Carbenoxolone induces apoptosis and inhibits survivin and survivin-AEx3 genes expression in human leukemia K562 cells

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

[Growth inhibitory and apoptotic effects of carbenoxolone in human leukemia K562 cell line]

Leukemia is a malignant and progressive disorder in which genetic defects in hematopoietic cells lead to uncontrolled proliferation of blood cells. Different drugs have been proposed for the treatment of leukemia but none of them resulted in complete remission. Recently, anti-cancer effects of carbenoxolone [CBX], that is a 3- hemisuccinate, have been reported in several cell lines. In the present study we evaluated the effects of CBX on K562 cell line as an experimental model of chronic myeloid leukemia [CML]. K562 cells were cultured and treated for various time intervals with different concentrations of CBX [50-300 micro M]. Trypan blue exclusion test and tetrazolium salt absorption test [MTT] were used to evaluate the growth inhibitory and apoptotic effects of the drug. Fluorescence microscopy and DNA fragmentation assay were used to study apoptosis. The results of this study showed that CBX induced growth inhibition of K562 cells in a dose- and time- dependent manner. For example, growth inhibition rates after 48 hours treatment with concentrations of 50 micro m, 100 micro m, 150 micro m, 200 micro m and 300 micro m were 11%, 41%, 59%, 79% and 92%, respectively. Furthermore results of fluorescence microscopy and DNA fragmentation assays indicated that apoptosis, is the cause of cell death induced by CBX. Considering the growth inhibitory and apoptotic effects of CBX on human myeloid leukemia K562 cells, the drug can be considered as a potential candidate for further studies on CML treatment

[Effect of adenosine 5-triphosphate on cell cycle of human acute myeloid leukemia KG1 cells during S phase]

Adenosine 5-triphosphate [ATP] not only is the current energy source of all cells but also plays critical role in triggering signaling pathways leading to apoptosis or differentiation. In recent years many investigations have reported anti-cancer activity of ATP on different cell lines. Also several mechanisms have been proposed for its mechanism of action and it appears that the mechanism depends on the cell type. In the present study the effects of ATP on human leukemia KG1 cell line as an experimental model of AML and mechanistic approach was studied. KG1 cells were cultured and treated with different concentrations of the ATP [50-1000 micro M] for various time intervals [24-72 hours]. The Effect of ATP on cell proliferation was studied by MTT assay. Apoptosis was studied by flowcytometery, fluorescent microscopy and DNA fragmentation assay. Cell cycle was analyzed using flowcytometery. The effects of ATP gamma S [un-degradable agonist of ATP] and degradable products of ATP such as AMP, ADP and adenosine were studied to evaluate its mechanism of action. Data were analyzed by student-t-test [p<0.05]. ATP inhibited growth and induced S-phase cell cycle arrest at 24 h to 72 h in concentration between 100-1000 micro m [p<0.05] along with apoptosis. In addition, results showed that these effects of ATP on KG1 cell line were made through P2X[7]receptors. Because current AML therapy methods based on chemotherapy are not so effective and have side effects, according to the results of present study ATP can be used as an effective compound alone or in combination of other drugs to treat of AML

[Apoptosis induction and S-phase cell cycle arrest by adenosine 5-triphosphate through its degradation to adenosine in human myeloid leukemia K562 cell line]

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