Prognostic impact of prognostic nutritional index in advanced (stage IIIB/IV) non-small cell lung cancer patients.

Prognostic nutritional index (PNI) is a parameter reflecting prognosis for various cancers, including resected lung cancer. However, there were few reports to study the relationship between the PNI and overall survival (OS) in patients with advanced (stage IIIB/IV) non-small lung cancer (NSCLC). In this study, we collected the clinical data of 315 patients with advanced (stage IIIB/IV) NSCLC who had received chemotherapy or epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) between January 2010 and June 2011. Survival curves were plotted using the Kaplan-Meier method. Multivariate analyses were used to evaluate prognostic significance of PNI in patients with advanced (stage IIIB/IV) NSCLC. In our analysis, we found that PNI (p=0.001) was significantly associated with OS in patients with advanced (stage IIIB/IV) NSCLC, so was smoking (p<0.001) and disease stage (p=0.005). We demonstrated that PNI could be utilized to predict survival outcomes in patients with advanced (stage IIIB/IV) NSCLC. Patients with a lower PNI may have worse prognosis.

Knockdown of human serine/threonine kinase 33 suppresses human small cell lung carcinoma by blocking RPS6/BAD signaling transduction.

Small cell lung cancer (SCLC) is characterized by rapid growth rate and a tendency to metastasize to distinct sites of patients' bodies. The human serine/threonine kinase 33 (STK33) gene has shown its potency as a therapeutic target for prevention of lung carcinomas including non-small cell lung cancer (NSCLC), but its function in the oncogenesis and development of SCLC remains unrevealed. In the current study, it was hypothesized that STK33 played a key role in the proliferation, survival, and invasion of SCLC cells. The expression of STK33 in human SCLC cell lines NCI-H466 and DMS153 was inhibited by specific shRNA. The cell proliferation, cell apoptosis, and cell invasion of the cells were assessed with a series of in vitro assays. To explore the mechanism through which STK33 gene exerted its function in the carcinogenesis of SCLC cells, the effect of STK33 knockdown on the activity of S6K1/RPS6/BAD signaling was detected. Then the results were further confirmed with STK33 inhibitor ML281 and in vivo assays. The results demonstrated that inhibition of STK33 in SCLC cells suppressed the cell proliferation and invasion while induced cell apoptosis. Associated with the change in the phenotypic features, knockdown of STK33 also decreased the phosphorylation of RPS6 and BAD while increased the expression of cleaved caspase 9, indicating that apoptosis induced by STK33 suppression was mediated via mitochondrial pathway. Similar to the results of STK33 knockdown, incubating NCI-H466 cells with STK33 inhibitor also reduced the cell viability by suppressing RPS6/BAD pathways. Additionally, STK33 knockdown also inhibited tumor growth and RPS6/BAD activity in mice models. Findings outlined in our study were different from that in NSCLC to some extent: knockdown of STK33 in SCLC cells induced the apoptosis through mitochondrial pathway but independent of S6K1 function, inferring that the function of STK33 might be cancer type specific.

Metabolic, idiosyncratic toxicity of drugs: overview of the hepatic toxicity induced by the anxiolytic, panadiplon.

Preclinical drug safety evaluation studies, typically conducted in two or more animal species, reveal and define dose-dependent toxicities and undesirable effects related to pharmacological mechanism of action. Idiosyncratic toxic responses are often not detected during this phase in development due to their relative rarity in incidence and differences in species sensitivity. This paper reviews and discusses the metabolic idiosyncratic toxicity and species differences observed for the experimental non-benzodiazepine anxiolytic, panadiplon. This compound produced evidence of hepatic toxicity in Phase 1 clinical trial volunteers that was not predicted by rat, dog or monkey preclinical studies. However, subsequent studies in Dutch-belted rabbits revealed a hepatic toxic syndrome consistent with a Reye's Syndrome-like idiosyncratic response. Investigations into the mechanism of toxicity using rabbits and cultured hepatocytes from several species, including human, provided a sketch of the complex pathway required to produce hepatic injury. This pathway includes drug metabolism to a carboxylic acid metabolite (cyclopropane carboxylic acid), inhibition of mitochondrial fatty acid beta-oxidation, and effects on intermediary metabolism including depletion of glycogen and disruption of glucose homeostasis. We also provide evidence suggesting that the carboxylic acid metabolite decreases the availability of liver CoA and carnitine secondary to the formation of unusual acyl derivatives. Hepatic toxicity could be ameliorated by administration of carnitine, and to a lesser extent by pantothenate. These hepatocellular pathway defects, though not directly resulting in cell death, rendered hepatocytes sensitive to secondary stress, which subsequently produced apoptosis and hepatocellular necrosis. Not all rabbits showed evidence of hepatic toxicity, suggesting that individual or species differences in any step along this pathway may account for idiosyncratic responses. These differences may be roughly applied to other metabolic idiosyncratic hepatotoxic responses and include variations in drug metabolism, effects on mitochondrial function, nutritional status, and health or underlying disease.

Disruption of mitochondrial activities in rabbit and human hepatocytes by a quinoxalinone anxiolytic and its carboxylic acid metabolite.

The quinoxalinone anxiolytic, panadiplon, was dropped from clinical development due to unexpected hepatic toxicity in human volunteers. Subsequent experimental studies in rabbits demonstrated a hepatic toxicity that resembled Reye's syndrome. In the present studies, we examined the effects of panadiplon and a metabolite, cyclopropane carboxylic acid (CPCA) on hepatic mitochondrial activities in vitro and ex vivo. Acute inhibition of beta-oidation of [14C]palmitate was observed in rabbit and human hepatocyte suspensions incubated with 100 microM panadiplon. Panadiplon (30 microM) also reduced mitochondrial uptake of rhodamine 123 (R123) in cultured rabbit and human, but not rat hepatocytes, following 18 h exposure. CPCA also impaired beta-oxidation and R123 uptake in rabbit and human hepatocytes. R123 uptake and beta-oxidation in cells from some donors was not impaired by either agent, and cell death was not observed in any experiment. Hepatocytes isolated from panadiplon-treated rabbits had reduced palmitate beta-oxidation rates and inhibited mitochondrial R123 uptake; R123 uptake remained inhibited until 48-72 h in culture. Rabbit mitochondrial respiration experiments revealed a slightly lower ratio of ATP formed/oxygen consumed in panadiplon-treated animals: direct exposure of normal rabbit liver mitochondria to panadiplon did not have this effect. Hepatocytes isolated from panadiplon-treated rabbits showed reduced respiratory control ratios and lower oxygen consumption compared to controls. Our results indicate that panadiplon induces a mitochondrial dysfunction in the liver, and suggest that this dysfunction may be attributed to the carboxylic acid metabolite.

Potentiation of hypoxic injury in cultured rabbit hepatocytes by the quinoxalinone anxiolytic, panadiplon.

The quinoxalinone anxiolytic, panadiplon, produces hepatic metabolic inhibition (mitochondrial impairment), microvesicular steatosis and centrilobular necrosis in rabbits. Metabolic inhibition occurs in cultured hepatocytes without cytotoxicity, suggesting that hepatic injury is influenced by additional factors. The present experiments were conducted to determine if metabolic inhibition by panadiplon predisposed hepatocytes to hypoxic injury. Injury (cell death) was evaluated by lactate dehydrogenase (LDH) release from cells; ATP and glycogen levels were also evaluated. Under hypoxic conditions, control cultures showed a 6.5-fold increase in LDH release compared to normoxic controls, with a coincident 80% decrease in ATP and 50% decrease in glycogen levels. Under normoxic conditions 10 microgram/ml panadiplon treatment for 48 h reduced ATP and glycogen levels by 40% but did not cause an increase in LDH leakage. Cells treated with panadiplon, then exposed to hypoxia conditions, showed a significant level of injury compared to normoxic control cultures, and a further reduction in ATP. No additional decrease in glycogen ws observed. In an attempt to prevent panadiplon-mediated injury, glycolytic substrates (dihydroxyacetone or pyruvate) were included during normoxic and hypoxic incubations. Both cotreatments reduced the level of LDH leakage produced by panadiplon during hypoxia. Cotreatment did not generally increase ATP or glycogen levels (compared to panadiplon treatment groups) during hypoxia, though individual experiments showed a slight increase in ATP levels. During normoxia both cotreatments with panadiplon resulted in significantly higher glycogen levels than in panadiplon cultures alone. These results suggest that cellular glycogen and subsequently ATP levels are reduced during panadiplon exposure, metabolically predisposing hepatocytes to hypoxic injury.

Biotransformation of lifibrol (U-83860) to mixed glyceride metabolites by rat and human hepatocytes in primary culture.

Lifibrol (U-83860), K12.148) is a lipid-lowering drug that has the potential to accumulate in the liver and induce hepatic peroxisome proliferation. To investigate the identity and potential human relevance of persistent lifibrol-related residues in rat liver, rat and human hepatocyte primary cultures were treated with 30 microM of [14C]lifibrol. After a steady uptake for 24 hr, cellular levels of radioactivity became stable for the next 24-48 hr. A nonradioactive lifibrol chase caused an efflux of intracellular radioactivity. Cellular autoradiography revealed the association of radioactivity with small lipid drops at 6 hr exposure and with large lipid drops at 24 hr exposure. HPLC analysis of media revealed that lifibrol acyl glucuronide and a t-butylcarboxylic acid metabolite (U-94613) were the major metabolites of rat and human hepatocytes, respectively. Using an HPLC method suitable for nonpolar metabolites, the analysis of rat and human cell extracts revealed a broad band of multiple, radioactive peaks that had a similar retention and UV spectrum to a synthetic standard of lifibrol cholesterol ester. Folch extracts of liver from rats treated with [14C]lifibrol or unlabeled lifibrol and [14C]acetate had a unique radioactive TLC band that had similar HPLC retention to hepatocyte residues. The group of nonpolar peaks from the hepatocytes was purified by HPLC. Conversion of the lifibrol sec-hydroxy group to a nicotinate ester afforded particle beam-electron impact mass spectra of the cholesterol ester standard and hepatocyte residues. The derivatized rat hepatocyte residue did not contain detectable lifibrol cholesterol ester (M+.816), but did contain molecular ion clusters corresponding to a mixed triglyceride of lifibrol and two fatty acids. Lifibrol-specific product ions of molecular ion clusters centered at M+.1021, 1047, and 1073 were observed at m/z 448, 430, and 310. The major lifibrol-containing triglycerides had a fatty acid composition of C16-C20 with 0-6 unsaturations.

Cultured hepatocytes as investigational models for hepatic toxicity: practical applications in drug discovery and development.

Drugs can fail at any phase during discovery, preclinical or clinical development due to unacceptable levels of toxicity, and liver is commonly the principle target organ. Investigational toxicology methods, using appropriate models and hypotheses, can often resolve problems, identify toxic chemical substituents and salvage therapeutic discovery programs. While in vivo models are used to investigate hepatic drug effects in the context of toxicokinetics and systemic influences, cell culture models provide in vitro systems for investigating specific mechanisms in a precisely controlled environment. Using primary hepatocytes isolated from laboratory animals, we have explored several drug-induced hepatic disorders that surfaced during different phases of drug discovery and development. Additionally, the use of human hepatocytes has allowed us to address concerns for human exposure, examine human relevance of animal data, and provide perspective on problems encountered in clinical trials.

Induction of a hepatic toxic syndrome in the Dutch-belted rabbit by a quinoxalinone anxiolytic.

The non-benzodiazepine anxiolytic, panadiplon, was discontinued from clinical development due to evidence of hepatic toxicity in human volunteers that was not predicted by rat or monkey preclinical development studies. The present study was conducted to examine potential toxicity in the rabbit. Three groups of female rabbits were administered vehicle, 10 mg/kg per day or 20 mg/kg per day of panadiplon by oral gavage for 14 days. Animals in the 20 mg/kg group lost weight, and 6/10 developed a profound lethargy. Hepatic toxicity was observed in treated animals, evidenced by dose- and time-related increases in serum transaminase activities, gross hepatic lesions and multifocal centrilobular necrosis. Hepatic microvesicular steatosis was evident in treated animals; lipid analysis revealed a 123% increase in hepatic triglyceride. A time-dependent increase in serum triglyceride levels was observed in the high-dose group beginning on day 4. Hepatic glycogen was reduced, and histochemical examination revealed the reduction to be heterogeneous across the lobule with some areas showing a complete absence of glycogen. One rabbit in each drug-treated group showed mild hypoglycemia at day 12, and 4/10 rabbits in the high-dose group showed hyperglycemia at days 12-14. We conclude that panadiplon produced a microvesicular steatosis and hepatic toxicity in the rabbit. The observed toxicity resembled a Reye's syndrome-like toxicity produced by a variety of mitochondrial fatty acid oxidation inhibitors.

Morphometric study of the prepubertal rabbit testis: germ cell numbers and seminiferous tubule dimensions.

Testes from rabbits aged 1-9 weeks were examined by light microscopy. Changes in seminiferous tubule dimensions, testicular volume, and volume fraction of tubules were assessed. Germ cells and Sertoli cells were counted in round tubular cross sections and total germ cell number in each testis was estimated. Mitotic, meiotic, and degenerative activities of germ cells as well as their basal or central positions within tubules were quantified. A marked, steady increase in testis volume and in tubular length and volume occurred over the prepubertal period; but diameter underwent no significant increase and in fact decreased until week 4. Overall, tubules lengthened 40-fold and testis volume increased 25-fold; the percentage volume of the testis occupied by tubules rose from one-third neonatally to three-fifths at the onset of spermatogenesis. The ratio of germ cells to total tubular (germ and Sertoli) cells was lowest at 3 weeks. However, the total number of germ cells increased little until 3 weeks, after which it rose at a sharp rate commensurate with testis volume. Percentage of germ cells in mitosis peaked sharply at 3 weeks, dropped in subsequent weeks, and then rose at 7 weeks at the initiation of spermatogenesis. Importantly, the surge in mitosis at 3 weeks was followed by a redistribution of germ cells to a predominantly basal location from 3 to 7 weeks. Meiotic activity was sparse at 7 weeks and became abundant by 9 weeks. Germ cell degeneration remained relatively constant during weeks 1 through 6, with an increase at 7 weeks.

Cryopreservation of cynomolgus monkey (Macaca fascicularis) hepatocytes for subsequent culture and protein synthesis studies.

A method is described for the preservation and subsequent recovery of hepatocytes obtained by collagenase perfusion of cynomolgus monkey (Macaca fascicularis) livers. The fresh cells are suspended in fetal bovine serum containing 10% dimethylsulfoxide and, using a microprocessor-controlled, liquid nitrogen freezing chamber and a specific cooling protocol, processed in such a way that they can be stored in liquid nitrogen for several months and still restored to active culture. When the cryopreserved cells were established in culture, they were found to actively synthesize and secrete both albumin and apolipoprotein A-I. That, taken together with morphologic evidence, was viewed as indication that the cells recovered in culture were in fact hepatocytes and not some other cell type from the monkey liver. The availability of this procedure for storing hepatocytes should contribute significantly to the efficient use of nonhuman primates as models with which to study hepatic metabolism.

Formation of the blood-testis barrier in the rabbit.

The time of establishment of the blood-testis barrier in the rabbit was studied by electron microscopy using lanthanum nitrate. This electron-dense tracer was present in the intercellular spaces in all regions of the seminiferous cords in 7- to 9-week-old animals. In 10- and 11-week-old rabbits, the penetration of lanthanum nitrate was restricted to the basal region of the seminiferous cords. Closer examination revealed the presence of numerous tight junctions between adjacent Sertoli cells. The morphological appearance of these junctions was similar to those described previously in other mammals. Entry of the tracer substance was restricted at these junctions. Pachytene germ cells, which reside beyond the junctions, were never surrounded by the tracer. Based on our observations it was concluded that the blood-testis barrier in the rabbit is formed between the 9th and 10th postnatal week, and that it is functionally effective by the 10th week.

Squash preparation studies of germ cells in human fetal testes.

Testes from human fetuses 21 to 41 weeks of gestation were investigated using a modified squash technique. At all of the gestational ages examined, 90 to 95% of the germ cells were in interphase. Preleptotene germ cells, which exhibited densely staining condensed clumps of chromosomes, were found in all samples studied and constituted 3 to 9% of the germ cells observed. In appearance they resembled preleptotene figures seen in the fetal ovary. Occasionally, germ cells containing slender chromosomes with a beaded appearance, but without prominent condensation, were encountered. These cells were interpreted as preleptotene germ cells in the early stages of degeneration. Germ cells in other meiotic stages were not observed in this study.

Proliferative activity in the rat epididymis during postnatal development.

The proliferative activity of the rat epididymis during postnatal development was investigated with the use of autoradiography. Animals at 14, 21, 28, 35 and 56 days of age were sacrificed 2 hours following the administration of 3H-thymidine. Cell types that showed a significant labeling index were columnar cells in 14- and 21-day-old animals; principal and basal cells in 28, 35, and 56 day old rats. During this period of development, the pattern of cellular activity in the initial segment differed from the middle and terminal segments in having a peak of activity on day 28. The middle and terminal segments had similar proliferative patterns. In two additional experiments, 10- and 21-day-old rats were given 3H-thymidine and killed 1 week later on day 16 and 28. Labeled narrow cells were present in day 16 animals, whereas labeled narrow, principal, and basal cells were found in day 28 rats. It was concluded that columnar cells are the precursor to narrow, principal and basal cells.

Morphological characteristics of cells with apical nuclei in the initial segment of the adult rat epididymis.

The cytology of epithelial cells with apical nuclei in the initial segment of the rat epididymis was studied with the light and electron microscopes. Two types of cells were distinguished and were designated apical cells and narrow cells. The apical cells are more numerous than the narrow cells and closely resemble principal cells except for the location of the nucleus. They probably correspond to the apical cells of Reid and Cleland ('57) and may represent a variation of the principal cell. The narrow cells differ markedly from the apical cells in both light microscopic appearance and fine structure. Narrow cells strain intensely with toluidine blue and are characterized by a slender shape, many mitochodria with tubular cristae, and a large number of apical cup-shaped cytoplasmic vesicles. The possible relationship of narrow cells to other cell types is discussed.

Development of cell types and of regional differences in the postnatal rat epididymis.

The development of cell types and regional differences in the rat epididymis was studied in specimens of the initial, middle and terminal segments prepared at intervals between birth and postnatal day 94. The development of the epididymis was divided into three phases: (1) an undifferentiated period; (2) a period of differentiation, and (3) a phase of expansion. During the undifferentiated period, from birth to day 15, the epithelial cells had a uniform appearance. Halo cells, which are believed to be migratory leukocytes, appeared on day 14. The period of differentiation extended from day 16 to day 44. Slender, densely staining cells, termed narrow cells, appeared in the epithelium of all three segments on day 16, constituting the first evidence of differentiation of cell types in the epididymal epithelium per se. In addition to their shape and apical nuclei, the narrow cells were distinguished from other epithelial cells by the presence of cup-shaped apical vacuoles and mitochondria with tubular cristae. Principal cells and basal cells were identified on day 28, which also marked the firsh distinction of differences in epithelial height among the different segments. Narrow cells persisted into the adult in the initial segment. In the middle and terminal segments, however, narrow cells disappeared by day 35, when light cells made their appearance. The major event of the period of expansion, from day 45 to 3 months, was the appearance of sperm in the lumen between days 45 and 52. A model for differentation of cell types in the epididymis is proposed and it is suggested that narrow cells are precursors to light cells in the middle and terminal segments. The development of ultrastructural features of adult cell types preceded the appearance of sperm in the lumen.