Dehydroleucodine inhibits tumor growth in a preclinical melanoma model by inducing cell cycle arrest, senescence and apoptosis.

Malignant melanoma represents the fastest growing public health risk of all cancer types worldwide. Several strategies and anti-cancer drugs have been used in an effort to improve treatments, but the development of resistance to anti-neoplastic drugs remains the major cause of chemotherapy failure in melanomas. Previously, we showed that the sesquiterpene lactone, dehydroleucodine (DhL), promotes the accumulation of DNA damage markers, such as H2AX and 53BP1, in human tumor cells. Also DhL was shown to trigger either cell senescence or apoptosis in a concentration-dependent manner in HeLa and MCF7 cells. Here, we evaluated the effects of DhL on B16F0 mouse melanoma cells in vitro and in a pre-clinical melanoma model. DhL inhibited the proliferation of B16F0 cells by inducing senescence or apoptosis in a concentration-dependent manner. Also, DhL reduced the expression of the cell cycle proteins cyclin D1 and B1 and the inhibitor of apoptosis protein, survivin. In melanomas generated by subcutaneous injection of B16F0 cells into C57/BL6 mice, the treatment with 20 mg DhL /Kg/day in preventive, simultaneous and therapeutic protocols reduced tumor volumes by 70%, 60% and 50%, respectively. DhL treatments reduced the number of proliferating, while increasing the number of senescent and apoptotic tumor cells. To estimate the long-term effects of DhL, a mathematical model was applied to fit experimental data. Extrapolation beyond experimental time points revealed that DhL administration following preventive and therapeutic protocols is predicted to be more effective than simultaneous treatments with DhL in restricting tumor growth.

Phospholipase A1: a novel virulence factor in Trypanosoma cruzi.

Phospholipase A1 (PLA1) has been described in the infective stages of Trypanosoma cruzi as a membrane-bound/secreted enzyme that significantly modified host cell lipid profile with generation of second lipid messengers and concomitant activation of protein kinase C. In the present work we determined higher levels of PLA1 expression in the infective amastigotes and trypomastigotes than in the non-infective epimastigotes of lethal RA strain. In addition, we found similar expression patterns but distinct PLA1 activity levels in bloodstream trypomastigotes from Cvd and RA (lethal) and K98 (non-lethal) T. cruzi strains, obtained at their corresponding parasitemia peaks. This fact was likely due to the presence of different levels of anti-T. cruzi PLA1 antibodies in sera of infected mice, that modulated the enzyme activity. Moreover, these antibodies significantly reduced in vitro parasite invasion indicating the participation of T. cruzi PLA1 in the early events of parasite-host cell interaction. We also demonstrated the presence of lysophospholipase activity in live infective stages that could account for self-protection against the toxic lysophospholipids generated by T. cruzi PLA1 action. At the genome level, we identified at least eight putative genes that codify for T. cruzi PLA1 with high amino acid sequence variability in their amino and carboxy-terminal regions; a putative PLA1 selected gene was cloned and expressed as a recombinant protein that possessed PLA1 activity. Collectively, the results presented here point out at T. cruzi PLA1 as a novel virulence factor implicated in parasite invasion.

The sesquiterpene lactone dehydroleucodine triggers senescence and apoptosis in association with accumulation of DNA damage markers.

Sesquiterpene lactones (SLs) are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL), a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser), a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models.

Electrophoretic analysis of protein selectively extracted with chaotropic agents from membranes of rat sperm

Sperm from rat cauda epididymis was washed, sonicated and centrifuged to obtain fractions sedimenting at 600 x g for 5 min, 27.000 x g for 5 min, and 100.000 x g for 40 min. All fractions were observed with the electron microscopy and assayed for cytochrome c oxidase activity. The 100.000 x g fraction contained only small membranous vesicles and less than 0.5 of the total enzymatic activity. This fraction was considered to represent sperm plasmalemma and it was extracted with Tris-HCl buffer before treating it with one of the following chemicals: acetate buffer, pH: 4.5; 0.6 M KCl; bicarbonate buffer, pH 11.0; Triton X-100, and Sodium Dodecyl Sulfate (SDS). After centrifuging, the residual sediments were solubilized in hot 2 SDS. The extracts and the solubilized sediments (hot SDS) were analyzed in SDS-PAGE. The extracts obtained with the first three chemicals contained 11,9, and 25 of total proteins respectively. The bicarbonate buffer solubilized 45, and the detergents 55 and 65 respectively. A total of 30 bands were seen in the extracts and sediments. Acid pH extracted a low number of bands of high mobility and low molecular weight. Instead, the KCl and bicarbonate buffer, extracted a great number of bands over a wide range of molecular weights (23, 38.5, 55, 100, and 140 KD). The detergents had similar effects: both solubilized four new bands. In residual sediments there were no new proteins and the bands corresponded to those extracted with the detergents, but they varied in staining intensity. According to the results obtained with the mild chaotropic agents of 0.6 M KCl and bicarbonate buffer, 50 of the mass of membraneous proteins may be peripheric. Proteins partially extracted with the detergents were also found in the residual sediment, and they may constitute the skeleton of sperm membrane.(AU)

Electrophoretic analysis of protein selectively extracted with chaotropic agents from membranes of rat sperm

Sperm from rat cauda epididymis was washed, sonicated and centrifuged to obtain fractions sedimenting at 600 x g for 5 min, 27.000 x g for 5 min, and 100.000 x g for 40 min. All fractions were observed with the electron microscopy and assayed for cytochrome c oxidase activity. The 100.000 x g fraction contained only small membranous vesicles and less than 0.5 of the total enzymatic activity. This fraction was considered to represent sperm plasmalemma and it was extracted with Tris-HCl buffer before treating it with one of the following chemicals: acetate buffer, pH: 4.5; 0.6 M KCl; bicarbonate buffer, pH 11.0; Triton X-100, and Sodium Dodecyl Sulfate (SDS). After centrifuging, the residual sediments were solubilized in hot 2 SDS. The extracts and the solubilized sediments (hot SDS) were analyzed in SDS-PAGE. The extracts obtained with the first three chemicals contained 11,9, and 25 of total proteins respectively. The bicarbonate buffer solubilized 45, and the detergents 55 and 65 respectively. A total of 30 bands were seen in the extracts and sediments. Acid pH extracted a low number of bands of high mobility and low molecular weight. Instead, the KCl and bicarbonate buffer, extracted a great number of bands over a wide range of molecular weights (23, 38.5, 55, 100, and 140 KD). The detergents had similar effects: both solubilized four new bands. In residual sediments there were no new proteins and the bands corresponded to those extracted with the detergents, but they varied in staining intensity. According to the results obtained with the mild chaotropic agents of 0.6 M KCl and bicarbonate buffer, 50 of the mass of membraneous proteins may be peripheric. Proteins partially extracted with the detergents were also found in the residual sediment, and they may constitute the skeleton of sperm membrane.