Cholecalciferol (Vitamin D<Subscript>3</Subscript>) Inhibits Growth and Invasion by Up-regulating Nuclear Receptors and 25-Hydroxylase (CYP27A1) in Human Prostate Cancer Cells

Epidemiological evidence suggests an inverse relationship between prostate cancer and serum vitamin D levels. We examined the ability of cholecalciferol (vitamin D₃), a calcitriol precursor, to inhibit or reverse cellular changes associated with malignant transformation and invasion and explored its mechanisms of action. The RWPE2-W99 human prostate epithelial cell line, which forms slow-growing tumors in nude mice, was used because it mimics the behavior of the majority of primary human prostate cancers. Cholecalciferol, at physiological levels: (i) inhibited anchorage-dependent and -independent growth; (ii) induced differentiation by decreasing vimentin expression with a concomitant decrease in motility/chemotaxis; (iii) decreased MMP-9 and MMP-2 activity with concomitant decrease in invasion; and (iv) exerted its effects by up-regulating vitamin D receptor (VDR), retinoid-X receptor-α (RXR-α), and androgen receptor (AR) in a dose-dependent manner. Furthermore, we found that RWPE2-W99 prostate cancer cells, similar to RWPE-1 cells (Tokar and Webber. Clin Exp Metast 2005; 22: 265–73), constitutively express the enzyme 25-hydroxylase CYP27A1 which is markedly up-regulated by cholecalciferol. Cholecalciferol has effects similar to those of calcitriol on growth, MMP activity, and VDR. The ability of CYP27A1 to catalyze the conversion of cholecalciferol to 25(OH)D₃ and of 25(OH)D₃ to calcitriol has been reported. RWPE2-W99 cells, similar to RWPE-1 cells, appear to have the rare ability to locally convert cholecalciferol to the active hormone calcitriol. Because it can inhibit cellular changes associated with malignant transformation and invasion, we propose that cholecalciferol may be an effective agent for the treatment of prostate cancer.     

Effects of propylene oxide exposure on rat nasal respiratory cell proliferation.

Long-term exposure of rodents to propylene oxide (PO) induced inflammation, respiratory cell hyperplasia, and nasal tumors at concentrations >/= 300 ppm, suggesting a possible role for cytotoxicity and compensatory cell proliferation in PO carcinogenesis. In this study, the effects of PO exposure on histopathology and cell proliferation in nasal and hepatic tissues were studied in male F344 rats exposed by inhalation for 3 or 20 days (0, 5, 25, 50, 300, and 500 ppm). Histopathology revealed an increase in mucous cell hyperplasia in the anterior nasal passages after 20 days of exposure (>/=300 ppm). This was associated with the formation of goblet cell nests. Cell proliferation was measured in the respiratory epithelium (NRE; mucociliary and transitional) lining the anterior nasal passages, the nasopharyngeal meatus (NPM), and the liver using BrdU administered with 3-day osmotic pumps. Significant increases in cell proliferation occurred (>3.6-fold) in the mucociliary epithelium lining the anterior nasal cavity at and above 300 ppm for both exposure periods. In the mucociliary epithelium, the 20-day labeling was commonly associated with nests of goblet cells. Significant increases in cell proliferation (>2.3-fold) were observed in the transitional epithelium at 500 ppm after 3 days of exposure and at 300 and 500 ppm after 20 days of exposure. Significant increases in cell proliferation in the NPM (>2.8-fold) were evident at 500 ppm PO after 3 days and at 300 and 500 ppm PO after 20 days of exposure. No exposure-related changes in cell proliferation were observed in the liver. These studies demonstrate a clear concordance between the site and exposure concentration for tumor induction and those causing significant increases in cell proliferation in the rat nose.     

Genotype-phenotype correlation and molecular heterogeneity in pyruvate kinase deficiency

Pyruvate kinase (PK) deficiency is a rare recessive congenital hemolytic anemia caused by mutations in the PKLR gene. This study reports the molecular features of 257 patients enrolled in the PKD Natural History Study. Of the 127 different pathogenic variants detected, 84 were missense and 43 non-missense, including 20 stop-gain, 11 affecting splicing, five large deletions, four in-frame indels, and three promoter variants. Within the 177 unrelated patients, 35 were homozygous and 142 compound heterozygous (77 for two missense, 48 for one missense and one non-missense, and 17 for two non-missense variants); the two most frequent mutations were p.R510Q in 23% and p.R486W in 9% of mutated alleles. Fifty-five (21%) patients were found to have at least one previously unreported variant with 45 newly described mutations. Patients with two non-missense mutations had lower hemoglobin levels, higher numbers of lifetime transfusions, and higher rates of complications including iron overload, extramedullary hematopoiesis, and pulmonary hypertension. Rare severe complications, including lower extremity ulcerations and hepatic failure, were seen more frequently in patients with non-missense mutations or with missense mutations characterized by severe protein instability. The PKLR genotype did not correlate with the frequency of complications in utero or in the newborn period. With ICCs ranging from 0.4 to 0.61, about the same degree of clinical similarity exists within siblings as it does between siblings, in terms of hemoglobin, total bilirubin, splenectomy status, and cholecystectomy status. Pregnancy outcomes were similar across genotypes in PK deficient women. This report confirms the wide genetic heterogeneity of PK deficiency.     

Interferon‐γ‐ and glucocorticoid‐mediated pathways synergize to enhance death of CD4+ CD8+ thymocytes during Salmonella enterica serovar Typhimurium infection

Thymic atrophy is known to occur during infections; however, there is limited understanding of its causes and of the cross-talk between different pathways. This study investigates mechanisms involved in thymic atrophy during a model of oral infection by Salmonella enterica serovar Typhimurium (S. typhimurium). Significant death of CD4⁺ CD8⁺ thymocytes, but not of single-positive thymocytes or peripheral lymphocytes, is observed at later stages during infection with live, but not heat-killed, bacteria. The death of CD4⁺ CD8⁺ thymocytes is Fas-independent as shown by infection studies with lpr mice. However, apoptosis occurs with lowering of mitochondrial potential and higher caspase-3 activity. The amounts of cortisol, a glucocorticoid, and interferon-γ (IFN-γ), an inflammatory cytokine, increase upon infection. To investigate the functional roles of these molecules, studies were performed using Ifnγ^−/− mice together with RU486, a glucocorticoid receptor antagonist. Treatment of C57BL/6 mice with RU486 does not affect colony-forming units (CFU), amounts of IFN-γ and mouse survival; however, there is partial rescue in thymocyte death. Upon infection, Ifnγ^−/− mice display higher CFU and lower survival but more surviving thymocytes are recovered. However, there is no difference in cortisol amounts in C57BL/6 and Ifnγ^−/− mice. Importantly, the number of CD4⁺ CD8⁺ thymocytes is significantly higher in Ifnγ^−/− mice treated with RU486 along with lower caspase-3 activity and mitochondrial damage. Hence, endogenous glucocorticoid and IFN-γ-mediated pathways are parallel but synergize in an additive manner to induce death of CD4⁺ CD8⁺ thymocytes during S. typhimurium infection. The implications of this study for host responses during infection are discussed.     

Predictors of mortality in children due to severe and very severe pneumonia

Background:

Mortality due to pneumonia in children is more than any other illness. Limited data is available to predict mortality in children with pneumonia from central India.

Aim:

To study predictors of mortality in children aged 1-59 months hospitalised with severe and very severe pneumonia.

Materials and Methods:

Present study was observational longitudinal study that was done in a tertiary care hospital of central India. Two hundred and ninety children, aged 1-59 months, presented with severe and very severe pneumonia were enrolled in this study. Outcome and predictors of mortality were studied. Data was analysed with Chi-square test, univariate and multivariate regression analysis.

Results:

Out of 270 enrolled study subjects, maximum (108, 37.24%) were belonged to 1-6-months age group. Proportion of mortality was maximum (16, 64.00%) in that age group. Overall case fatality rate was 8.62%. Among significant variables, delayed hospital referral [adjusted odds ratio (OR)-52.09, 95% confidence interval (CI)- 6.74-402.39], incomplete immunisation (OR-12.28, 95% CI-2.15-69.93), severe malnutrition (Z score < −3) (OR-15.51, 95% CI- 2.04-117.83), refusal to feed (OR- 30.57, 95% CI- 2.47-378.26), and hypoglycaemia (OR- 6.98, 95% CI- 1.05-46.30) were found significant independently on multivariate regression analysis. Conclusion: Delayed hospital referral, incomplete immunisation, severe malnutrition, refusal to feed, and hypoglycaemia were independent predictors of mortality in children with severe and very severe pneumonia.     

Genetic modifiers of EGFR dependence in non-small cell lung cancer

Lung adenocarcinomas harboring activating mutations in the epidermal growth factor receptor (EGFR) represent a common molecular subset of non-small cell lung cancer (NSCLC) cases. EGFR mutations predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and thus represent a dependency in NSCLCs harboring these alterations, but the genetic basis of EGFR dependence is not fully understood. Here, we applied an unbiased, ORF-based screen to identify genetic modifiers of EGFR dependence in EGFR-mutant NSCLC cells. This approach identified 18 kinase and kinase-related genes whose overexpression can substitute for EGFR in EGFR-dependent PC9 cells, and these genes include seven of nine Src family kinase genes, FGFR1, FGFR2, ITK, NTRK1, NTRK2, MOS, MST1R, and RAF1. A subset of these genes can complement loss of EGFR activity across multiple EGFR-dependent models. Unbiased gene-expression profiling of cells overexpressing EGFR bypass genes, together with targeted validation studies, reveals EGFR-independent activation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT pathways. Combined inhibition of PI3K-mTOR and MEK restores EGFR dependence in cells expressing each of the 18 EGFR bypass genes. Together, these data uncover a broad spectrum of kinases capable of overcoming dependence on EGFR and underscore their convergence on the PI3K-AKT and MEK-ERK signaling axes in sustaining EGFR-independent survival.The term “oncogene addiction” has been used to describe the phenomenon whereby tumor cells exhibit singular reliance on an oncogene or oncogenic pathway for their survival, despite the accumulation of multiple genetic lesions (1). In non-small cell lung cancer (NSCLC), this principle is perhaps best exemplified with the finding that epidermal growth factor receptor (EGFR) mutations predict response to EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, and thus represent a dependency in the subset of tumors harboring these alterations (2–6). However, though EGFR-mutant NSCLCs typically respond dramatically to EGFR TKIs, clinical responses are not universal, even within this genetically defined cohort, with the rate of objective response estimated to be ∼71% (5, 6). Furthermore, the overwhelming majority of patients who initially respond to EGFR inhibitors ultimately develop resistance to therapy (7). A deeper understanding of the genetic underpinnings of EGFR addiction, and how EGFR-mutant cells can overcome reliance on EGFR, may improve clinical outcomes.Here, we have applied an unbiased screening approach to identify genetic modifiers of EGFR dependence in NSCLC. Mounting evidence supports the existence of several genetic modifiers of EGFR dependence in EGFR-mutant NSCLC, which can reduce the degree to which these tumors rely on EGFR and thereby contribute to EGFR TKI resistance (8). Examples include amplification of the MET receptor tyrosine kinase (RTK) (9), activation of the NF-κB pathway (8), amplification of the HER2 (ERBB2) RTK (10), amplification of the CRKL gene (11), and activation of the AXL kinase (12). Notably, MET bypass can be reciprocally achieved via EGFR activation in MET-dependent cells (13), and analogous examples of reciprocal kinase switching have been reported in other kinase-driven cancer models (14, 15). These and other findings suggest that compensatory kinase switching may be a more general way in which oncogene-dependent cancers overcome reliance on their primary driver kinase (14, 16), but the full-range of kinases capable of mediating EGFR bypass has not been systematically studied.Recent advances in large-scale functional genetic libraries have made it possible to query a wide range of genetic perturbations for their ability to modulate specific cellular phenotypes in mammalian systems (17, 18). Using the model of EGFR-mutant, erlotinib-sensitive NSCLC cells, we have performed a systematic ORF-based screen to identify kinase and kinase-related genes whose overexpression can complement loss of EGFR activity in an EGFR-dependent context. Our findings indicate broad potential for EGFR substitution in the setting of EGFR dependence, with compensatory mechanisms commonly conferring EGFR-independent activation of the PI3K-AKT and MEK-ERK signaling pathways. Importantly, this approach has recovered known mechanisms of erlotinib resistance as well as identified novel mediators of EGFR bypass in EGFR-mutant NSCLC. These data support the idea that the EGFR-dependent state can be redundantly driven by diverse genetic inputs that commonly converge on shared downstream signaling nodes.     

Solid pseudopapillary tumor of the pancreas: ‘Experiences’ and ‘Lessons’ at a tertiary‐care oncology center

Solid pseudopapillary tumor of the pancreas (SPT) is a rare and fascinating entity of elusive histogenesis and unpredictable biology. It has a peculiar proclivity to afflict young females and involve the pancreatic body‐tail region. Cytology diagnosis of these rare neoplasms remains a challenge. We analyzed the cytology features of all SPT cases diagnosed on fine needle aspiration cytology (FNAC) from 2003 to 2009 along with their histopathology slides. Nineteen consecutive cases were diagnosed as SPT on FNAC. Fifteen out of nineteen cases were confirmed as true SPT on histopathology. Amongst the true SPT, all except one occurred in females. Age ranged from 14 to 50 years. Pseudopapillae bearing stout branches terminating in bulbous tips and enclosing transgressing vessels, separated from a collar of tumor cells by a clear zone of myxohyaline coat were pathognomonic of SPT. Singly dispersed monomorphic tumor cells with bland chromatin formed the second diagnostic component of SPT. Nuclear grooves and hyaline globules were in addition helpful in segregating SPT from its close differentials. In four cases diagnosed as SPT on FNAC, histopathology revealed a different final diagnosis (one case each of paraganglioma, extragastrointestinal stromal tumor, metastatic papillary renal cell carcinoma and inflammatory myofibroblastic tumor). Conversely, one case of SPT had been erroneously diagnosed as neuroendocrine tumor on FNAC. Six cases (40%) developed metastasis; commonest site being liver. In conclusion, cytology in conjunction with clinico‐radiologic findings plays a key role in making a correct diagnosis. Awareness of unique cytomorphological features is important in distinguishing this tumor from its diverse mimics. Diagn. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.