(c) The effect of Indo alone (light columns) and combination with ATO 2? em /em M (dark columns) on COX-2 expression. 3.4. IC50 as single agent, we hypothesized that combination of these drugs would be more efficient to suppress the growth of the cells. We also tested the effect of Dex on the cytotoxic effect of ATO. Thus, cells were treated with combinations of Indo (1, 10, and 50? 0.001 and * 0.05, compared to ATO alone). These results suggest that noneffective dose of Indo (10? 0.001) and Indo 50?= 4; * 0.05, ** 0.01 and *** 0.001). 3.3. ATO Decreases the Expression of COX-2 mRNA Dose-Dependently Considering the role of COX-2 and COX inhibition in lung cancer [26], we have assessed the mRNA expression of COX-2 with different concentrations of ATO as well as ATO 2?= 3). (c) The effect of Indo alone (light columns) and combination with ATO 2? em /em M (dark columns) on COX-2 expression. 3.4. Expression of Cox-2, Akt, ERK1/2, p38, JNK, and Bax Proteins in the Cells Treated with ATO, Indo, Dex, ATO/Indo, and ATO/Dex Combinations To address the role of proteins involved in the apoptosis and survival, the expression of Akt, ERK1/2, p38, JNK, and Bax proteins was determined by western blotting analysis. The expression of COX-2 protein decreased dose-dependently by ATO especially in the dose of 50? em /em M (Figure 5). Indo alone did not change the expression of COX-2 protein. However, combination of ATO 2? em /em M and Indo (2 and 10? em /em M) decreased the COX-2 protein expression. ERK1/2 and p38 proteins levels were decreased with 50? em /em M ATO treatment but remained unchanged with other treatments. Akt, Bax, and JNK seemed to be unchanged with different treatments. Open in a separate window Figure 5 Western blot analysis of COX-2, Akt, ERK1/2, p38, JNK, and Bax proteins in A549 cells treated with ATO, Indo, Dex, ATO + Indo, and ATO + Dex combinations. Dex alone and in combination with ATO decreased expression of COX-2 protein completely. Furthermore, Dex decreased p38 and ERK1/2 proteins expressions dose-dependently which remained unaltered in combination with ATO. 3.5. ERK and p38 Proteins Were Highly Phosphorylated in the Cells Treated with ATO/Indo Combination Since the change in the total ERK and p38 protein expressions was remarkable, we investigated the phosphorylation of ERK and p38 proteins in the ATO, Indo and ATO/Indo treatments. As shown in Figure 6, treatment of A549 cells with ATO and Indo alone lowered the phospho-ERK at 24?hrs; however, in cells treated with both ATO/Indo, the phosphorylation of ERK was increased and reached maximum level at 24?hr. Phosphorylation of p38 did not change in ATO and Indo single treatments. However, combination of ATO/Indo induced phosphorylation of p38 at 4?hrs and increased phospho-p38 to a remarkable level at 24?hr, suggesting a synergistic effect of combination treatment on p38 pathway activation. Open in a separate window Figure 6 Phosphorylation of p38 and ERK in A549 cells treated with ATO, Indo, and ATO/Indo combination. 3.6. Both ATO and Indo Activate Caspase-3 To address the part of caspase-3 in the cytotoxicity of ATO, Indo, and ATO/Indo combination, the caspase-3 activity was measured. As demonstrated in Number 7, caspase-3 activity improved 1.2 and 1.6 collapse with ATO 2? em /em M and Indo 10? em /em M, respectively. Increase in the caspase-3 activity in the cells treated with combination of ATO 2? em /em M and Indo 10? em /em M was related to that of Indo 10? em /em M. Caspase-3 inhibitor-treated cell lysate control showed that maybe some other caspases are becoming triggered in the treated cells. Open in a separate window Number 7 Activation of caspase-3 in A549 cells treated with ATO, Indo, and ATO/Indo combination. 4. Discussion Combination therapy with providers that hire different signaling pathways is definitely a promising strategy for overcoming drug Gw274150 resistance in cancerous cells and for increasing treatment effectiveness and/or decreasing drug toxicity [27]. The present findings show that Indo increases the cytotoxicity of ATO in human being lung malignancy cell collection A549. This event is definitely associated with activation of ERK and p38 MAPK signaling pathways. Our study showed that ATO, Cel, and Indo dose-dependently decreased cell viability (IC50s are 68.7, 98.2, and 396.5? em /em M, resp.). Considering this effect, Indo produces very weak effect (IC50 = 396.5? em /em M) compared to two additional medicines. It has been demonstrated that ATO mediates its cytotoxic effect by induction of apoptosis via disruption of mitochondrial transmembrane potentials and caspase-3 activation in various cell lines and human being lymphocytes [18, 28]. Consequently, the cytotoxic effect of arsenic is related to apoptotic pathways since caspase-3 has been.Akt, Bax, and JNK Gw274150 seemed to be unchanged with different treatments. Open in a separate window Figure 5 Western blot analysis of COX-2, Akt, ERK1/2, p38, JNK, and Bax proteins in A549 cells treated with ATO, Indo, Dex, ATO + Indo, and ATO + Dex combinations. Dex only and in combination with ATO decreased manifestation of COX-2 protein completely. with Either Agent Only Because ATO, Cel, and Indo experienced fairly high IC50 as solitary agent, we hypothesized that combination of these medicines would be more efficient to suppress the growth of the cells. We also tested the effect of Dex within Rabbit Polyclonal to CYSLTR1 the cytotoxic effect of ATO. Therefore, cells were treated with mixtures of Indo (1, 10, and 50? 0.001 and * 0.05, compared to ATO alone). These results suggest that noneffective dose of Indo (10? 0.001) and Indo 50?= 4; * 0.05, ** 0.01 and *** 0.001). 3.3. ATO Decreases the Manifestation of COX-2 mRNA Dose-Dependently Considering the part of COX-2 and COX inhibition in lung malignancy [26], we have assessed the mRNA manifestation of COX-2 with different concentrations of ATO as well as ATO 2?= 3). (c) The effect of Indo only (light columns) and combination with ATO 2? em /em M (dark columns) on COX-2 manifestation. 3.4. Manifestation of Cox-2, Akt, ERK1/2, p38, JNK, and Bax Proteins in the Cells Treated with ATO, Indo, Dex, ATO/Indo, and ATO/Dex Mixtures To address the part of proteins involved in the apoptosis and survival, the manifestation of Akt, ERK1/2, p38, JNK, and Bax proteins was determined by western blotting analysis. The manifestation of COX-2 protein decreased dose-dependently by ATO especially in the dose of 50? em /em M (Number 5). Indo only did not switch the manifestation of COX-2 protein. However, combination of ATO 2? em /em M and Indo (2 and 10? em /em M) decreased the COX-2 protein manifestation. ERK1/2 and p38 proteins levels were decreased with 50? em /em M ATO treatment but remained unchanged with additional treatments. Akt, Bax, and JNK seemed to be unchanged with different treatments. Open in a separate window Number 5 Western blot analysis of COX-2, Akt, ERK1/2, p38, JNK, and Bax proteins in A549 cells treated with ATO, Indo, Dex, ATO + Indo, and ATO + Dex mixtures. Dex only and in combination with ATO decreased manifestation of COX-2 protein completely. Furthermore, Dex decreased p38 and ERK1/2 proteins expressions dose-dependently which remained unaltered in combination with ATO. 3.5. ERK and p38 Proteins Were Highly Phosphorylated in the Cells Treated with ATO/Indo Combination Since the switch in the total ERK and p38 protein expressions was impressive, we investigated the phosphorylation of ERK and p38 proteins in the ATO, Indo and ATO/Indo treatments. As demonstrated in Number 6, treatment of A549 cells with ATO and Indo only lowered the phospho-ERK at Gw274150 24?hrs; however, in cells treated with both ATO/Indo, the phosphorylation of ERK was improved and reached maximum level at 24?hr. Phosphorylation of p38 did not switch in ATO and Indo solitary treatments. However, combination of ATO/Indo induced phosphorylation of p38 at 4?hrs and increased phospho-p38 to a remarkable level at 24?hr, suggesting a synergistic effect of combination treatment about p38 pathway activation. Open in a separate window Number 6 Phosphorylation of p38 and ERK in A549 cells treated with ATO, Indo, and ATO/Indo combination. 3.6. Both ATO and Indo Activate Caspase-3 To address the part of caspase-3 in the cytotoxicity of ATO, Indo, and ATO/Indo combination, the caspase-3 activity was measured. As demonstrated in Number 7, caspase-3 activity improved 1.2 and 1.6 collapse with ATO 2? em /em M and Indo 10? Gw274150 em /em M, respectively. Increase in the caspase-3 activity in the cells treated with combination of ATO 2? em /em M and Indo 10? em /em M was related to that of Indo 10? em /em M. Caspase-3 inhibitor-treated cell lysate control.