In vitro and in vivo antimelanoma effect of ethyl ester cyclohexyl analog of ethylenediamine dipropanoic acid.

Melanoma, an aggressive skin tumor with high metastatic potential, is associated with high mortality and increasing morbidity. Multiple available chemotherapeutic and immunotherapeutic modalities failed to improve survival in advanced disease, and the search for new agents is ongoing. The aim of this study was to investigate antimelanoma effects of O,O-diethyl-(S,S)-ethylenediamine-N,N'di-2-(3-cyclohexyl) propanoate dihydrochloride (EE), a previously synthesized and characterized organic compound. Mouse melanoma B16 cell viability was assessed using acid phosphatase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, sulforhodamine B, and lactate dehydrogenase assays. Apoptosis and autophagy were investigated using flow cytometry, fluorescence and electron microscopy, and western blotting. In vivo antitumor potential was assessed in subcutaneous mouse melanoma model after 14 days of treatment with EE. Tumor mass and volume were measured, and RT-PCR was used for investigating the expression of autophagy-related, proapoptotic, and antiapoptotic molecules in tumor tissue. Investigated organic compound exerts significant cytotoxic effect against B16 cells. EE induced apoptosis, as confirmed by phosphatidyl serine externalisation, caspase activation, and ultrastructural features typical for apoptosis seen on fluorescence and electron microscopes. The apoptotic mechanism included prompt disruption of mitochondrial membrane potential and oxidative stress. No autophagy was observed. Antimelanoma action and apoptosis induction were confirmed in vivo, as EE decreased mass and volume of tumors, and increased expression of several proapoptotic genes. EE possesses significant antimelanoma action and causes caspase-dependent apoptosis mediated by mitochondrial damage and reactive oxygen species production. Decrease in tumor growth and increase in expression of proapoptotic genes in tumor tissue suggest that EE warrants further investigation as a candidate agent in treating melanoma.

Protective effect of autophagy in laser-induced glioma cell death in vitro.

BACKGROUND AND OBJECTIVE: Laser phototherapy could be potentially used for cancer treatment, but the mechanisms of laser-induced cell death are not completely understood. Autophagy is the process in which the damaged cellular proteins and organelles are engulfed by and destroyed in acidified multiple-membrane vesicles. The aim of the present study was to investigate the role of autophagy in laser-induced tumor cell death in vitro. STUDY DESIGN/MATERIALS AND METHODS: The monolayers of U251 human glioma tumor cells were exposed to 532 nm laser light from a single mode frequency-doubled Nd-YVO4 laser. A flattened Gaussian radial profile of laser beam (0.5-4 W) was used to uniformly illuminate entire colony of cells for various amounts of time (15-120 seconds) in the absence of cell culture medium. The cells were grown for 24 hours and the cell viability was determined by crystal violet or MTT assay. The presence of autophagy was assessed after 16 hours by fluorescence microscopy/flow cytometric analysis of acridine orange-stained autophagolysosomes and Western blot analysis of the autophagosome-associated LC3-II protein. The concentration of the principal pro-autophagic protein beclin-1 was determined after 6 hours by cell-based ELISA. RESULTS: The intracytoplasmic accumulation of autophagic vesicles, increase in LC3-II and up-regulation of beclin-1 expression were clearly observed under irradiation conditions that caused approximately 50% cytotoxicity. Post-irradiation addition of three different autophagy inhibitors (bafilomycin A1, chloroquine, or wortmannin) further increased the laser-induced cytotoxicity, without affecting non-irradiated cells. CONCLUSIONS: These data indicate that beclin-1-dependent induction of autophagy can protect glioma cells from laser-mediated cytotoxicity.

Diagnosis of Trichomonas vaginalis infection: The sensitivities and specificities of microscopy, culture and PCR assay.

OBJECTIVES: The aim of this study was to compare wet mount-, Giemsa stain-, acridine orange fluorescent stain-, cultivation- and polymerase chain reaction (PCR)-based approaches to establish which method or combination of methods was most effective in the laboratory diagnosis of trichomoniasis. STUDY DESIGN: Out of 200 investigated patients with various gynecological complaints, Trichomonas vaginalis infection was detected in 27 (13.5%) by any of methods investigated. Among women with trichomonads, a typical clinical finding was presented in only nine. For analysis of sensitivity and specificity of the methods used, the receiver operating characteristic (ROC) curve concept with culture as a gold standard was applied. RESULTS: Infection was diagnosed by wet mount in 14 (7.0%) women, by Giemsa stain in 11 (5.5%) and by acridine orange stain in 16 (8.0%) women. In 21 (10.5%) women, it was diagnosed by culture in Diamond's medium, and in 22 (11.0%) by PCR. For the initial diagnosis of trichomoniasis, wet preparation is the test that is widely available in most STD clinics, but its sensitivity is poor (66.67%). Giemsa stain shows a low sensitivity of 52.38%. Acridine orange shows reasonable sensitivity and specificity of 71.43% and 99.44%, respectively. The sensitivity and specificity of PCR (80.95% and 97.21%) did not exceed that of culture. CONCLUSION: With regard to the fact that trichomoniasis can have an atypical or even asymptomatic course, in order to accurately diagnose this disease, microbiological investigation is necessary. Comparison of different methods showed that at least two techniques, such as culture and acridine orange staining, have the potential for better diagnosis of T. vaginalis infection. PCR detection of infection has been demonstrated to be highly specific and sensitive, but its availability and cost effectiveness are in question. PCR could provide an alternative for laboratory diagnosis of trichomoniasis by culture.