Dietary polyphenols influence antimetabolite agents: methotrexate, 6-mercaptopurine and 5-fluorouracil in leukemia cell lines

Abstract

Polyphenols have been previously shown to sensitize leukemia cell lines to topoisomerase inhibitors. Here, we assess the effects of five polyphenols when used alone and in combination with antimetabolites: methotrexate, 6-mercaptopurine and 5-fluorouracil; in lymphoid and myeloid leukemia cells lines, and non-tumor control cells. The effects of combined treatments were investigated on ATP and glutathione levels, cell-cycle progression, DNA damage and apoptosis. Polyphenols antagonized methotrexate and 6-mercaptopurine induced cell-cycle arrest and apoptosis in most leukemia cell lines. This was associated with reduced DNA damage and increased glutathione levels, greater than that seen following individual treatments alone. In contrast, 5-fluorouracil when combined with quercetin, apigenin and rhein caused synergistic decrease in ATP levels, induction of cell-cycle arrest and apoptosis in some leukemia cell lines. However, antagonistic effects were observed when 5-fluorouracil was combined with rhein and cis-stilbene in myeloid cell lines. The effects were dependant on polyphenol type and chemotherapy agent investigated, and cell type treated. Interestingly treatment of non-tumor control cells with polyphenols protected cells from antimetabolite treatments. This suggests that polyphenols modulate the action of antimetabolite agents; more importantly they antagonized methotrexate and 6-mercaptopurine actions, thus suggesting the requirement of polyphenol-exclusion during their use.

Keywords: 5-fluorouracil; 6-mercaptopurine; leukemia; methotrexate; polyphenols.

Figure 1

Effect of methotrexate, 6-mercaptopurine and 5-fluorouracil alone on ATP levels (A, B & C) and caspase 3 activity (D, E & F) in two lymphoid (CCRF-CEM and Jurkat), two myeloid (THP-1 and KG-1a) leukemia cell lines, and two non-tumor control cells (CD133⁺ HSC and CD34⁺ HSC). The lowest significant doses (LSD): which caused a significant reduction on ATP levels and induction of caspase 3 activity when compared to the vehicle control were determined for each anti-metabolite agent in each cell lines. The ^* indicated for LSD in each cell line.

Figure 2. The effect of methotrexate (MTX), 6-mercaptopurine (6-MP) and 5-fluorouracil (5-FLU) when used in combination with apigenin (AP) on ATP levels: in two lymphoid (Jurkat and CCRF-CEM) and two myeloid (THP-1 and KG-1a) leukemia cell lines, evaluated by CellTiter-Glo® assay

Cells were treated with MTX, 6-MP or 5-FLU and apigenin alone and in combination for 24 hr using their lowest-significant doses (LSD); together with a vehicle control. All data was normalised to the vehicle control which was assigned 100% cell viability. The data was expressed as medians and ranges (n=4). Effects of combination treatments were statistically classified as synergistic (^*) causing a decrease in ATP levels or antagonistic (^#) causing an increase in ATP levels; when compared to vehicle control, drugs alone and expected values of combination treatments. Statistical significant was set at P≤0.05.

Figure 3. The effect of methotrexate (MTX), 6-mercaptopurine (6-MP) and 5-fluorouracil (5-FLU) when used in combination with rhein (RH) on ATP levels: in two lymphoid (Jurkat and CCRF-CEM) and two myeloid (THP-1 and KG-1a) leukemia cell lines

This was evaluated by CellTiter-Glo® assay. Cells were treated with MTX, 6-MP or 5-FLU and rhein alone and in combination for 24 hr using their lowest-significant doses (LSD); together with a vehicle control. All data was normalised to the vehicle control which was assigned 100% cell viability. The data was expressed as medians and ranges (n=4). Effects of combination treatments were statistically classified as synergistic (^*) causing a decrease in ATP levels or antagonistic (^#) causing an increase in ATP levels; when compared to vehicle control, drugs alone and expected values of combination treatments. Statistical significant was set at P≤0.05.

Figure 4. The effect of methotrexate (MTX), 6-mercaptopurine (6-MP) and 5-fluorouracil (5-FLU) when used in combination with apigenin (AP) on caspase 3 activity: in two lymphoid (Jurkat and CCRF-CEM) and two myeloid (THP-1 and KG-1a) leukemia cell lines

This was evaluated by NucView caspase 3 activity assay. Cells were treated with MTX, 6-MP or 5-FLU and apigenin alone and in combination for 24 hr using their lowest-significant doses (LSD); together with a vehicle control. All data was normalised to the vehicle control which was assigned 0% apoptosis. The data was expressed as medians and ranges (n=4). Effects of combination treatments were statistically classified as synergistic (^*) causing an increase in caspase 3 activity or antagonistic (^#) causing an decrease in caspase 3 activity; when compared to vehicle control, drugs alone and expected values of combination treatments. Statistical significant was set at P≤0.05.

Figure 5. The effect of methotrexate (MTX), 6-mercaptopurine (6-MP) and 5-fluorouracil (5-FLU) when used in combination with rhein (RH) on caspase 3 activity: in two lymphoid (Jurkat and CCRF-CEM) and two myeloid (THP-1 and KG-1a) leukemia cell lines

This was evaluated by NucView caspase 3 activity assay. Cells were treated with MTX, 6-MP or 5-FLU and rhein alone and in combination for 24 hr using their lowest-significant doses (LSD); together with a vehicle control. All data was normalised to the vehicle control which was assigned 0% apoptosis. The data was expressed as medians and ranges (n=4). Effects of combination treatments were statistically classified as synergistic (^*) causing an increase in caspase 3 activity or antagonistic (^#) causing an decrease in caspase 3 activity; when compared to vehicle control, drugs alone and expected values of combination treatments. Statistical significant was set at P≤0.05.

Figure 6. The effect of methotrexate (MTX), 6-mercaptopurine (6-MP) and 5-fluorouracil (5-FLU) on cell cycle progression, when used in combination with apigenin (AP): in two lymphoid leukemia cell lines (Jurkat and CCRF-CEM) and two myeloid leukemia cell lines (THP-1 and KG-1a)

This was analysed by flow cytometry following propidium iodide staining. Cells were treated with MTX, 6-MP or 5-FLU and apigenin alone and in combination for 24 hrs using their lowest-significant doses (LSD) as determined by CellTiter-Glo assay, together with a vehicle control. The percentage of cells in each phase was analysed with FlowJo software using Waston pragmatic model. The data was expressed as medians with ranges (n=4). Statistical significance of combination treatments were determined and compared with the vehicle control and the individual treatments alone. The green asterisk (^*) represents significant increase in cell accumulation in a phase of the cell cycle; whilst the black asterisk (^*) indicates a significant decrease in cell accumulation in a phase of the cell cycle. Statistical significant was set at P≤0.05.

Figure 7. The effect of methotrexate (MTX), 6-mercaptopurine (6-MP) and 5-fluorouracil (5-FLU) on cell cycle progression, when used in combination with rhein (RH): in two lymphoid leukemia cell lines (Jurkat and CCRF-CEM) and two myeloid leukemia cell lines (THP-1 and KG-1a)

This was analysed by flow cytometry following propidium iodide staining. Cells were treated with MTX, 6-MP or 5-FLU and rhein alone and in combination for 24 hrs using their lowest-significant doses (LSD) as determined by CellTiter-Glo assay, together with a vehicle control. The percentage of cells in each phase was analysed with FlowJo software using Waston pragmatic model. The data was expressed as medians with ranges (n=4). Statistical significance of combination treatments were determined and compared with the vehicle control and the individual treatments alone. The green asterisk (^*) represents significant increase in cell accumulation in a phase of the cell cycle; whilst the black asterisk (^*) indicates a significant decrease in cell accumulation in a phase of the cell cycle. Statistical significant was set at P≤0.05.

Figure 8. The effect of methotrexate (MTX) and 6-mercaptopurine (6-MP) when used in combination with quercetin (QUE), apigenin (AP), emodin (EMO), rhein, (RH) or cis-stilbene (CIS) on glutathione (GSH) levels: in two lymphoid (Jurkat and CCRF-CEM) and two myeloid (THP-1 and KG-1a) leukemia cell lines

GSH levels were evaluated by the GSH-Glo™ Glutathione. Cells were treated with MTX or 6-MP and polyphenols alone and in combination for 24 hrs using their lowest-significant doses (LSD). Data was normalised to the vehicle control which was assigned 100% of GSH levels. The data was expressed as medians and ranges (n=4). Effects of combination treatments were statistically classified as synergistic (^*) causing a decrease in GSH levels or antagonistic (^#) causing an increase in GSH levels; when compared to vehicle control, drugs alone and expected values of combination treatments. Statistical significant was set at P≤0.05.

Figure 9. The effect of methotrexate (MTX) and 6-mercaptopurine (6-MP) when used in combination with quercetin (QUE), apigenin (AP), emodin (EMO), rhein, (RH) or cis-stilbene (CIS) on γ-H2AX foci formation (DNA damage marker): in two lymphoid (Jurkat and CCRF-CEM) and two myeloid (THP-1 and KG-1a) leukemia cell lines

This was evaluated by the immunofluorescent staining using Alexa Fluor® 647 Mouse anti-H2AX (pS139). Cells were treated with MTX or 6-MP and polyphenols alone and in combination for 24 hrs using their lowest-significant doses (LSD). The data was expressed as medians and ranges (n=4). Data was normalised to the vehicle control which was assigned 0% of γ-H2AX foci formation (DNA damage marker). Effects of combination treatments were statistically classified as synergistic (^*) causing an increase in the percentage of cells with γ-H2AX foci or antagonistic (^#) causing an decrease in the percentage of cells with γ-H2AX foci; when compared to vehicle control, drugs alone and expected values of combination treatments. Statistical significant was set at P≤0.05.