Phytosphingosine induces apoptotic cell death via caspase 8 activation and Bax translocation in human cancer cells.

PURPOSE: Sphingolipid metabolites, such as sphingosine and ceramide, are highly bioactive compounds and are involved in diverse cell processes, including cell-cell interaction, cell proliferation, differentiation, and apoptosis. However, the physiological roles of phytosphingosine are poorly understood. In this study, we report that phytosphingosine can potently induce apoptotic cell death in human cancer cells via caspase activation and caspase-independent cytochrome c release. EXPERIMENTAL DESIGN: Phytosphingosine-induced apoptosis was determined by Hoechst 33258 staining, flow cytometric analysis, and DNA fragmentation assay. Involvement of caspases was determined by immunoblot analysis and cell death detection assays after treatment with synthetic inhibitor z-Val-Ala-Asp-fluoromethyl ketone, z-DEVD-fmk, or z-IETD-fmk. Death receptor (DR) dependency was analyzed by examining expression of DRs (Fas, DR4, DR5, TNFR1, and R2), and interaction of Fas-associated death domain and caspase 8. Involvement of the mitochondria pathway was examined by monitoring of the mitochondria membrane potential, cytochrome c release, and Bax translocation. RESULTS: Phytosphingosine-treated cells displayed several features of apoptosis, including increase of sub-G(1) population, DNA fragmentation, and poly(ADP-ribose) polymerase cleavage. We observed that phytosphingosine cause activation of caspase 8 in a DR-independent fashion. Phytosphingosine also induced activation of caspase 9 and 3, loss of mitochondrial membrane potential, and the cytochrome c release from mitochondria. However, we failed to detect Bid cleavage. Moreover, caspase 8 inhibitor z-IETD-fmk did not affect phytosphingosine-induced cytochrome c release and caspase 9 activation, suggesting that phytosphingosine-induced cytochrome c release is caused by caspase 8-independent manner. Phytosphingosine induced mitochondrial translocation of Bax from the cytosol without changes in the protein levels of Bcl-2, Bcl-xL, and Bax. In addition, Bcl-2/Bax interaction was diminished after addition of phytosphingosine. CONCLUSION: These findings indicate that phytosphingosine induces apoptotic cell death in human cancer cells by direct activation of caspase 8, and by mitochondrial translocation of Bax and subsequent release of cytochrome c into cytoplasm, providing a potential mechanism for the anticancer activity of phytosphingosine.     

Neuronal apoptosis after CNS injury: the roles of glutamate and calcium

While a role has been well established for excitotoxic necrosis in the pathogenesis of traumatic or ischemic damage to the CNS, accumulating evidence now suggests that apoptosis may also be a prominent contributor. In this review we focus on the role of glutamate and attendant intracellular calcium influx in triggering or modifying excitotoxic necrosis and apoptosis, raising the possibility that calcium influx may affect these two death pathways in opposite directions. Incorporating consideration of both pathways will probably be needed to develop the most effective neuroprotective treatments for CNS injury.     

High glucose inhibits glucose uptake in renal proximal tubule cells by oxidative stress and protein kinase C

BACKGROUND: High glucose has been considered to play an important role in alteration of renal proximal tubule transporter's activity. This study examined the mechanism by which high glucose modulates alpha-methyl-D-glucopyranoside (alpha-MG) uptake in primary cultured rabbit renal proximal tubule cells (PTCs). METHODS: PTCs were incubated with 25 mmol/L glucose alone or combined with taurine, ascorbic acid, catalase, staurosporine, and bisindolylmaleimide I. Then alpha-MG uptake and lipid peroxide (LPO) formation were examined. RESULTS: Twenty-five mmol/L glucose from four hours, but not 25 mmol/L mannitol, inhibited alpha-MG uptake by 23% compared with 5 mmol/L glucose (control). In the study to examine the relationship of oxidative stress in the high-glucose-induced inhibition of alpha-MG uptake, 25 mmol/L glucose significantly increased LPO by 27% compared with control. However, 10 mmol/L glucose did not affect alpha-MG uptake and LPO formation. Taurine (2 mmol/L), ascorbic acid (1 mmol/L), endogenous antioxidants, or catalase (600 U/mL) significantly blocked 25 mmol/L glucose-induced increase of LPO formation and inhibition of alpha-MG uptake. In the experiment to examine the effects of protein kinase C on LPO formation, 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 ng/mL) increased LPO formation, and staurosporine (10(-7) mol/L) and bisindolylmaleimide I (10(-6) mol/L) totally blocked 25 mmol/L glucose-induced increase of LPO formation and inhibition of alpha-MG uptake. In addition, taurine reduced TPA-induced increase of LPO formation and inhibition of alpha-MG uptake. CONCLUSION: High glucose induces, in part, the inhibition of alpha-MG uptake through LPO formation, and activation of protein kinase C may play a role in high-glucose-induced LPO formation in the primary cultured rabbit renal PTCs.     

Expression of senescence-associated beta-galactosidase in enlarged prostates from men with benign prostatic hyperplasia

OBJECTIVES: Cellular senescence is a unique cellular response pathway thought to be closely associated with the aging process. The senescent phenotype is characterized by the loss of a cell's ability to respond to proliferative and apoptotic stimuli even while normal metabolic activity and vitality is maintained. Recently, a novel biomarker, senescent-associated beta-galactosidase (SA-beta-gal), was found to identify cells with the senescent phenotype. In the present study, we examined whether human prostatic epithelial cells adopt a senescence-associated phenotype after prolonged culture and analyzed a series of human benign prostatic hyperplasia (BPH) specimens to determine whether the cellular senescence process might be a factor in the development of BPH. METHODS: A primary culture of epithelial cells was established from the normal tissue of the peripheral zone of a radical prostatectomy specimen and was serially passaged until senescence. Forty-three human prostate specimens were obtained subsequent to radical prostatectomy or transrectal ultrasound-guided biopsy. The cultured cells and tissue specimens were histochemically stained to reveal the expression of SA-beta-gal, the cellular senescence biomarker. RESULTS: As has been reported for other types of cultured cells, human prostatic epithelial cells demonstrated widespread expression of the cellular senescence marker, SA-beta-gal, on prolonged culture. In our survey of hypertrophied human prostate tissues, 17 specimens (40%) of the 43 analyzed demonstrated positive staining for SA-beta-gal. In these tissues, SA-beta-gal expression was noted only in the epithelial cells. No statistical correlation (P = 0.42) between the chronologic age of the patient donor and SA-beta-gal expression was found. However, a high prostate weight (greater than 55 g) was found to correlate strongly with the expression of the SA-beta-gal biomarker (P = 0. 0001). CONCLUSIONS: Cultured prostatic epithelial cells expressed SA-beta-gal on reaching replicative senescence in vitro. The survey of human BPH specimens for the senescent marker showed that prostatic epithelial cells in patients with BPH with more advanced enlargement of the prostate (greater than 55 g prostate weight) expressed SA-beta-gal, and the prostates from patients with BPH that weighed less than 55 g tended to lack senescent epithelial cells. On the basis of these results, we propose that the accumulation of senescent epithelial cells may play a role in the development of the prostatic enlargement associated with BPH.     

Actual half-life of alpha-fetoprotein as a prognostic tool in pediatric malignant tumors

In a retrospective study, the prognostic value of monitoring the decay of alpha-fetoprotein (AFP) was assessed. Serum AFP was determined serially in 18 children with malignant germ-cell or hepatic tumors: 7 endodermal sinus tumor, 3 embryonal carcinoma, 5 malignant teratoma, 2 hepatoblastomas, and 1 hepatocellular carcinoma. The actual half-life (AHL) of AFP was computed after surgical resection of the tumor. In group 1, which had complete resection and no recurrence during follow-up (n = 13), the AHL of AFP was 4.0 ± 0.9 days. In group 2, which had incomplete resection or recurrence during follow-up (n = 5), the AHL of AFP was 24.8 ± 20 days, significantly longer than that of group 1 (P = 0.0026). The increased AHL of AFP indicated residual active tumor after surgical resection. The AHL of AFP may be more sensitive than serial monitoring of AFP in detecting preclinical recurrence after surgical resection of AFP-secreting tumors. Treatment strategies can be based on AFP clearance, and prospective clinical trials are warranted.     

Radiation Therapy Results for Patients Undergoing Inappropriate Surgery in the Presence of Invasive Cervical Carcinoma

Total vaginal or abdominal hysterectomy was considered an inadequate treatment method for invasive uterine cervix cancer. Usually the procedure was inadvertently performed on patients who were thought preoperatively to have benign or premalignant conditions. Between 1985 and 1993, 64 patients undergoing hysterectomy in the presence of invasive cervical cancer were treated with external radiation therapy and/or intracavitary radiotherapy. Preoperative diagnoses were carcinomain situ(36), severe dysplasia (2), and early invasive cancer (14), and others were benign disease. Overall 5-year survival and relapse-free survival rates were 75.8 and 77.5%, respectively. For patients in retrospective stage IA, IB, and IIB (gross residual after surgery), overall 5-year survival rates were 90.9, 88.8, and 27.9%, respectively. Thirteen patients developed treatment failure; most of them (10/13) were patients with gross residual disease. Patients with early invasive cervical cancer (stage IA) had no treatment-related failure. Prognostic factors affecting survival by univariate analysis were retrospective stage (P= 0.0000) and preoperative diagnosis (P= 0.0021). Tumor histology was marginally significant factor (P= 0.0938). By multivariate analysis, only retrospective stage was significant prognostic factor (P= 0.0001). Adjuvant radiotherapy appears to be an effective treatment method for patients with presumed stage IA and IB after inadvertent hysterectomy. Survival for patients with gross disease remaining after inappropriate hysterectomy is poor. So, early cancer detection and proper management with precise pretreatment staging is necessary to avoid inadherent hysterectomy, especially in cases of gross residual disease.     

Validity and cost-effectiveness of diagnostic procedures in CS2 poisoning

To determine relatively useful diagnostic procedures of carbon disulfide (CS2) poisoning in terms of validity and cost-effectiveness, several diagnostic tests are evaluated on 1,552 people by prevalence ratio (exposed/nonexposed), dose-response relationship, sensitivity and specificity, and the cost of the tests. Several symptoms with high kappa indices are found to be useful in various combinations, showing a consistent dose-response relationship and high exposed-nonexposed ratio. In clinicopathologic tests on functions of the kidney, liver and hematopoietic systems, eight items out of 22 have been shown to have significant dose-response relationship, mostly in liver function tests. A thorough dental examination failed to identify any useful indicator peculiar to the CS2 poisoning. Unlike Western people, the prevalence of coronary heart disease among Koreans was too low to be useful in the diagnosis of CS2 poisoning. Among four elective tests, i.e., Minnesota Multiphasic Personality Inventory (MMPI), Brain magnetic resonance imaging (MRI), nerve conduction velocity (NCV), and fluorescent angiography (FAG), the NCV appeared to be a more sensitive and specific test than the others are. Combinations of the tests improved the probability of diagnosing CS2 poisoning cases when any one test out of four was positive. Addition of other valid tests increased the probability of excluding non-cases. It was concluded that diagnosis of CS2 poisoning could be made validly and inexpensively if the diagnostic tests were carefully chosen step by step.     

Turkish pepper (extra hot)

A 38 year old female office worker was admitted with a newly discovered blood pressure of 250/110 mm Hg. Evaluation for secondary forms of hypertension was negative and treatment was begun. Sodium excretion was markedly reduced, plasma aldosterone was normal, and plasma renin activity was low. Therefore, presence of an aldosterone-like activity was suspected. Eventually, the patient confessed to abusing "Turkish Pepper", a brand of Scandinavian liquorice candies and "Fisherman's Friend", another brand of liquorice candies, concurrently. After eliminating liquorice from her diet, the hypertension disappeared thus allowing her antihypertensive treatment to be stopped.