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.

New anti-cancer characteristics of jatrophane diterpenes from Euphorbia dendroides.

Jatrophane diterpenes were shown to be inhibitors of P-glycoprotein (P-gp). There are also evidences on their microtubule-interacting activity in cancer cells. We evaluated new anti-cancer characteristics of two jatrophane type compounds from Euphorbia dendroides. For that purpose, the model system of sensitive non-small cell lung cancer cell line (NCI-H460) and its resistant counterpart (NCI-H460/R) was used. Although both jatrophanes showed inhibitory effect on cancer cell growth, they were non-toxic for peripheral blood mononuclear cells (PBMC). We examined their effects in combination with paclitaxel (PTX), a well-known mitotic spindle interacting chemotherapeutic. Jatrophanes overcome PTX resistance in concentration-dependent manner in MDR cancer cell line (NCI-H460/R). We observed that this synergistic effect is not caused merely by P-gp inhibition. In combination with PTX, jatrophanes induce cell killing and change cell cycle distribution leading to G2/M arrest. Furthermore, they exert an anti-angiogenic effect by decreasing the vascular endothelial growth factor (VEGF) secretion. The reduction of the level of mdr1 mRNA expression in sensitive cells, suggests that these compounds could not contribute to the development of resistance. In conclusion, present study provides a rational basis for the new cancer treatment approach with jatrophanes that are non-toxic to normal cells and have new favorable anti-cancer characteristics.

Isolation and biological evaluation of jatrophane diterpenoids from Euphorbia dendroides.

From the Montenegrin spurge Euphorbia dendroides, seven new diterpenoids [jatrophanes (1-6) and a tigliane (7)] were isolated and their structures elucidated by spectroscopic techniques. The biological activity of the new compounds was studied against four human cancer cell lines. The most effective jatrophane-type compound (2) and its structurally closely related derivative (1) were evaluated for their interactions with paclitaxel and doxorubicin using a multi-drug-resistant cancer cell line. Both compounds exerted a strong reversal potential resulting from inhibition of P-glycoprotein transport.

Sulfinosine enhances doxorubicin efficacy through synergism and by reversing multidrug resistance in the human non-small cell lung carcinoma cell line (NCI-H460/R).

A resistant non-small cell lung carcinoma cell line-NSCLC (NCI-H460/R) was established in order to investigate the potential of sulfinosine (SF) to overcome multidrug resistance (MDR). The cytotoxicity of doxorubicin (DOX) in NCI-H460/R cells was enhanced by interaction with SF. SF improved the sensitivity of resistant cells to DOX when NCI-H460/R cells were pretreated with SF. Synergism was accompanied by the accumulation of cells in S and G(2)/M phases. Pretreatment with SF was more potent in improving the sensitivity to DOX than verapamil (VER). The decrease of mdr1 and topo II alpha expression (assessed by RT-PCR), was consistent with the DOX accumulation assay and cell cycle analysis. Also, SF significantly decreased intracellular glutathione (GSH) concentration. These results point to SF as a potential agent of MDR reversal and a valuable drug for improving chemotherapy of NSCLC.

Synergistic effects of the purine analog sulfinosine and curcumin on the multidrug resistant human non-small cell lung carcinoma cell line (NCI-H460/R).

Multidrug resistance (MDR) is the main obstacle to a successful chemotherapy of lung cancer. We tested the potential of sulfinosine and curcumin, alone and in combination, for modulating MDR in the human resistant, non-small cell lung carcinoma cell line (NCI-H460/R). First, we determined the mutational status of the p53 gene in NCI-H460/R cells by PCR-SSCP and DNA sequencing and identified mutations which could at least partially contribute to the development of the MDR phenotype. The effects of sulfinosine and curcumin were studied, both separately and in combination, at the level of cytotoxicity, cell cycle distribution and gene expression. Sulfinosine displayed dose-dependent growth inhibition in both resistant and control sensitive cell lines, whereas curcumin considerably inhibited their growth only at relatively high doses. When sulfinosine was combined with a low dose of curcumin the drugs exerted a synergistic cytotoxic effect in NCI-H460/R cells. The expression of MDR-related genes mdr1, gst-pi and topo IIalpha, was altered by sulfinosine and curcumin. The most pronounced effect was observed when the agents were applied together. Sulfinosine and curcumin caused perturbations in cell cycle distribution in the NCI-H460/R cell line. The combination of the two drugs induced a more pronounced cell cycle arrest in S and G(2)/M in NCI-H460/R cells. Our results show that sulfinosine and curcumin overcome MDR in non-small cell lung carcinoma cell line (NSCLC), especially in combination despite the presence of a mutated p53 gene.

The kinetics of hypoxanthine transport across the perfused choroid plexus of the sheep.

The uptake of principal salvageable nucleobase hypoxanthine was investigated across the basolateral membrane of the sheep choroid plexus (CP) perfused in situ. The results suggest that hypoxanthine uptake was Na+-independent, which means that transport system on the basolateral membrane can mediate the transport in both directions. Although the unlabelled nucleosides adenosine and inosine markedly reduce the transport it seems that this inhibition was due to nucleoside degradation into nucleobases in the cells, since non-metabolised nucleoside analogue NBTI did not inhibit the transport. The presence of adenine also inhibits hypoxanthine uptake while the addition of the pyrimidines does not show any effect, so it seems that the transport of purine nucleobases through basolateral membrane is mediated via a common transporter which is different from the nucleoside transporters. The inclusion of allopurinol in the perfusion fluid did not change the value and general shape of the curve for the uptake which suggest that degradation of hypoxanthine into xanthine and uric acid does not occur in the CP. The capacity of the CP basolateral membrane to transport hypoxanthine is high (90.63+/-3.79 nM/min/g) and close to the values obtained for some essential amino acids by the CP and blood-brain barrier, while the free diffusion is negligible. The derived value of Km (20.72+/-2.42 microM) is higher than the concentration of hypoxanthine in the sheep plasma (15.61+/-2.28 microM) but less than a half of the concentration in the CSF, which indicates that the transport system at basolateral membrane mostly mediates the efflux of hypoxanthine from the cerebrospinal fluid in vivo.