Double labeling with calcein (green) and demeclocycline (orange/brown) of trabeculae at 400x magnification in PTH-infused WT and KO mice. <b>B.</b> Histomorphometric analysis of distal femurs. Bars are means ± SEM for 7 WT and 7 KO mice treated with vehicle and 8 WT and 8 KO mice treated with PTH. ^aSignificant effect of PTH, p<0.01; ^bp<0.05. ^cSignificant effect of genotype, p<0.01.</p

Cortical morphometry by μCT in the midshaft femoral region of WT and <i>Cox2</i> KO mice after infusion with vehicle or PTH for 12 days.

<p>Data are means ± SEM for (n) mice.</p><p>^aSignificant effect of PTH, p<0.01.</p><p>^bSignificant effect of genotype, p<0.05.</p><p>Cortical morphometry by μCT in the midshaft femoral region of WT and <i>Cox2</i> KO mice after infusion with vehicle or PTH for 12 days.</p

Body weight, serum calcium (Ca) and serum PGE₂ in wild type (WT) and <i>Cox2</i> KO mice infused with vehicle or PTH for 12 days.

<p>Data are means ± SEM for (n) mice.</p><p>^aSignificant effect of PTH, p<0.01.</p><p>^bSignificant effect of genotype, p<0.01.</p><p>*Three of the 6 samples were below the limit of detectability of the assay (3.25 pg/ml) and the mean was calculated from the remaining 3 samples. Hence, the value is an overestimate of true value. UD = undetectable. 352 pg/ml = 1 nM PGE₂</p><p>Body weight, serum calcium (Ca) and serum PGE₂ in wild type (WT) and <i>Cox2</i> KO mice infused with vehicle or PTH for 12 days.</p