Sinalização de morte celular via NA+, K+-ATPase como alvo de drogas anticâncer: estudos com o álcool perílico / Signaling of cell death via NA+, K+ -atpase as a target of anticancer drugs: studies with perillyl alcohol

A Na+, K+ ATPase (NKA) atua na manutenção do potencial de membrana dascélulas e em mecanismos de transdução de sinal. Alterações na atividade da NKAsão importantes em muitos processos biológicos e patológicos. A NKA pode serinibida pelo álcool perílico (POH), um monoterpeno utilizado no tratamento detumores, incluindo cerebrais. Neste trabalho, determinamos que o POH tambématua sobre cascatas de sinalização, moduladas via NKA, que controlam aproliferação e/ou morte celular. Foi avaliado o efeito do POH e do PA seu principalmetabólito, sobre a atividade da NKA em duas linhagens de células de glioblastoma(GBM) humano (U87 e U251), células não tumorais de astrócitos de camundongo eda linhagem (VERO). Nossos resultados, baseados na avaliação da atividade daNKA por incorporação do Rb+ , o qual mimetiza o K+ , mostraram uma sensibilidade àinibição pelo POH semelhante entre os quatro tipos de células (IC50 U87 2 mM;U251 1,8 mM; VERO 2,4 mM e astrócitos de camundongo 1,4 mM), enquanto o PAnão apresentou efeito. Sabe-se que nos GBMs existe uma superexpressão dasubunidade α1 da NKA, situada na estrutura das cavéolas que é provavelmenteresponsável pelo papel sinalizador atribuído a essa enzima, especialmente emrelação aos mecanismos apoptóticos. Comparamos a viabilidade celular,determinando a atividade da enzima lactato desidrogenase presente nosobrenadante das células tratadas por 24 h com POH e PA. O PA não alcançouefeito citotóxico igual ou superior a 30 por cento nas células mesmo na concentraçãoelevada de 4 mM. Já o POH reduziu, de maneira dependente da concentração, aviabilidade das células (IC50 U87 1,1 mM; U251 1,4 mM; VERO 0,9 mM e astrócitosde camundongo 1,4 mM). Na análise por western blot, 1,5 mM de POH ativou aproteína c-Jun N-terminal quinase (JNK), nas células U87, U251 e nos astrócitos decamundongo (incubação de 30 min)...

The Na+, K+ ATPase (NKA) acts in keeping the cell membrane potential and in signaltransduction mechanisms. Modifications in the activity of this enzyme are important inphysiological and pathological processes. The NKA is inhibited by perillyl alcohol(POH), a monotherpene used in the treatment of tumors, including brain tumors. Inthis work, we also show that POH acts in signaling cascades associated to NKA,controlling cell proliferation and/or cellular death. We evaluated the effect of POH andof its main metabolite (perillic acid - PA) on the NKA activity in cultured glioblastomacells (GBM) U87 and U251 and on non-tumor cells (mouse astrocytes and VEROcells). NAK activity was measured by non-radioactive Rb+incorporation by cells (Rb+is a K+substitute). Our results showed a similar sensitivity for the four cells typestested (IC50 U87 – 2 mM; U251 - 1,8 mM; VERO - 2,4 mM and mouse astrocytes -1,4 mM). Perillic acid did not show any effect in any cell type. In GBMs, it is knownthat NKA α1 subunit is super expressed. This isoform is embedded in caveolarstructures and is probably responsible by the signaling properties of this enzyme inapoptosis mechanisms. Cell viability was measured by lactate dehydrogenase in cellsupernatants of POH treated cells. The maximum PA cytotoxic effect obtained was30 percent even at 4 mm. However, POH reduced dose dependently cell viability, (IC50 U87- 1,1 mM; U251 - 1,4 mM; VERO - 0,9 mM and mouse astrocyte - 1,4 mM).Considering the western blot analysis, 1,5 mM POH activated the c-Jun N-terminalKinase (JNK), on U87, U251 and in mouse astrocytes after 30min incubation...

Variation in bioactive principles of Artemisia amygdalina Decne. in wild and tissue culture regenerants

Wild and tissue culture raised regenerants of Artemisia amygdalina, a critically endangered and endemic plant of Kashmir and North West Frontier Provinces of Pakistan were screened for the amount of bioactive principles and in particular antimalarial compound artemesinin. Phytochemical screening of extracts revealed the presence of terpenes, alkaloids, phenolics, tannins [polyphenolics], cardiac glycosides and steroids in wild [aerial, inflorescence] and tissue culture regenerants [in vitro grown plant, callus and green house acclimatized plants]. HPLC of Artemisia amygdalina revealed the presence of artemesinin in petroleum ether extracts of wild aerial part, tissue culture raised plant and green house acclimatized plants. Acetonitrile and water in 70:30 ratios at flow rate of 1ml/min was standardised as mobile phase. Retention time for standard chromatogram was 6.7. Wild inflorescences and callus does not produce artemesinin. This is the first report of phytochemical screening and artemesinin estimation of wild and tissue culture raised regenerants of Artemisia amygdalina

Toxic and sub-lethal effects of oleandrin on biochemical parameters of fresh water air breathing murrel, Channa punctatus (Bloch.).

Active compound oleandrin extracted from Nerium indicum (Lal Kaner) leaf has potent piscicidal activity. The piscicidal activity of oleandrin on freshwater fish C. punctatus was both time and dose dependent. Exposure to sub-lethal doses of oleandrin for 24hr and 96hr to fish caused significant alteration in the level of total protein, total free amino acid, nucleic acid, glycogen, pyruvate, lactate and enzyme protease, phosphatases, alanine aminotransferase, aspartate aminotransferase and acetylcholinesterase activity in liver and muscle tissues. The alterations in all the above biochemical parameters were also significantly time and dose dependent. The results show a significant recovery in all the above biochemical parameters, in both liver and muscle tissues of fish after the 7th day of the withdrawal of treatment. Toxicity persistence test of oleandrin on juvenile Labeo rohita shows that fish seed of common culturing carp can be released into rearing ponds after three days of oleandrin treatment. It supports the view that the oleandrin is safer and may be useful substitute of other piscicides for removing the unwanted freshwater fishes from aquaculture ponds.

Cardiac glycosides isolated from the Indian-snuff, Maquira sclerophylla Ducke

The hydroalcoholic extract of the powdered bark of the Indian-snuff Maquira sclerophylla Ducke was purified by column chromatography in silica-gel and the major cardenolide isolated from preparative TLC was identified by 1H-NMR, 1 2 C-NMR and IR analyses. The spectra showed that the active substance has strophanthidin as aglicone.