Chloroquine-Mediated Lysosomal Dysfunction Enhances the Anticancer Effect of Nutrient Deprivation

Purpose

To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation.

Methods

Serum-deprived U251 glioma, B16 melanoma and L929 fibrosarcoma cells were treated with chloroquine in vitro. Cell viability was measured by crystal violet and MTT assay. Oxidative stress, apoptosis/necrosis and intracellular acidification were analyzed by flow cytometry. Cell morphology was examined by light and electron microscopy. Activation of AMP-activated protein kinase (AMPK) and autophagy were monitored by immunoblotting. RNA interference was used for AMPK and LC3b knockdown. The anticancer efficiency of intraperitoneal chloroquine in calorie-restricted mice was assessed using a B16 mouse melanoma model.

Results

Chloroquine rapidly killed serum-starved cancer cells in vitro. This effect was not mimicked by autophagy inhibitors or LC3b shRNA, indicating autophagy-independent mechanism. Chloroquine-induced lysosomal accumulation and oxidative stress, leading to mitochondrial depolarization, caspase activation and mixed apoptotic/necrotic cell death, were prevented by lysosomal acidification inhibitor bafilomycin. AMPK downregulation participated in chloroquine action, as AMPK activation reduced, and AMPK shRNA mimicked chloroquine toxicity. Chloroquine inhibited melanoma growth in calorie-restricted mice, causing lysosomal accumulation, mitochondrial disintegration and selective necrosis of tumor cells.

Conclusion

Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy.     

Early postnatal castration affects thymic and thymocyte noradrenaline levels and beta-adrenoceptor-mediated influence on the thymopoiesis in adult rats

The interactions among the nervous, endocrine and immune system were studied by examining: i) thymic and thymocyte catecholamine levels in adult rats castrated (Cx) at postnatal day 3 and ii) effects of 14-day-long propranolol (P) treatment on main thymocyte differentiational molecule expression in adult non-Cx and Cx rat. The results demonstrated that castration in early postnatal period lowers levels of both neurally- and thymocyte-derived noradrenaline in adult rats, and thereby diminishes beta-adrenoceptor-mediated fine tuning of the T-cell differentiation/maturation. In non-Cx rats P affected TCRalphabeta-dependent stages of thymocyte differentiation/maturation decreasing frequency of CD4+8+ double positive (DP) TCRalphabeta(low) cells entering selection processes and increasing relative number of positively selected DP TCRalphabeta(high) (most likely due to an increased thymocyte surface density of Thy-1 that is involved in negative control of TCRalphabeta-mediated signaling/selection thresholds) and the most mature CD4+8- TCRalphabeta(high) cells (including CD4+25+ regulatory cells). However, in Cx rats P failed to produce any significant changes in thymocyte subset composition.     

Significance of Helicobacter pylori infection and nonsteroidal anti-inflammatory drugs in patients with bleeding from upper part of the gastrointestinal tract

BACKGROUND/AIM: Helicobacter pylori (H. pylori) infection and nonsteroidal anti-inflammatory drugs (NSAIDs) use are considered to be the most important risk factors having influence on the onset of bleeding gastroduodenal lesions. Whether there is an interaction between H. pylori infection and the use of NSAIDs in the development of peptic ulcer disease is still controversial. The aim of the present study was to evaluate the prevalence of NSAIDs use and H. pylori infection in patients presented with bleeding gastroduodenal lesions. METHODS: During the period from January 2003 December 2003 we prospectively obtained data of all the patients (n=106) presented with signs of upper gastrointestinal bleeding. All the patients were admitted to the intensive care unit, with the endoscopy performed within 12 hours after admission. Histologic analysis was used for the detection of H. pylori infection. The NSAIDs and aspirin use data were obtained by anamnesis. RESULTS: The results of our study revealed that the most common sources of upper gastrointestinal bleeding were duodenal (57 patients, 53.77%) and ventricular (36 patients, 33.96%) ulcers. The majority of the examined cases were associated with both H. pylori infection and NSAIDs use. A statistically significant difference among the studied groups of patients was proven. CONCLUSION: The majority of bleeding gastroduodenal lesions were associated with the coexistance of H. pylori infection and NSAIDs use, while their independent influences were statistically less important. Eradication of H. pylori infection in patients using NSAIDs might prevent upper gastrointestinal hemorrhage and reduce peptic ulcer bleeding risk.     

Hemodialysis-related headaches

BACKGROUND/AIM: Hemodialysis (HD) is a therapeutic procedure used to partially correct homeostatic disorders and prevent complications of uremia to appear in the terminal stage of renal insufficiency. The aim of this study was to evaluate and analyze the incidence and features of haedaches in patients undergoing hemodialysis. METHODS: A total of 143 patients, 50 women and 93 men, undergoing hemodialysis, were questionned about their problems with headache using a questionnary designend according to the diagnostic criteria of the Intemational Headache Classification of Headache Disorders from 2004. The patients were separated into two groups: the patients without headache and the patients with repeated headaches. Afterwards, the patients with headaches were further divided into subgroups: the patients who had the headaches before the beginning of HD and patients who experienced repeated headaches with the beginning of HD headache (HDH). In the group of patients with headaches we analyzed characteristics of headache according to which diagnoses of headaches were made, as well as the effects of HD on headaches. We also analyzed features of HDH. The patients with headache were compared to the patients without haedache regarding sex, age, duration of HD, causes of end-stage renal disease, arterial diastolic and systolic blood pressure and serum concentration of hemoglobin, urea nitrogen, creatinine, sodium and potassium. The results were statistically compared. RESULTS: In the group of 143 patients examined, 27 (18.9%) patients had headaches. There were no statistically significant differences between the group of patients with headaches and those without headache regarding to sex, age, duration of HD, causes of end-stage renal disease, serum concentration of hemoglobin, urea nitrogen, creatinine, sodium and potassium. The patients with headaches showed significantlly higher mean values of systolic blood pressure during HD in comparison to the patients without headaches (p = 0.029). There was no statistically significant difference between the two groups regarding the mean values of diastolic blood pressure. Nineteen (13.3%) patients had had headache before starting HD. HD did not have any effect on the characteristics of headaches in more than a half of these patients. In 8 (5.6%) patients we diagnozed HDH using the diagnostic criteria of the International Headache Classification of Headache Disorders from 2004. HDH showed similar caracteristics in all the patients: it appeared mostly in men, during the 4th hour of HD, lasted less than four hours, it was localized bilaterally in the frontal parts of the head, strong in intensity, throbbing and without the associated symptoms. CONCLUSION: The results of our study clearly showed that HDH was a particular entity of headache, not only because of its connenction with HD, but because it had similar characteristics in all the patients in wich it had appeared. Finding out the pathophysiological mechanisms of their occurrence would significantly improve the quality of life style of patients on hemodialysis.     

Expression of nuclear Ki-67 antigen in prostatic high grade intraepithelial neoplasia and prostatic carcinoma

BACKGROUND/AIM: Prostatic intraepithelial high grade neoplasia (PINHG) is accepted as preneoplastic lesion in prostatic carcinoma. One of the fundamental events in early oncogenesis is the disruption of proliferative activity. One of the numerous regulatory proteins is Ki-67 expressed in all proliferating cells. Index Ki-67 is considered to have prognostic significance. The aim of the study was to compare the level of proliferation in hyperplastic epithelium, prostatic carcinoma (Gleason score > 6) and PINHG. METHODS: Micromorphological examination was done in 85 patients. Pathohistological analysis was performed on standard histologic specimens with the estimation of Gleason score and the presence of PINHG in its surroundings. Nuclear proliferative activity was analyzed immunohistochemically in 19 cases, using a monoclonal anti-Ki-67 antibody. RESULTS: PINHG was found in prostatic carcinoma surrounding in 30% of the patients. In hyperplastic epithelia Ki-67 proliferative activity was 1,08, in PINHG 2,25 (p < 0,05), while in prostatic cancer, Ki-67 index was 17,64. Proliferative activity in prostatic carcinoma was significantly higher than in PINHG (p < 0,001) and hyperplasia (p < 0,001). CONCLUSION: This study confirmed that high grade PIN lesion predominately appears in the surrounding of poor or moderately differentiated prostate carcinoma, and that it represents progressive disorder of proliferation in preneoplastic and neoplastic prostatic epithelium.     

Damage of tubule cells in diabetic nephropathy type 2: urinary N-acetyl-beta-D-glucosaminidasis and gamma-glutamil-transferasis

BACKGROUND/AIM: A damage of tubular epithelial cells is followed by the release of cell enzymes and production of proinflammatory compounds, which lead to the tubulointerstitial damage. The aim of this study was to examine the function of renal tubules in the patients with diabetes mellitus type 2 (DM type 2) and the various proteinuria degrees, to establish the damage of the proximal tubule cells caused by DM type 2 by determining urinary N-acetyl-beta-D-glucosaminidasis (beta-NAG) and gamma-glutamil-transferasis (gamma-GT) activity in urine, as well as to compare the obtained results in the examined groups of patients with the values in the healthy examinees. METHODS: A complete examination of renal function and selective enzymuria was performed in 37 patients with DM type 2, and 14 healthy examinees as the controls. The patients were divided in three groups according to the degree of proteinuria. The first group consisted of the patients with diabetes without microalbuminuria; the second one consisted of the patients with proteinuria of < 300 mg/24 h, and microalbuminuria of > 20 mg/24 h, while the third one included the patients with proteinuria of > 300 mg/24 h. RESULTS: In the patients with DM type 2 and the preserved global renal function, fractional excretion of sodium, potassium and phosphates, as well as renal threshold of phosphates concentration, were not sensitive parameters for discovering the damage of the renal tubule function. The determination of beta-NAG activity proved to be the most sensitive parameter for early discovering of tubule cells damages. The difference among the examined groups was statistically highly significant. CONCLUSION: The increased presence of beta-NAG in the urine of DM type 2 patients, pointed out an early tubular disorder and damage of cells, while gamma-GT was a less sensitive indicator of this damage.     

Divergent Squamous Differentiation in Upper Urothelial Carcinoma—Comparative Clinicopathological and Molecular Study

Upper urothelial carcinoma (UUC) has a plasticity to demonstrate divergent differentiation with squamous metaplastic elements. There was no previous study exploring profiling of molecular markers in metaplastic squamous upper urothelial carcinoma (SUUC) and conventional upper urothelial carcinoma (CUUC). The aims of this study was to compare expression of the phenotypic characteristics of tumors and molecular markers (p53, p16, cyclin D1, E-cadherin, HER-2, Ki-67, Bcl-2, Bax) in SUUC and CUUC. SUUC was detected in 20% of 44 patients. There was significant difference between SUUC and CUUC in the pathological stage, grade, growth and presence of lymphovasular invasion (p < 0.05; 0.05; 0.05; 0.01 respectively). The mean Ki-67 and p53 labeling index was significantly higher in SUUC than in CUUC (p < 0.05; 0.05). There was no significant difference in the expression of p16, cyclin D1, E-cadherin, HER-2, Bcl-2 and Bax between SUUC and CUUC. Univariant model showed that SUUC was significantly associated with lymphovascular invasion (p = 0.007), Ki-67 activity (p = 0.016) and growth (p = 0.026). Exploration of UUC with squamous divergent differentiation showed changes in phenotypic characteristics and Ki-67, as well as similar molecular profile with CUUC.