Chemopreventive activity of fluphenazine and its analogues in human lymphocyte cultures preincubated with ceramide synthase inhibitor

Wstęp. Ceramid (cer) jest zazwyczaj produkowany na drodze hydrolizy sfingomieliny przez sfingomielinazy (SMase). Zbadano, że aktywność chemoprewencyjna — propoptotyczna i chemouwrażliwiająca — piperazynowej pochodnej fenotiazyny (Pht), flufenazyny (FPh) i jej nowo syntezowanych analogów — związków 1b i 3f — zależy od stymulacji lizosomalnej, kwaśnej sfingomielinazy (aSMase), niezależnej od Zn2+, jednego z enzymów uczestniczących w powsta­waniu endogennego cer. Innym ważnym szlakiem tworzenia cer w komórce jest synteza de novo z udziałem syntetazy ceramidowej (CerS). Celem pracy była ocena aktywności chemoprewencyjnej FPh i jej analogów: związków 1b i 3f po nieodwracalnym zablokowaniu CerS. Materiał i metody. Aktywność chemoprewencyjną badanych związków (10 μM, 2 godz.) oceniano w hodowlach limfocytów ludzkich uszkodzonych genotoksycznie przez inkubację z benzo[a]pirenem (+B[a]P; 7,5 μM, 48 godz.) i po preinkubacji z selektywnym inhibitorem CerS — fumonizyną B1 (+FB1; 20 μM, 1,5 godz.). W ocenie efektów badanych związków zastosowano metody: mikroskopii fluorescencyjnej, spektrofotometrii i spektrofluorymetrii. Istotność statystyczną uzyskanych rezultatów sprawdzono za pomocą testu t-Studenta. Wyniki. W hodowlach limfocytów w obecności inhibitora CerS (+B[a]P; +FB1) inkubacja hodowli z analogami 1b i 3f prowadziła do istotnie niższego (p < 0,05) odsetka komórek w apoptozie i akumulacji rodaminy 123 (Rod-123) w po­równaniu z hodowlą bez inhibitora CerS (+B[a]P; -FB1). W przypadku macierzystego związku FPh wpływ na hodowle limfocytów nie zależał od obecności inhibitora CerS. Wnioski. Efekty proapoptotyczny i chemouwrażliwiający analogów 1b i 3f są uwarunkowane aktywacją CerS. Nato­miast dla macierzystej FPh wykazano, że jej aktywność chemoprewencyjna nie zależy od stymulacji szlaku syntezy de novo cer z udziałem CerS.Introduction. The ceramide (cer) is generated mainly via hydrolysis of sphingomyelin by sphingomeylinase (SMase). It was previously documented that chemopreventive activity — proapoptotic and chemosensitizng of piperazine phenothiazine derivative, fluphenazine (FPh) and its newly synthesized analogues, 1b and 3f compounds, depends on the activity of lysosomal acidic sphingomyelinase (aSMase), Zn2+-independent, one of the enzymes participating in intracellular ceramide generation. Another important pathway of intracellular formation of ceramide is de novo synthesis with participation of ceramide synthase (CerS). The aim of this research is to assess the chemopreventive activity of FPh and 1b and 3f compounds after irreversible CerS blockade. Material and methods. Chemopreventive activity of the tested compounds (10 μM, 2 h) was evaluated in hu­man lymphocyte cultures, genotoxically damaged by benzo[a]pyrene (+B[a]P; 7,5 μM, 48 h) and preincubated with a selective inhibitor CerS — fumonisin B1 (+FB1; 20 μM, 1,5 h) and inspected with fluorescence microscopy, as well as with spectrophotometric and spectrofluorimetric methods. Student’s t-test was used for testing the statistical significance of the results. Results. It was established that in the presence of the CerS inhibitor (+B[a]P; +FB1) the effects of 1b and 3f analogues on apoptotic cell frequency in lymphocyte cultures and on accumulation of rhodamine 123 (Rod-123), were signifi­cantly (p < 0.05) decreased when compared to the cultures carried out in the absence of the CerS inhibitor (+B[a]P; -FB1). Whereas in the case of the parent compound: the FPh, the presence of the inhibitor did not influence on FPh chemopreventive activity. Conclusions. The proapoptotic and chemosensitizing effects of 1b and 3f compounds were determined in major part by CerS activation. Also the chemopreventive activity of the parent compound: the FPh, was independent of the cer de novo synthesis pathway conducted by CerS

Compact Wireless Microscope for In-Situ Time Course Study of Large Scale Cell Dynamics within an Incubator

Imaging of live cells in a region of interest is essential to life science research. Unlike the traditional way that mounts CO2 incubator onto a bulky microscope for observation, here we propose a wireless microscope (termed w-SCOPE) that is based on the "microscope-in-incubator" concept and can be easily housed into a standard CO2 incubator for prolonged on-site observation of the cells. The w-SCOPE is capable of tunable magnification, remote control and wireless image transmission. At the same time, it is compact, measuring only ~10 cm in each dimension, and cost-effective. With the enhancement of compressive sensing computation, the acquired images can achieve a wide field of view (FOV) of ~113 mm(2) as well as a cellular resolution of ~3 μm, which enables various forms of follow-up image-based cell analysis. We performed 12 hours time-lapse study on paclitaxel-treated MCF-7 and HEK293T cell lines using w-SCOPE. The analytic results, such as the calculated viability and therapeutic window, from our device were validated by standard cell detection assays and imaging-based cytometer. In addition to those end-point detection methods, w-SCOPE further uncovered the time course of the cell's response to the drug treatment over the whole period of drug exposure