Effect of andrographolide and TP on cell proliferation
When applied separately, both andrographolide and TP exhibited antiproliferative effects on U937 cell line in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) of 22.25 μM with andro treatment and 0.65 μM with TP treatment at 24 h (Figures 1 and 2). Cell proliferation upon treatment with various combinations of TP and andrographolide was then assessed. Co-treating the cells with both andrographolide (10 μM) and TP simultaneously at various concentrations did not exhibit any significant changes in cell proliferation when compared to treating the cells with both drugs separately (Figure 2). However, pretreating the cells with andrographolide (10 μM) for 24 h prior to TP addition (0.05, 0.1, 0.15, and 0.3 μM) led to a significant decrease in cell proliferation (P≤0.0001) as compared to treatment with the compounds separately. The optimal conditions that caused a significant reduction in proliferation were the pretreatment combination of 10 μM of andrographolide prior to 0.3 μM of TP, leading to a decrease in cell proliferation from 67% to 39% with P ≤0.0001 (Figure 3).
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Effect of andrographolide and TP on cell-cycle arrest
After determining the optimal combination range that has inhibited cell proliferation in U937 upon pretreatment with andrographolide before TP application, flow cytometry was used to assess the effect of this combination on cell-cycle arrest. Cells were pretreated with 10 μM of andrographolide followed by 0.05, 0.1, 0.15, and 0.3 μM of TP. After fixation with ethanol, the cells were stained with PI stain and then the DNA content of cells was analyzed using the C6 flow cytometer and cells were accordingly assigned to their respective phases: pre-G1 cells were <2n, G0/G1 cells were 2n, and S/M phase cells were >2n. When applied separately on the cells, TP caused a specific cell-cycle arrest at the S phase in addition to an increase in the percentage of cells in the pre-G1 phase at concentrations ≥0.3 μM (Figure 4A). These alterations in cell-cycle progression were further enhanced upon andrographolide pretreatment, even though andrographolide when applied alone did not show any significant change in the distribution of cells at the various stages of the cell cycle. Again, the pronounced synergistic effect was observed upon pretreatment with andrographolide followed by 0.3 μM of TP, which led to an increase in the percentage of cells in the S phase, reaching 40.4%, compared to 19.3% upon treatment with TP alone (Figure 4B).
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Effect of andrographolide and TP on apoptosis
Annexin V/PI staining was used to determine if U937 cells died of apoptosis or necrosis upon the application of both compounds separately and after pretreatment using the same TP concentrations (0.05, 0.1, 0.15, and 0.3 μM). Upon combination of TP with andrographolide, the proapoptotic effect was further observed even though andrographolide alone did not show any alteration in cell survival. However, pretreating the cells with andrographolide prior to 0.3 μM of TP treatment resulted in a significant increase in the number of apoptotic cells from 16% to 57% (Figure 5).
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Effect of andrographolide and TP on the proapoptotic and antiproliferative pathways
To determine the metabolic pathway through which the combination of andrographolide and TP is inhibiting the cell proliferation of U937, Western blot analysis was performed to assess the expression of a series of proteins related to different pathways. The cells were treated with either TP only at various concentrations (0.05, 0.1, 0.15, and 0.3 μM) or pretreated with andrographolide followed by the same concentrations of TP. Beta-actin was used to ensure equal loading. The expression of both p53 and cytochrome c did not change with TP alone, while it showed a dose-dependent upregulation upon the pretreatment with andrographolide. The proapoptotic effect of andrographolide and TP was assessed by measuring the expression of cleaved PARP, caspase-9, pro-caspase-3, and cleaved caspase-3. Cleaved PARP and caspase-9 showed a significant upregulation upon the separate treatment with TP and pretreatment with andrographolide, but the upregulation was more intense upon the combination of both compounds. Pro-caspase-3 appeared to be cleaved into its active form with increasing doses in both conditions with a more significant effect upon combination. Also, the upregulation in the expression of Bax was observed to be higher upon pretreating the cells with andrographolide; Bcl2 expression was downregulated with TP alone but upregulated with the combination. These results confirm that apoptosis was induced upon TP treatment through an intrinsic pathway and was further enhanced with the pretreatment of andrographolide (Figure 6).
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