Background Surgical treatment of neuromas involves excision of neuromas proximally to the level of grossly "normal" fascicles; however, proximal changes at the axonal level may have both functional and therapeutic implications with regard to amputated nerves. In order to better understand the retrograde "zone of injury" that occurs after nerve transection, we investigated the gross and histologic changes in transected nerves using a rabbit forelimb amputation model. Methods Four New Zealand White rabbits underwent a forelimb amputation with transection and preservation of the median, radial, and ulnar nerves. After 8 weeks, serial sections of the amputated nerves were then obtained in a distal-to-proximal direction toward the brachial plexus. Quantitative histomorphometric analysis was performed on all nerve specimens. Results All nerves demonstrated statistically significant increases in nerve cross-sectional area between treatment and control limbs at the distal nerve end, but these differences were not observed 10 mm more proximal to the neuroma bulb. At the axonal level, an increased number of myelinated fibers were seen at the distal end of all amputated nerves. The number of myelinated fibers progressively decreased in proximal sections, normalizing at 15 mm proximally, or the level of the brachial plexus. The cross-sectional area of myelinated fibers was significantly decreased in all sections of the treatment nerves, indicating that atrophic axonal changes proceed proximally at least to the level of the brachial plexus. Conclusions Morphologic changes at the axonal level extend beyond the region of gross neuroma formation in a distal-to-proximal fashion after nerve transection. This discrepancy between gross and histologic neuromas signifies the need for improved standardization among neuroma models, while also providing a fresh perspective on how we should view neuromas during peripheral nerve surgery.

Background. The flavonoid apigenin exhibits anti-proliferative and anti-angiogenic activities. Our objective was to evaluate the effect of apigenin on hypoxia responsive genes important in pancreatic cancer cell proliferation.Materials and Methods. Immunohistochemistry for GLUT-1 expression was conducted on human pancreatic cancer samples and adjacent controls. Real-time RT-PCR, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA) were conducted on CD18 and S2-013 human pancreatic cancer cells treated with apigenin (0-50 mM) in normoxic and hypoxic conditions to evaluate HIF-1 alpha, GLUT-1, and VEGF mRNA and protein expression and secretion.Results. GLUT-1 expression was significantly increased in pancreatic adenocarcinoma samples versus adjacent controls (P < 0.001). Hypoxic conditions induced HIF-1 alpha, GLUT-1, and VEGF protein expression in both CD18 and S2-013 pancreatic cancer cells. Apigenin (50 mM) blocked hypoxia induced up-regulation of all three proteins in both cell lines. Apigenin also impeded hypoxia-mediated induction of GLUT-1 and VEGF mRNA in both cell lines (P < 0.05).Conclusions. Apigenin inhibits HIF-1 alpha, GLUT-1, and VEGF mRNA and protein expression in pancreatic cancer cells in both normoxic and hypoxic conditions. This may account for the mechanism of apigenin's anti-proliferative and anti-angiogenic effects and further supports the potential of apigenin as a future chemopreventive agent for pancreatic cancer. (C) 2011 Published by Elsevier Inc.

Pancreatic cancer has proven to be one of the most difficult diseases to detect, diagnose, and treat due to both the location of the pancreas in the abdomen and the lack of overt symptoms before cancer dissemination. Thus, it is critical to have a variety of modeling systems that can be employed to evaluate drug responses/mechanisms while providing a relatively simple format for drug screening. Cell culture serves this purpose. In this context, it is necessary to review the available pancreatic cancer cell lines, culture techniques, predominant signaling pathways, and types of analyses that can be utilized to assess the effectiveness of drugs on pancreatic cancer cells, including aspects of pharmocotherapeutic strategies for the development of novel methodologies. Special consideration will be given to different signal transduction pathways like MAPK, JAK/STAT, PI3K/AKT, RTKs, VEGF, and NF-kappa B, which are involved in various aspects of pancreatic cancer development and progression, complete with a repertoire of chemical inhibition at several levels within a cascade. Ultimately, abrogation of these cell signals can be detected through measurable variables at the cellular level, including apoptosis, proliferation, altered cell phenotype, the ability to invade and/or metastasize, and changes in cell cycle parameters. Evaluation of drugs at this level can set the stage for future strategies as well as exploration of novel compounds that inhibit other vital cancer signaling pathways.

Objectives: To investigate the effects and mechanism of Frondanol-A5P, a polar extract from Cucumaria frondosa, on growth inhibition and apoptosis in S2013 and AsPC-1 human pancreatic cancer cells. Methods: The effects of Frondanol-A5P on proliferation, cell cycle, expression of cell cycle proteins and p21waf1, phosphorylation of MAP kinases, annexin V binding, and caspase-3 activation were examined. Results: Frondanol-A5P inhibited proliferation and induced G2/M phase cell cycle arrest in both cell lines with decreased expression of cyclin A, cyclin B, and cdc25c. Frondanol-A5P induced phosphorylation of stress-activated protein kinase and Janus kinase (SAPK/JAK) and p38 mitogen-activated protein kinase (MAP) within 5 minutes. Frondanol-A5P markedly increased expression of p21waf1 messenger RNA and protein at 3 hours in both cell lines. This effect was reduced by the p38 kinase inhibitor, SB203580. Frondanol-A5P markedly increased annexin V binding and activated caspase-3. Conclusions: Frondanol-A5 causes cell cycle arrest and apoptosis in human pancreatic cancer cells. These changes are associated with decreased expression of cyclin A, cyclin B, and cdc25c and increased expression of p21waf1 that, at least in part, is mediated by a p38 kinase-dependent mechanism. Because Frondanol-A5P is derived from an edible, nontoxic, sea cucumber, it may be valuable for nutritional therapy or prevention of pancreatic cancer.

Objectives: The aim of the study was to evaluate the effect of combination therapy of apigenin and gemcitabine on cell proliferation, the cell cycle, and gemcitabine resistance in human pancreatic cancer cells.Methods: Cell counting was used to assess the effect of single-agent and combination treatment on the proliferation of CD18 and AsPC-1 pancreatic cancer cells. Flow cytometry was performed to assess the effect of combination treatment on cell cycle progression and induction of apoptosis. Western blot analysis was used to evaluate phosporylated AKT (pAkt) and cell cycle proteins. The effect of apigenin on gemcitabine-resistant AsPC-1 cells was assessed via thymidine incorporation.Results: Apigenin in combination with gemcitabine inhibited pancreatic cancer cell proliferation more than either agent alone. Combination treatment induced both S and G2/M phase arrest and increased apoptosis. Apigenin down-regulated pAkt expression and abrogated gemcitabine-mediated pAkt induction. In gemcitabine-resistant AsPC-1 cells, apigenin significantly inhibited cell proliferation in a dose-dependent manner.Conclusion: Combination treatment with apigenin and gemcitabine inhibited pancreatic cancer cell growth via cell cycle arrest, down-regulation of the prosurvival factor pAkt, and induction of apoptosis. Combination therapy may prove useful for the treatment of pancreatic cancer.

Purpose: Arachidonic acid metabolism via the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways modulates cell growth and apoptosis. Many studies have examined the effects of COX inhibitors on human colorectal cancer, but the role of 5-LOX in colonic cancer development has not been well studied. The purpose of this study was to evaluate the expression of 5-LOX in colonic polyps and cancer and the effect of 5-LOX inhibition on colon cancer cell proliferation. Experimental Design: Colonic polyps, cancer, and normal mucosa were evaluated for 5-LOX expression by immunohistochemistry. Reverse transcription-PCR was used to establish 5-LOX expression in colon cancer cells. Thymidine incorporation and cell counts were used to determine the effect of the nonspecific LOX inhibitor Nordihydroguaiaretic Acid and the 5-LOX inhibitor Rev5901 on DNA synthesis. A heterotopic xenograft model in athymic mice using HT29 and LoVo human colon cancer cells was used to evaluate the effect of the 5-LOX inhibitor zileuton on tumor growth. Results: 5-LOX is overexpressed in adenomatous polyps and cancer compared with that of normal colonic mucosa. LOX inhibition and 5-LOX inhibition decreased DNA synthesis in a concentration- and time-dependent manner in the Lovo cell line (P < 0.05). Inhibition of 5-LOX in an in vivo colon cancer xenograft model inhibited tumor growth compared with that of controls (P < 0.05). Conclusions: This study showed that 5-LOX is up-regulated in adenomatous colon polyps and cancer compared with normal colonic mucosa. The blockade of 5-LOX inhibits colon cancer cell proliferation both in vitro and in vivo and may prove a beneficial chemopreventive therapy in colon cancer.

Augmented expression of connective tissue growth factor (CTGF/CCN2) is observed in healing wounds and in a variety of fibrotic disorders. It appears to enhance many of the effects of transforming growth factor-beta and has been shown to have independent fibrogenic functions. Despite these observations, its importance to dermal wound healing and the transition from wound to scar remains poorly defined. In this study, we use established rabbit models to evaluate the roles of CTGF in dermal wound healing and hypertrophic scarring. We show that CTGF mRNA demonstrates persistent up-regulation in hypertrophic scars. Treatment of wounds with antisense oligonucleotides to CTGF has no measurable effect on early wound closure. However, antisense therapy significantly limits subsequent hypertrophic scarring. Inhibition of CTGF is associated with a marked reduction in the number of myofibroblasts in scars and decreased transcription of TIMP-1 and types I and III collagen. These findings confirm CTGF to be a key mediator of hypertrophic scarring in this model. Its effect on myofibroblasts in this setting suggests a mechanism whereby it plays this role. Its limited participation in early healing implies that it may be a useful and specific target for modulating hypertrophic scarring following injury.

Pancreatic cancer is a devastating disease in which current therapies are inadequate. Separate lines of research have identified the 5-lipoxygenase/leukotriene B-4 receptor pathway and the PPAR. pathway as potential targets for prevention or treatment of this disease. LY293111 was originally designed as a potent leukotriene B4 receptor antagonist for treatment of inflammatory conditions. LY293111 was also known to have inhibitory effects on 5-lipoxygenase, which is upstream of the production of leukotrienes. LY293111 was shown to have potent anticancer effects in pancreatic cancer and several other solid malignancies, where it caused cell cycle arrest and marked apoptosis. Subsequently, it came to light that LY293111 exhibited PPAR. agonist activity in addition to its effects on the 5-lipoxygenase pathway. This raises the question of which of the two targets is of greatest importance with regard to the anticancer effects of this agent. The evidence to date is not conclusive, but suggests that the effects of LY293111 may be mediated by both LTB4 receptors and PPAR gamma. Copyright (C) 2008 Thomas E. Adrian et al.

Introduction: Despite attempts of curative resection and adjuvant therapy, patients diagnosed with pancreatic cancer continue to have a poor prognosis. Novel therapeutics are currently under investigation. The flavonoid apigenin exhibits growth inhibition in several cancer cell lines. We have previously shown that apigenin inhibits pancreatic cancer cell proliferation through the induction of G2/M cell cycle arrest. In the present study we evaluated the effects of combination therapy with apigenin and gemcitabine on cell proliferation, the cell cycle and the pro-survival factor pAkt. Further, we evaluate the effect of Apiginen in Gemcitabine-resistant pancreatic cancer cells. Methods: Cell counting and thymidine incorporation were utilized to assess the combination effects of apigenin and gemcitabine on AsPC-1 and CD18 human pancreatic cancer cell proliferation. Flow cytometry was used to evaluate the effects of apigenin and gemcitabine treatment on the cell cycle. Western blot analysis was used to evaluate pAkt, cyclin A, cyclin B, cyclin E and CDK2 expression as a function of apigenin alone, gemcitabine alone or in combination in CD 18, AsPC-1 and AsPC-1 Gemcitabine-resistant cells. Results: Combination treatment with apigenin (25 μM) and gemcitabine (10 μM) significantly inhibited cell number and cell proliferation compared to controls and gemcitabine alone in both CD18 and AsPC-1 human pancreatic cancer cells (P<0.05). Apigenin down-regulated pAkt expression and prevented gemcitabine mediated pAkt induction in both CD18 and AsPC-1 cell lines. As determined by flow cytometry, combination treatment induced cells to accumulate in the S phase. Gemcitabine induced the expression of cyclin A, cyclin B, cyclin E and CDK2. In the combination treatment, apigenin blocked gemcitabine-induced upregulation of the aforementioned cell cycle proteins. Furthermore, apigenin treatment (0-100 μM) inhibited cell proliferation of AsPC-1 cells resistant to gemcitabine (P<0.01). Conclusions: Combination treatment with apigenin and gemcitabine inhibited cell growth in human pancreatic cells, causing cell cycle arrest in the S phase. Apigenin blocked gemcitabine’s induction of cell cycle regulatory proteins as well as pro-survival factor p-Akt. These results indicate combination therapy may prove useful for the treatment of pancreatic cancer.

Pancreatic adeniocarcinoma is among the deadliest of human cancers. Apigenin, an antitumor flavonoid, inhibits pancreatic cancer cell proliferation in vitro. Geminin is a recently identified novel protein that plays a critical role in preventing abnormal DNA replication by binding to and inhibiting the essential replication factor Cdt1. Microarray analysis identified geminin to be downregulated in pancreatic cancer cells treated with apigenin, Therefore, we investigated the effects of apigenin on geminin expression and other proteins involved in replication (Cdc6, Cdt1, and MCM7) in pancreatic cancer cell lines CD18 and S2013. Real time RT-PCR and western blotting analysis showed that geminin expression is downregulated by apigenin at both mRNA and protein levels. Furthermore, treatment of cells with proteosome inhibitor MG132 reversed the downregulation of geminin by apigenin, supporting our hypothesis that the degradation pathway is another mechanism by which apigenin affects geminin expression. Apigenin treatment also resulted in downregulation of Cdc6 at both mRNA and protein levels. However, Cdt1 and MCM7 expression was not affected in apigenin-treated cells. The effect of apigenin treatment on geminin promoter activity was measured by transient transfection of Hela cells with a reporter gene, demonstrating that apigenin inhibited geminin promoter activity. Geminin expression was also evaluated in human pancreatic tissue (n = 15) by immunohistochemistry and showed that geminin is overexpressed in human pancreatic cancer compared to normal adjacent pancreatic tissue. In conclusion, our studies demonstrated that geminin is overexpressed in human pancreatic cancer and downregulated by apigenin which may contribute to the antitumor effect of this natural flavonoid. (C) 2008 Wiley-Liss, Inc.