Functional evaluation of a fluorescent schweinfurthin: mechanism of cytotoxicity and intracellular quantification.

Schweinfurthins are potent inhibitors of cancer cell growth, especially against human central nervous system tumor lines such as SF-295 cells. However, the mechanisms through which these compounds impede cell growth are not fully understood. In an effort to understand the basis for the effects of schweinfurthins, we present a fluorescent schweinfurthin, 3-deoxyschweinfurthin B-like p-nitro-bis-stilbene (3dSB-PNBS), which displays biological activity similar to that of 3-deoxyschweinfurthin B (3dSB). These two schweinfurthins retain the unique differential activity of the natural schweinfurthins, as evidenced by the spindle-like morphological changes induced in SF-295 cells and the unaltered appearance of human lung carcinoma A549 cells. We demonstrate that incubation with 3dSB or 3dSB-PNBS results in cleavage of poly-ADP-ribose polymerase (PARP) and caspase-9, both markers of apoptosis. Coincubation of 3dSB or 3dSB-PNBS with the caspase-9 inhibitor (Z)-Leu-Glu(O-methyl)-His-Asp(O-methyl)-fluoromethylketone prevents PARP cleavage. Therapeutic agents that induce apoptosis often activate cellular stress pathways. A marker for multiple stress pathways is the phosphorylation of eukaryotic initiation factor 2α, which is phosphorylated in response to 3dSB and 3dSB-PNBS treatment. Glucose-regulated protein 78 and protein disulfide isomerase, both endoplasmic reticulum chaperones, are up-regulated with schweinfurthin exposure. Using the fluorescent properties of 3dSB-PNBS and dimethoxyphenyl-p-nitro-bis-stilbene (DMP-PNBS), a control compound, we show that the intracellular levels of 3dSB-PNBS are higher than those of Rhodamine 123 or DMP-PNBS in SF-295 and A549 cells.

A retrospective and correlative analysis of academic and nonacademic predictors of COMLEX level 1 performance.

CONTEXT: National licensing exams (NLEs) including the Comprehensive Osteopathic Medical Licensing Examination (COMLEX) Level 1 evaluate student achievement. Scores have historically been utilized to stratify medical student applicants for residency. Grade point average (GPA), number of practice questions completed, and performance on practice exams have been shown to be predictive of NLE performance. Test anxiety and acute stress have been shown to negatively impact NLE performance. The role of study behaviors and other nonacademic factors in COMLEX Level 1 performance is unknown. OBJECTIVES: This study aims to evaluate academic and nonacademic factors and to correlate them with COMLEX Level 1 performance. Additional analysis is conducted to associate COMLEX Level 1 performance with academic and nonacademic factors when controlling for GPA. METHODS: An anonymous online survey was administered to third- (OMS III) and fourth-year (OMS IV) osteopathic medical students at Kansas City University that had completed the COMLEX Level 1 examination. In total, 72 students responded to the survey. Survey results were linked to student records of GPA and COMLEX Level 1 scores, resulting in 59 complete responses for analysis. Independent-sample t-tests and linear ordinary least squares regression were utilized to analyze the results. RESULTS: The majority of participants are male (62.7%) and OMS III (98.3%) with an average age of 27.14 ± 2.58 (mean ± standard deviation). Further demographic data reveal hours per week spent for personal time during dedicated study (n=46, 19.7 ± 18.53), hours of sleep per night during dedicated study (7.34 ± 0.92), and money spent on board preparation ($1,319.12 ± $689.17). High ($1,600-$3,000), average ($1,000-$1,500), and low ($100-$900) spenders do not statistically differ and COMLEX Level 1 performance is not related to the number of resources utilized (F statistics <1; p>0.05). Pearson correlations reveal a statistically significant relationship between COMLEX Level 1 scores with GPA (0.73, p<0.001), number of practice exams completed (0.39, p<0.001), number of questions completed (0.46, p<0.001), number of weeks of study (0.55, p<0.001), and preparation cost (0.28, p<0.05). The regression analysis revealed that money spent on board preparation, number of questions completed, and time spent studying accounted for 75.8% of the variance in COMLEX Level 1 scores after controlling for GPA. CONCLUSIONS: The data show the association of money spent on board preparation, numbers of questions competed, and time spent studying with a student's COMLEX Level 1 score. Additionally, these results highlight the amount of money students spend on extracurricular materials to prepare for COMLEX Level 1, yet the data show that the number of resources that students utilized is not related to a student's COMLEX Level 1 performance.

RABL6A inhibits tumor-suppressive PP2A/AKT signaling to drive pancreatic neuroendocrine tumor growth.

Hyperactivated AKT/mTOR signaling is a hallmark of pancreatic neuroendocrine tumors (PNETs). Drugs targeting this pathway are used clinically, but tumor resistance invariably develops. A better understanding of factors regulating AKT/mTOR signaling and PNET pathogenesis is needed to improve current therapies. We discovered that RABL6A, a new oncogenic driver of PNET proliferation, is required for AKT activity. Silencing RABL6A caused PNET cell-cycle arrest that coincided with selective loss of AKT-S473 (not T308) phosphorylation and AKT/mTOR inactivation. Restoration of AKT phosphorylation rescued the G1 phase block triggered by RABL6A silencing. Mechanistically, loss of AKT-S473 phosphorylation in RABL6A-depleted cells was the result of increased protein phosphatase 2A (PP2A) activity. Inhibition of PP2A restored phosphorylation of AKT-S473 in RABL6A-depleted cells, whereas PP2A reactivation using a specific small-molecule activator of PP2A (SMAP) abolished that phosphorylation. Moreover, SMAP treatment effectively killed PNET cells in a RABL6A-dependent manner and suppressed PNET growth in vivo. The present work identifies RABL6A as a new inhibitor of the PP2A tumor suppressor and an essential activator of AKT in PNET cells. Our findings offer what we believe is a novel strategy of PP2A reactivation for treatment of PNETs as well as other human cancers driven by RABL6A overexpression and PP2A inactivation.

Calcium and P-glycoprotein independent synergism between schweinfurthins and verapamil.

Schweinfurthins are intriguing natural products with anti-cancer activities and as yet incompletely understood mechanisms of action. We investigated whether inhibitors of P-glycoprotein (Pgp), in a manner analogous to other natural products, might enhance schweinfurthins' growth inhibitory actions by increasing intracellular schweinfurthin levels. Both the schweinfurthin-sensitive glioblastoma multiforme cell line SF-295 and relatively insensitive lung carcinoma cell line A549 were treated with 2 schweinfurthin analogs: 3-deoxyschweinfurthin B-p-nitro bis-stilbene (3dSB-PNBS) and 5'-methylschweinfurthin G (methyl-G). There was a synergistic enhancement of growth inhibition with the combination of the Pgp inhibitor verapamil and both analogs in SF-295 cells. Methyl-G, verapamil, and the combination did not result in alterations to intracellular calcium concentration. Verapamil increased the intracellular concentration of 3dSB-PNBS in both SF-295 and A549 cells in a Pgp-independent manner. Methyl-G, verapamil, and the combination do not result in increased ER stress. Methyl-G increased the intracellular concentration of a known Pgp substrate, Rhodamine 123 in SF-295 cells. Reduction of cellular cholesterol leads to the accumulation of Pgp substrates, as Pgp requires cholesterol for proper function. Since 3dSB enhances lovastatin-induced upregulation of the cholesterol efflux pump ABCA1, it is intriguing that co-treatment with cholesterol rescued the methyl-G-induced increase in Rhodamine 123 intracellular concentration. These studies support the hypothesis that verapamil potentiates the schweinfurthin growth inhibitory effect by increasing its intracellular concentration.