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Survivorship navigation is a relatively new concept in the field of patient navigation but an important one. This article highlights the essential functions of the survivorship navigator and defines core outcomes and measures for navigation in the survivorship period. Barriers to access to care experienced by patients during active cancer treatment can continue into the post-treatment period, affecting quality follow-up care for survivors. These barriers to care can be particularly acute for non-English speakers, immigrants, the uninsured, the underinsured, and other vulnerable populations. The survivorship navigator can help reduce barriers and facilitate access to survivorship care and services through communication and information exchange for patients. Survivorship navigation may improve appropriate health care utilization through education and care coordination, potentially improving health outcomes and quality of life of survivors while reducing cost to the health care system. Survivorship navigators can also educate survivors on how to improve their overall wellness, thereby directly impacting the health of a growing population of cancer survivors. [ABSTRACT FROM AUTHOR]

Some forms of hexavalent chromium [Cr(VI)] are known to cause damage to respiratory tract tissue, and are thought to be human lung carcinogens. Because Cr(VI) is mutagenic and carcinogenic at doses that evoke cell toxicity, the objective of these experiments was to examine the effect of Cr(VI) on the growth, survival, and mode of cell death in normal human lung fibroblasts (HLF cells). DNA adduct formation was monitored as a marker for bioavailability of genotoxic chromium. We also examined the modulation of these endpoints by vitamins C and E. Long-term Cr(VI) exposures were employed, which decreased clonogenic cell survival by 25% to 95% in a dose-dependent manner. The predominant cellular response to Cr(VI) was growth arrest. We found that Cr(VI) caused up to 20% of HLF cells to undergo apoptosis, and documented apoptotic morphology and the phagocytosis of apoptotic bodies by neighboring cells. P53 levels increased 4- to 6-fold in chromium-treated cells. In contrast with previous studies using CHO cells, the present study using HLFs found that pretreatment with either vitamin C or E did not exhibit a significant effect on Cr-induced apoptosis or clonogenic survival. In addition, pretreatment with vitamin C did not affect the p53 induction observed after chromium treatment. Neither vitamin had any effect on Cr-DNA adduct formation. These data indicate that although pretreatment with vitamin C or E alters the spectrum of cellular and/or genetic lesions induced by chromium(VI), neither vitamin altered the initiation or progression of apoptosis in diploid human lung cells. [ABSTRACT FROM PUBLISHER]

Abstract Background Diminished expression or activity of prostate apoptosis response protein 4 (Par-4) has been demonstrated in a number of cancers, although reports on Par-4 expression during colon cancer progression are lacking. An understanding of the molecular events in conjunction with the genetic networks affected by Par-4 is warranted. Results Colon cancer specimens derived from patients have significantly diminished expression of Par-4 mRNA relative to paired normal colon. Hence, the functional consequences of reintroducing Par-4 into HT29 colon cancer cells were assessed. Overexpression augmented the interaction of Par-4 with NFκB in the cytosol but not nucleus, and facilitated apoptosis in the presence of 5-fluorouracil (5-FU). Analogous findings were obtained when AKT1 pro-survival signaling was inhibited. Transcriptome profiling identified ~700 genes differentially regulated by Par-4 overexpression in HT29 cells. Nearly all Par-4-regulated genes were shown by promoter analysis to contain cis-binding sequences for NFκB, and meta-analysis of patient expression data revealed that one-third of these genes exist as a recurrent co-regulated network in colon cancer specimens. Sets of genes involved in programmed cell death, cell cycle regulation and interestingly the microRNA pathway were found overrepresented in the network. Noteworthy, Par-4 overexpression decreased NFκB occupancy at the promoter of one particular network gene DROSHA, encoding a microRNA processing enzyme. The resulting down-regulation of DROSHA was associated with expression changes in a cohort of microRNAs. Many of these microRNAs are predicted to target mRNAs encoding proteins with apoptosis-related functions. Western and functional analyses were employed to validate several predictions. For instance, miR-34a up-regulation corresponded with a down-regulation of BCL2 protein. Treating Par-4-overexpressing HT29 cells with a miR-34a antagomir functionally reversed both BCL2 down-regulation and apoptosis by 5-FU. Conversely, bypassing Par-4 overexpression by direct knockdown of DROSHA expression in native HT29 cells increased miR-34a expression and 5-FU sensitivity. Conclusion Our findings suggest that the initiation of apoptotic sensitivity in colon cancer cells can be mediated by Par-4 binding to NFκB in the cytoplasm with consequential changes in the expression of microRNA pathway components.

In addition to its primary role in hemostasis and blood coagulation, thrombin is a potent mitogen capable of inducing cellular functions. Therefore, it should come as no surprise that thrombin has proved to be of importance in the behavior of cancer. In this review, we focus on the ability of tissue factor (TF) and thrombin to influence tumor angiogenesis. Both exert their influence on angiogenesis through clotting-dependent and clotting-independent mechanisms: (1). directly affecting signaling pathways that mediate cell functions, and (2). mediating clot formation, thereby providing a growth media for tumor cells. Therefore, anticoagulant drugs may prove efficacious in cancer treatment due to their ability to reduce the characteristic hypercoagulability of cancer and alter the fundamental biology of cancer. [ABSTRACT FROM AUTHOR]

Background: Gastric cancer (GC) accounts for the greatest disparity in cancer mortality between Black and White Americans. Although clinical trials have shown that Helicobacter pylori (Hp) treatment reduces risk of GC, Hp testing and treatment is not consistently performed in the US, and may offer an opportunity to improve survival., Methods: In a diverse retrospective cohort of 99 GC cases diagnosed at Duke University from 2002-2020 (57% Black; 43% white), we examined the association of Hp testing and treatment prior to or at cancer diagnosis with overall survival using Cox regression analyses to calculate adjusted hazards ratios (HRs) and 95% confidence intervals (CIs)., Results: Overall, 62% of patients were tested for Hp prior to or at GC diagnosis. Of those, 25% tested positive and were treated < 1 year prior to or at diagnosis, 15% tested positive and were treated ≥ 1 year prior to diagnosis, 6% tested positive without evidence of treatment, and 54% tested negative. Compared to never tested, Hp testing and treatment < 1 year prior to or at diagnosis was associated with a significantly reduced likelihood of death (HR 0.21, 95% CI 0.08-0.58). The benefit of any Hp test and treat prior to or at GC diagnosis was significant even among stage IV patients only (HR, 0.22; 95% CI 0.05-0.96)., Conclusions: These findings support Hp testing and treatment for patients at risk of or diagnosed with GC, and suggest Hp treatment may provide an opportunity to reduce GC mortality disparities in the US., (© 2023. The Author(s) under exclusive licence to The International Gastric Cancer Association and The Japanese Gastric Cancer Association.)

Purpose: Prostate cancer (PCa) screening can lead to potential over-diagnosis/over-treatment of indolent cancers. There is a need to optimize practices to better risk-stratify patients. We examined initial longitudinal outcomes of mid-life men with an elevated baseline prostate-specific antigen (PSA) following initiation of a novel screening program within a system-wide network., Materials and Methods: We assessed our primary care network patients ages 40 to 49 years with a PSA measured following implementation of an electronic health record screening algorithm from 2/2/2017-2/21/2018. The multidisciplinary algorithm was developed taking factors including age, race, family history, and PSA into consideration to provide a personalized approach to urology referral to be used with shared decision-making. Outcomes of men with PSA ≥1.5 ng/mL were evaluated through 7/2021. Statistical analyses identified factors associated with PCa detection. Clinically significant PCa (csPCa) was defined as Gleason Grade Group (GGG) ≥2 or GGG1 with PSA ≥10 ng/mL., Results: The study cohort contained 564 patients, with 330 (58.5%) referred to urology for elevated PSA. Forty-nine (8.7%) underwent biopsy; of these, 20 (40.8%) returned with PCa. Eleven (2.0% of total cohort and 55% of PCa diagnoses) had csPCa. Early referral timing (odds ratio [OR], 4.58) and higher PSA (OR, 1.07) were significantly associated with PCa at biopsy on multivariable analysis (both p<0.05), while other risk factors were not. Referred patients had higher mean PSAs (2.97 vs. 1.98, p=0.001)., Conclusions: Preliminary outcomes following implementation of a multidisciplinary screening algorithm identified PCa in a small, important percentage of men in their forties. These results provide insight into baseline PSA measurement to provide early risk stratification and detection of csPCa in patients with otherwise extended life expectancy. Further follow-up is needed to possibly determine the prognostic significance of such mid-life screening and optimize primary care physician-urologist coordination., Competing Interests: The authors have nothing to disclose., (Copyright © 2023 Korean Society for Sexual Medicine and Andrology.)

Purpose: Mobile health interventions can improve patient care. We developed the Digital Supportive Care Awareness and Navigation (D-SCAN) application (app) to facilitate symptom monitoring and use/awareness of cancer supportive care resources. This study tested feasibility, usability/satisfaction, and preliminary efficacy of D-SCAN., Methods: We randomized 50 patients with advanced cancer to receive the D-SCAN intervention or usual care; 10 caregivers also received D-SCAN. The primary feasibility outcome was determined by weekly symptom survey completion and end of study procedures. We assessed secondary outcomes including usability/satisfaction, awareness/use of supportive care resources, patient activation, and quality of life via various questionnaires including the Net Promoter Score (NPS), Patient Activation Measure (PAM-13), Functional Assessment of Cancer Therapy-General (FACT-G), and Caregiver Oncology Quality of Life (CarGOQOL) questionnaire., Results: Seventy-six percent of intervention patients met feasibility criteria, exceeding our pre-determined threshold of 75%. Usability/satisfaction by NPS was high, at 14.3% and 12.5% for patients and caregivers, respectively. Intervention patient and caregiver resource awareness increased by a mean of 3.7 (p = 0.27) and 4.1 items, respectively. Supportive care resource utilization increased by a mean of 0.8 items for intervention patients (p = 0.70) and 0.6 for caregivers. PAM-13 increased by a mean of 1.6 for intervention patients (p = 0.65). FACT-G increased by a mean of 1.1 for intervention patients (p = 0.91), and CarGOQoL increased by a mean of 2.2 (p = 0.41)., Conclusion: D-SCAN is a feasible, usable, and satisfactory intervention for augmenting patient and caregiver supportive care. Further testing is necessary to formally assess D-SCAN's efficacy and impact on patients and caregivers., Clinical Trial Registration Number: NCT03628794. Registered on August 14th, 2018., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Implementation methods of risk-stratified cancer screening guidance throughout a health care system remains understudied., Objective: Conduct a preliminary analysis of the implementation of a risk-stratified prostate cancer screening algorithm in a single health care system., Design: Comparison of men seen pre-implementation (2/1/2016-2/1/2017) vs. post-implementation (2/2/2017-2/21/2018)., Participants: Men, aged 40-75 years, without a history of prostate cancer, who were seen by a primary care provider., Interventions: The algorithm was integrated into two components in the electronic health record (EHR): in Health Maintenance as a personalized screening reminder and in tailored messages to providers that accompanied prostate-specific antigen (PSA) results., Main Measures: Primary outcomes: percent of men who met screening algorithm criteria; percent of men with a PSA result. Logistic repeated measures mixed models were used to test for differences in the proportion of individuals that met screening criteria in the pre- and post-implementation periods with age, race, family history, and PSA level included as covariates., Key Results: During the pre- and post-implementation periods, 49,053 and 49,980 men, respectively, were seen across 26 clinics (20.6% African American). The proportion of men who met screening algorithm criteria increased from 49.3% (pre-implementation) to 68.0% (post-implementation) (p < 0.001); this increase was observed across all races, age groups, and primary care clinics. Importantly, the percent of men who had a PSA did not change: 55.3% pre-implementation, 55.0% post-implementation. The adjusted odds of meeting algorithm-based screening was 6.5-times higher in the post-implementation period than in the pre-implementation period (95% confidence interval, 5.97 to 7.05)., Conclusions: In this preliminary analysis, following implementation of an EHR-based algorithm, we observed a rapid change in practice with an increase in screening in higher-risk groups balanced with a decrease in screening in low-risk groups. Future efforts will evaluate costs and downstream outcomes of this strategy.

Background: Approximately 15% of all cancers are due to infection. The bacteria Helicobacter pylori is the single leading carcinogenic infectious agent and the main cause of stomach cancer. Prevalence of H. pylori, and, correspondingly, stomach cancer incidence and mortality, is significantly greater among African Americans than whites in the United States. In the present study, we conducted a pilot community-engaged H. pylori education and screening study in partnership with a predominantly African American church in Durham, North Carolina., Methods: Initially, we consulted with community advisory boards and convened stakeholder meetings with local community members and primary care physicians. We then developed this pilot study through an iterative collaboration with church partners. Our main outcomes were feasibility and acceptability as measured by participation in a one-day H. pylori screening initiative, and participation in follow-up for those who tested positive. We also sought to determine prevalence and determinants of active H. pylori infection in this population., Results: Community engagement informed the event logistics, messaging, educational materials provided, and follow-up plans. A total of 92 individuals participated in the primary study event, 25% of whom had a current H. pylori infection. Of those, 87% returned for the follow-up events, among whom 70% had successfully cleared their infection., Conclusions: Through community engagement, community-based H. pylori screening and stomach cancer prevention is feasible and acceptable. This is a necessary step in order to move stomach cancer prevention forward to population-based precision H. pylori screening and eradication.

Objective: Circulating tumor cells (CTCs) have known prognostic implications in metastatic castration-resistant prostate cancer, but little is known regarding its utility in biochemical recurrence (BR) of prostate cancer. The primary objectives were to determine whether CTCs are measurable in patients with BR and whether it can reliably predict prostate-specific antigen (PSA) increase and PSA doubling times (PSADTs)., Methods: BR was identified in patients after prostatectomy or radiation or both, with a PSA increase of ≥ 0.2 for prior prostatectomy or > 2 mg/dL increase for post-nadir in prior radiotherapy. CTCs were enumerated at baseline at the time of study entry using the CellSearch (Janssen Diagnostics, Raritan, NJ) test., Results: The median age for all 36 patients accrued was 69.5 years (range, 51-91) with a median PSA of 1.65 ng/mL (range, 0.2-65.8). Gleason scores ranged from 5 to 9 (median, 7). The majority had prostatectomy (n = 25), external beam radiotherapy (n = 9), CyberKnife (Accuray, Sunnyvale, CA) (n = 1), and combined radiohormonal therapy (n = 1). PSADT ranged from 0.35 to 55 months, with a median of 7.43 months. The incidence of positive CTCs was 8.3% (3 patients), of whom 2 had biopsy-proven bony lesions on presenting with equivocal scans and PSADTs of 2.27 and 3.08 months, respectively. The third CTC-positive patient had a PSADT of 4.99 months., Conclusions: Obtaining CTCs in unselected patients presenting with BR has a relatively low yield. However, obtaining a positive CTC raises the suspicion of the presence of metastatic disease and may have utility for longitudinal follow-ups of patients with BR., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Patient navigation--the provision of logistical, educational, and emotional support needed to help patients "navigate around" barriers to high-quality cancer treatment offers promise. No patient-reported outcome measures currently exist that assess patient navigation from the patient's perspective. We use a partial independence item response theory model to report on the psychometric properties of the Patient Satisfaction with Navigation, Logistical measure developed for this purpose., Methods: We used data from an ethnically diverse sample (n = 1873) from the National Cancer Institute Patient Navigation Research Program. We included individuals with the presence of an abnormal breast, cervical, colorectal, or prostate cancer finding., Results: The partial independence item response theory model fit well. Results indicated that scores derived from responses provide extremely precise and reliable measurement between -2.5 SD below and 2 SD above the mean and acceptably precise and reliable measurement across nearly the entire range., Conclusions: Our findings provide evidence in support of the Patient Satisfaction with Navigation, Logistical. Scale users should utilize 1 of the 2 described methods to create scores.

Background: The Patient Navigation Research Program (PNRP) is a cooperative effort of nine research projects, with similar clinical criteria but with different study designs. To evaluate projects such as PNRP, it is desirable to perform a pooled analysis to increase power relative to the individual projects. There is no agreed-upon prospective methodology, however, for analyzing combined data arising from different study designs. Expert opinions were thus solicited from the members of the PNRP Design and Analysis Committee., Purpose: To review possible methodologies for analyzing combined data arising from heterogeneous study designs., Methods: The Design and Analysis Committee critically reviewed the pros and cons of five potential methods for analyzing combined PNRP project data. The conclusions were based on simple consensus. The five approaches reviewed included the following: (1) analyzing and reporting each project separately, (2) combining data from all projects and performing an individual-level analysis, (3) pooling data from projects having similar study designs, (4) analyzing pooled data using a prospective meta-analytic technique, and (5) analyzing pooled data utilizing a novel simulated group-randomized design., Results: Methodologies varied in their ability to incorporate data from all PNRP projects, to appropriately account for differing study designs, and to accommodate differing project sample sizes., Limitations: The conclusions reached were based on expert opinion and not derived from actual analyses performed., Conclusions: The ability to analyze pooled data arising from differing study designs may provide pertinent information to inform programmatic, budgetary, and policy perspectives. Multisite community-based research may not lend itself well to the more stringent explanatory and pragmatic standards of a randomized controlled trial design. Given our growing interest in community-based population research, the challenges inherent in the analysis of heterogeneous study design are likely to become more salient. Discussion of the analytic issues faced by the PNRP and the methodological approaches we considered may be of value to other prospective community-based research programs.

Background & Aims: The μ opioid receptor (μOR) undergoes rapid endocytosis after acute stimulation with opioids and most opiates, but not with morphine. We investigated whether prolonged activation of μOR affects morphine's ability to induce receptor endocytosis in enteric neurons., Methods: We compared the effects of morphine, a poor μOR-internalizing opiate, and (D-Ala2,MePhe4,Gly-ol5) enkephalin (DAMGO), a potent μOR-internalizing agonist, on μOR trafficking in enteric neurons and on the expression of dynamin and β-arrestin immunoreactivity in the ileum of guinea pigs rendered tolerant by chronic administration of morphine., Results: Morphine (100 μmol/L) strongly induced endocytosis of μOR in tolerant but not naive neurons (55.7% ± 9.3% vs 24.2% ± 7.3%; P < .001) whereas DAMGO (10 μmol/L) strongly induced internalization of μOR in neurons from tolerant and naive animals (63.6% ± 8.4% and 66.5% ± 3.6%). Morphine- or DAMGO-induced μOR endocytosis resulted from direct interactions between the ligand and the μOR because endocytosis was not affected by tetrodotoxin, a blocker of endogenous neurotransmitter release. Ligand-induced μOR internalization was inhibited by pretreatment with the dynamin inhibitor, dynasore. Chronic morphine administration resulted in a significant increase and translocation of dynamin immunoreactivity from the intracellular pool to the plasma membrane, but did not affect β-arrestin immunoreactivity., Conclusions: Chronic activation of μORs increases the ability of morphine to induce μOR endocytosis in enteric neurons, which depends on the level and cellular localization of dynamin, a regulatory protein that has an important role in receptor-mediated signal transduction in cells., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objectives: The Sexual Health Inventory for Men (SHIM) is a widely used scale for the screening and diagnosis of erectile dysfunction (ED). Our objective was to incorporate the SHIM into our prostate cancer screening program to estimate the prevalence of ED among men screened for prostate cancer., Methods: During September 2006, men younger than 75 years of age living in the Washington, DC area were invited to participate in the George Washington University Prostate Cancer Screening Program. The SHIM questionnaire was administered to all participants. Information regarding primary care physician use, phosphodiesterase-5 inhibitor use, serum prostate-specific antigen levels, and digital rectal examination findings was also obtained. Those who registered SHIM scores of 17 or less or who were taking a phosphodiesterase-5 inhibitor were considered to have ED., Results: Overall, 333 men attended the program. Of the 328 men, 123 (37.5%) met our definition of ED; 30 (9%) were using a phosphodiesterase-5 inhibitor and 93 (28%) had an SHIM score of 17 or less. Univariate analysis suggested a significant difference in the prevalence of ED between African-American men and non-African-American men, with 25% and 41%, respectively, found to have a SHIM score of 17 or less (P < .01); however, this difference was not significant once we controlled for age (P > .05). Among our participants, 33% lacked a primary care physician. Of these, 22% had a SHIM score of 17 or less., Conclusions: The results of our study have shown that ED increases in a nonlinear fashion with age, consistent with the findings of previous reports. Of greater concern, however, given the strong association between ED and cardiovascular disease, was the number of those with ED who lacked a primary care physician.

Background & Aims: The neurokinin 1 receptors (NK(1)Rs) and substance P (SP) have been implicated in the stress and/or pain pathways involved in chronic pain conditions. Here we examined the participation of NK(1)Rs in sustained visceral hyperalgesia observed in rats exposed to chronic psychological stress., Methods: Male Wistar rats were exposed to daily 1-hour water avoidance stress (WA) or sham WA for 10 consecutive days. We tested intraperitoneal or intrathecal injection of the NK(1)R antagonist SR140333 on the visceromotor reflex to colorectal distention in both groups at day 11. Real-time reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry were used to assess the expression of NK(1)Rs and/or SP in samples of colon, spinal cord, and dorsal root ganglia., Results: Both intraperitoneal and intrathecal SR140333 injection diminished the enhanced visceromotor reflex to colorectal distention at day 11 in stressed rats but did not affect the response in control animals. Real-time polymerase chain reaction and Western blotting demonstrated stress-induced up-regulation of spinal NK(1)Rs. Immunohistochemistry showed an increased number of NK(1)R-expressing neurons in the laminae I of the dorsal horn in stressed rats. The expression of NK(1)Rs was decreased in colon from stressed rats compared with control. The expression of SP gene precursor in dorsal root ganglia was unchanged in stressed rats compared with controls., Conclusions: Stress-induced increased NK(1)R expression on spinal neurons and the inhibitory effect of intrathecal NK(1)R antagonist on visceral hyperalgesia support the key contribution of spinal NK(1)Rs in the molecular pathways involved in the maintenance of visceral hyperalgesia observed after chronic WA.

Background & Aims: We tested the hypothesis that N-methyl-D-aspartate (NMDA) receptors mediate surgery-induced opioid release in enteric neurons., Methods: We used mu opioid receptor (muOR) internalization as a measure of opioid release with immunohistochemistry and confocal microscopy. MuOR internalization was quantified in enteric neurons from nondenervated and denervated ileal segments of guinea pig after abdominal laparotomy with and without pretreatment with NMDA-receptor antagonists acting at different recognition sites (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,b] cyclohepten-5,10-imine (MK-801) or (D) 2-amino-5-phosphopenoic acid (AP-5) at .5, 1 mg/kg; 8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazinol [4,5-]quinoline-5-oxide choline (MRZ 2/576) or 8-chloro-1,4-dioxo-1,2,3,4-tetrahydropyridazinol [4,5-]quinoline choline salt (MRZ 2/596) at .3, 1 mg/kg, or with an antagonist for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (1, 3 mg/kg). To determine whether NMDA stimulation induces opioid release, (1) ilea were exposed to NMDA (100 micromol/L) and D-serine (10 micromol/L) with or without the antagonist MK-801 or AP-5 (50 micromol/L); and (2) neuromuscular preparations of the ileum were stimulated electrically (20 Hz, 20 min) with or without MK-801 or AP-5 (50 micromol/L)., Results: MuOR endocytosis induced by abdominal laparotomy was inhibited significantly by NMDA-receptor antagonists in nondenervated and denervated ileal segments, but not by the AMPA-receptor antagonist. MuOR endocytosis in neurons exposed to NMDA or electrical stimulation was prevented by NMDA-R antagonists., Conclusions: Abdominal laparotomy evokes local release of glutamate that results in endogenous opioid release through the activation of peripheral NMDA receptors. This suggests an interaction between the glutamatergic and opioid systems in response to the noxious and perhaps mechanosensory stimulation of surgery.

Immunohistochemistry and confocal microscopy were used to investigate mu opioid receptor (muOR) internalization in enteric neurons of the guinea-pig ileum following abdominal surgery. The following surgical procedures were performed under halothane or isofluorane anesthesia: a) midline abdominal skin incision, b) laparotomy or c) laparotomy with intestinal manipulation. Gastrointestinal transit was evaluated by using a non-absorbable marker and measuring fecal pellet output. In neurons from normal and control (anesthesia alone) animals, muOR was predominantly at the cell surface. muOR endocytosis following skin incision was not significantly different from controls (21.2+/-3.5% vs. 13.7+/-2.1%, mean+/-S.E.M.), whereas it was significantly increased by laparotomy (46.5+/-6.1%; P<0.01 vs. controls) or laparotomy plus intestinal manipulation (40.5+/-6.1%; P<0.01 vs. controls) 30 min following surgery compared with controls. muOR endocytosis remained elevated at 4 h (38.6+/-1.2%; P<0.01 vs. controls), whereas it was similar to controls at 6 and 12 h (17.5+/-5.8% and 11.2+/-3.0%). muOR endocytosis occurred in cholinergic and nitrergic neurons. Gastrointestinal transit was significantly delayed by laparotomy or laparotomy plus intestinal manipulation (12.8+/-1.2 and 13.8+/-0.6 h vs. 7.0+/-0.5 in controls; P<0.01), but was not significantly changed by skin incision (8.2+/-0.6 h). The findings of the present study support the concept that the noxious stimulation caused by abdominal surgery induces release of endogenous opioids thus resulting in muOR endocytosis in neurochemically distinct enteric neurons. muOR internalization can serve as indirect evidence of opioid release and as a means to visualize neuronal pathways activated by opioids.

The mu-opioid receptor (muOR), which mediates many of the opioid effects in the nervous system, is expressed by enteric neurons. The aims of this study were to determine whether 1) different classes of myenteric neurons in the guinea pig ileum contain muOR immunoreactivity by using double- and triple-labeling immunofluorescence and confocal microscopy, 2) muOR immunoreactivity is localized to enteric neurons immunoreactive for the endogenous opioid enkephalin, and 3) muOR immunoreactivity is localized to interstitial cells of Cajal visualized by c-kit. In the myenteric plexus, 50% of muOR-immunoreactive neurons contained choline acetyltransferase (ChAT) immunoreactivity, whereas about 43% of ChAT-immunoreactive neurons were muOR immunoreactive. Approximately 46% of muOR myenteric neurons were immunoreactive for vasoactive intestinal polypeptide (VIP), and about 31% were immunoreactive for nitric oxide synthase (NOS). MuOR immunoreactivity was found in about 68% of VIP-containing neurons and 60% of NOS-immunoreactive neurons. Triple labeling showed that about 32% of muOR neurons contained VIP and ChAT immunoreactivities. The endogenous opioid enkephalin (ENK) was observed in about 30% of muOR neurons; conversely, 48% of ENK neurons contained muOR immunoreactivity. MuOR was not detected in neurons containing calbindin, nor in interstitial cells of Cajal. MuOR-immunoreactive fibers formed a dense network around interstitial cells of Cajal in the deep muscular plexus. This study demonstrates that muOR is expressed by neurochemically distinct classes of myenteric neurons that are likely to differ functionally, is colocalized with the endogenous opioid ENK, and is not expressed by interstitial cells of Cajal., (Copyright 2003 Wiley-Liss, Inc.)

Immunohistochemistry and confocal microscopy were used to investigate endocytosis and recycling of the native mu opioid receptor (muOR) in enteric neurons. Isolated segments of the guinea-pig ileum were exposed to increasing concentrations of muOR agonists at 4 degrees C to allow ligand binding and warming to 37 degrees C for 0 min (baseline) to 6 h in ligand-free medium to allow receptor internalization and recycling. The endogenous ligand, [Met]enkephalin, and [D-Ala(2),MePhe(4),Gly-ol(5)] enkephalin (DAMGO), an opioid analog, and the alkaloids, etorphine and fentanyl, induced rapid internalization of muOR immunoreactivity in enteric neurons, whereas morphine did not. muOR internalization was prevented by muOR antagonists. Basal levels of muOR immunoreactivity in the cytoplasm were 10.52+/-2.05%. DAMGO (1 nM-100 microM) induced a concentration-dependent increase of muOR immunofluorescence density in the cytoplasm to a maximum of 84.37+/-2.26%. Translocation of muOR immunoreactivity in the cytoplasm was detected at 2 min, reached the maximum at 15-30 min, remained at similar levels for 2 h, began decreasing at 4 h, and was at baseline values at 6 h. A second exposure to DAMGO (100 nM) following recovery of internalized muOR immunoreactivity at the cell surface induced a translocation of muOR immunoreactivity in the cytoplasm comparable to the one observed following the first exposure (46.89+/-3.11% versus 43.31+/-3.80%). muOR internalization was prevented by hyperosmolar sucrose, phenylarsine oxide or potassium depletion, which inhibit clathrin-mediated endocytosis. muOR recycling was prevented by pre-treatment with bafilomycin A1, an acidotropic agent that inhibits endosomal acidification, but not by the protein synthesis inhibitor, cycloheximide. This study shows that native muOR in enteric neurons undergoes ligand-selective endocytosis, which is primarily clathrin-mediated, and recycles following endosomal acidification. Following recycling, muOR is activated and internalized by DAMGO indicating that recycled receptors are functional.

The cellular responses to carcinogen exposure influence cellular fate, which in turn modulates the neoplastic response. Certain hexavalent chromium [Cr(VI)] compounds are implicated as occupational respiratory carcinogens at doses that are both genotoxic and cytotoxic. We examined the mechanism of Cr(VI)-induced apoptosis in normal human fibroblasts (BJ) immortalized by human telomerase gene transfection (BJ-hTERT), and we assessed the spectrum of cumulative cellular fates [(a) regaining of replicative potential; (b) terminal growth arrest; or (c) apoptosis] for a narrow range of increasingly genotoxic doses of Cr(VI). Exposure of BJ-hTERT cells to Cr(VI) resulted in a dose-dependent increase in apoptosis that involved mitochondrial disruption as evidenced by mitochondrial membrane depolarization and cytochrome c release. The initial response to Cr(VI) exposure was inhibition of cell cycle progression. At the lowest dose tested (1 microM; 32% clonogenic survival), the cell cycle inhibition led to terminal growth arrest but no apoptosis. The fraction of terminally growth arrested cells increased as the dose was increased to 3 microM but then decreased at 4, 5, and 6 microM as apoptosis became the predominant cell fate. Our results suggest that cell populations exposed to Cr(VI) have a different spectrum of responses, depending on the extent of DNA damage, and that the regaining of replicative potential after relatively higher genotoxic exposures may be attributable to either escape from, or resistance to, terminal growth arrest or apoptosis.

Physiological stress conditions associated with the tumor microenvironment play a role in resistance to anticancer therapy. In this study, treatment of EMT6 mouse mammary tumor cells with hypoxia or the chemical stress agents brefeldin A (BFA) or okadaic acid (OA) causes the development of resistance to the topoisomerase II inhibitor etoposide. The mechanism of physiological stress-induced drug resistance may involve the activation of stress-responsive proteins and transcription factors. Our previous work shows that treatment with BFA or OA causes activation of the nuclear transcription factor NF-kappa B. Pretreatment with the proteasome inhibitor carbobenzyoxyl-leucinyl-leucinyl-leucinal inhibits stress-induced NF-kappa B activation and reverses BFA-induced drug resistance. To test whether NF-kappa B specifically mediates stress-induced drug resistance, an inducible phosphorylation site-deficient mutant of I kappa B alpha (I kappa B alpha M, S32/36A) was introduced into EMT6 cells. In this study, we show that I kappa B alpha M expression inhibits stress-induced NF-kappa B activation and prevents BFA-, hypoxia-, and OA-induced resistance to etoposide. These results indicate that NF-kappa B activation mediates both chemical and physiological drug resistance to etoposide. Furthermore, they imply that coadministration of agents that inhibit NF-kappa B may enhance the efficacy of topoisomerase II inhibitors in clinical cancer chemotherapy.

Hexavalent chromium (Cr (VI)) is reduced intracellularly to Cr (V), Cr (IV) and Cr (III) by ascorbate (Asc), cysteine and glutathione (GSH). These metabolites induce a spectrum of genomic DNA damage resulting in the inhibition of DNA replication. Our previous studies have shown that treatment of DNA with Cr (III) or Cr (VI) plus Asc results in the formation of DNA-Cr-DNA crosslinks (Cr-DDC) and guanine-specific arrests of both prokaryotic and mammalian DNA polymerases. GSH not only acts as a reductant of Cr (VI) but also becomes crosslinked to DNA by Cr, thus, the focus of the present study was to examine the role of GSH in Cr-induced DNA damage and polymerase arrests. Co-incubation of Cr (III) with plasmid DNA in the presence of GSH led to the crosslinking of GSH to DNA. GSH co-treatment with Cr (III) also led to a decrease in the degree of Cr-induced DNA interstrand crosslinks relative to Cr (III) alone, without affecting total Cr DNA binding. DNA polymerase arrests were observed following treatment of DNA with Cr (III) alone, but were markedly reduced when GSH was added to the reaction mixture. Pre-formed polymerase-arresting lesions (Cr-DDC) were not removed by subsequent addition of GSH. Treatment of DNA with Cr (VI), in the presence of GSH, resulted in crosslinking of GSH to DNA, but failed to produce detectable DNA interstrand crosslinks or polymerase arrests. The inhibitory effect of GSH on Cr-induced polymerase arrest was further confirmed in human genomic DNA using quantitative PCR (QPCR) analysis. Treatment of genomic DNA with Cr (III) resulted in a marked inhibition of the amplification of a 1.6 kb target fragment of the p53 gene by Taq polymerase. This was almost completely prevented by co-treatment with GSH and Cr (III). These results indicate that Cr-induced DNA interstrand crosslinks, and not DNA-Cr-GSH crosslinks, are the principal lesions responsible for blocking DNA replication. Moreover, the formation of DNA-Cr-GSH crosslinks may actually preclude the formation of the polymerase arresting lesions.

Eicosanoids modulate the interaction of tumor cells with various host components in cancer metastasis. Their synthesis involves the release of arachidonic acid (AA) from cellular phospholipids by phospholipase A2 (PLA2), followed by metabolism by cyclooxygenases (COXs) and lipooxygenases (LOXs). This study aimed to identify the pathway(s) of AA metabolism that are required for the invasion of prostate tumor cells. DU-145 and PC-3 human prostate cancer cell lines were used to test the effect of inhibitors of PLA2, COX, or LOX on the invasion of prostate tumor cells through Matrigel in vitro using the Boyden chamber assay and fibroblast-conditioned medium as the chemoattractant. We used nontoxic doses that did not inhibit simple cell motility and did not decrease clonogenic survival. All of the inhibitors caused a significant reduction in AA release from treated cells compared with control cells, which indicated that the treatments were biochemically active. Invasion through Matrigel was inhibited by the PLA2 inhibitor 4-bromophenacyl bromide (4-BPB), the general COX inhibitor ibuprofen (IB), and the highly selective COX-2 inhibitor NS398. Inhibition of cell invasiveness by 4-BPB (1.0 microM), IB (10.0 microM), and NS398 (10.0 microM) was reversed by the addition of prostaglandin E2 (PGE2). PGE2 alone, however, did not stimulate invasiveness, which suggests that its production is necessary for rendering the cells invasive-permissive but not sufficient for inducing invasiveness. In contrast, we found no significant inhibition of invasion of prostate tumor cells treated with esculetin (1.0 microM) or nordihydroguiaretic acid (1.0 microM), which are specific inhibitors of LOX. We also tested the effect of 4-BPB, IB, NS398, and esculetin on the secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), as key enzymes in the proteolysis of Matrigel during invasion, using gelatin zymograms and Western blots. Cells that received 4-BPB, IB, or NS398, but not esculetin showed a significant reduction in the levels of proMMP-2, MMP-9, and proMMP-9 in the culture medium. DU-145 cells did not secrete TIMP-1, and the drugs did not alter the secretion of TIMP-2. This work highlights the role played by COX in disturbing the balance between MMPs and TIMPs in prostate cancer cells, and it points to the potential use of COX inibitors, especially COX-2 selective inhibitors, in the prevention and therapy of prostate cancer invasion.

Some forms of hexavalent chromium [Cr(VI)] are known to cause damage to respiratory-tract tissue and DNA and are thought to be human lung carcinogens. In general, Cr(VI) is mutagenic and carcinogenic at doses that also evoke some cell death, and we previously showed that the predominant mode of death is apoptosis. Because p53 has been shown to initiate apoptosis after genotoxic insults, the objective of these experiments was to determine whether p53 is activated in and necessary for apoptosis of normal diploid human lung fibroblasts (HLF cells) after chromium exposure. By using annexin(V) staining and fluorescent microscopy, we found that Cr(VI) caused up to 14% of HLF cells to undergo apoptosis within 24 h after exposure. In addition, by using western blotting, we found that p53 protein levels increased fourfold to sixfold after exposure to sodium chromate. Because the major function of p53 is as a transcription factor, it must be translocated from the cytoplasm to the nucleus after chromate exposure to be active. Immunofluorescence studies using an antibody against p53 showed that, after chromate exposure, p53 was located in the nucleus of the treated HLF cells. The necessity of p53 for chromium-induced apoptosis was examined in two ways. One approach used dermal fibroblasts from p53 wild-type, heterozygous, and null mice, and the other approach used HLF cells that were transiently transfected with the human papilloma virus E6 gene, which targets p53 for degradation and creates a functional p53-null cell. These studies showed that chromium-induced apoptosis was p53 dependent. Mol. Carcinog. 28:111-118, 2000., (Copyright 2000 Wiley-Liss, Inc.)