ABSTRACT
Leukemia is a malignant disorder of the blood progenitor/stem cells which is characterized by abnormal proliferation of white blood cells. Although anti-cancer drugs induce apoptosis in cancerous cells, drug resistance is the significant problem mainly due to over-expression of inhibitors of apoptosis proteins [lAPs] such as survivin. In this content, it has been reported that an anti-inflammatory drug, Carbenoxolone [CBX], could induce apoptosis and growth inhibition in several types of cancerous cells. In the present study, effects of CBX on apoptosis and level of the expression of survivin gene and its deltaEx3 splicing variant have were evaluated in K562 cells. K562 cells were cultured and treated with different concentrations of CBX: [50-300 microM] at different time intervals [12-48 hrs]. Trypan blue exclusion test was used to evaluate cell viability. Fluorescent microscopy [Acridine Orange/Ethidium Bromide double staining] and DNA fragmentation assay were used to study apoptosis. The expression level of survivin and its deltaEx3 splice variant were studied by RT- PCR. It was found that both growth inhibition and apoptosis occurred in K562 cells. In addition, down-regulation of survivin and survin-deltaEx3 were observed, after 2-4 hrs treatment with 150 microM of CBX. However, the expression level of survivin and its deltaEx3 splice variant increased in subsequent time [6-12 hrs] nearly to the level of control cells. From the results of this study, it may be concluded that CBX can be considered as a candidate for further studies in CML treatment, especially in the case of drug- resistant leukemia cells
ABSTRACT
Adenosine 5-triphosphate [ATP] is not only the current energy sources for all cells but also plays a critical role in triggering signaling pathways leading to apoptosis or differentiation. During the past years many investigations have been reported the anti-cancer activity of ATP on different cell lines. Also several mechanisms have been proposed for its action and it appears that its mechanism of action depends on the cell type. In the present study effects of ATP on human leukemia K562 cell line as an experimental model of CML and it mechanism of action were studied. K562 cells were cultured and treated with different concentrations of ATP [50-1000 microM] at various time intervals [24-72 h]. Effect of ATP on cell proliferation was studied by MTT assay. Apoptosis was studied by flow cytometry and DNA fragmentation assay. Cell cycle and DNA contents were analyzed by flow cytometry. To evaluate the mechanism of action of ATP, effects of ATPgammaS [an undegradable agonist of ATP] and products of ATP degradation such as AMP, ADP and adenosine were studied. ATP with doses of 100-1000 microM inhibited growth of the cells and induced S-phase cell cycle arrest at the time intervals of 24-72 hour. These effects of ATP led to cell death by apoptosis. In addition, the results showed that these effects of ATP were through its degradation to adenosine and eventually induction of pyrimidine starvation. Because current CML therapy methods which are based on chemotherapy are not very effective and have side effects such as drug resistance, ATP can be proposed as an effective compound alone or in combination with other drugs for treatment of CML