Polyphenols act synergistically with doxorubicin and etoposide in leukaemia cell lines

Abstract

The study aimed to assess the effects of polyphenols when used in combination with doxorubicin and etoposide, and to determine whether polyphenols sensitised leukaemia cells, causing inhibition of cell proliferation, cell cycle arrest and induction of apoptosis. This study is based on findings in solid cancer tumours, which have shown that polyphenols can sensitize cells to chemotherapy, and induce apoptosis and/or cell-cycle arrest. This could enable a reduction of chemotherapy dose and off-target effects, whilst maintaining treatment efficacy. Quercetin, apigenin, emodin, rhein and cis-stilbene were investigated alone and in combination with etoposide and doxorubicin in two lymphoid and two myeloid leukaemia cells lines. Measurements were made of ATP levels (using CellTiter-Glo assay) as an indication of total cell number, cell cycle progression (using propidium iodide staining and flow cytometry) and apoptosis (NucView caspase 3 assay and Hoechst 33342/propidium iodide staining). Effects of combination treatments on caspases 3, 8 and 9 activity were determined using Glo luminescent assays, glutathione levels were measured using the GSH-Glo Glutathione Assay and DNA damage determined by anti-γH2AX staining. Doxorubicin and etoposide in combination with polyphenols synergistically reduced ATP levels, induced apoptosis and increased S and/or G2/M phase cell cycle arrest in lymphoid leukaemia cell lines. However, in the myeloid cell lines the effects of the combination treatments varied; doxorubicin had a synergistic or additive effect when combined with quercetin, apigenin, emodin, and cis-stilbene, but had an antagonistic effect when combined with rhein. Combination treatment caused a synergistic downregulation of glutathione levels and increased DNA damage, driving apoptosis via caspase 8 and 9 activation. However, in myeloid cells where antagonistic effects were observed, this was associated with increased glutathione levels and a reduction in DNA damage and apoptosis. This study has demonstrated that doxorubicin and etoposide activity were enhanced by polyphenols in lymphoid leukaemia cells, however, differential responses were seen in myeloid cells with antagonistic responses seen in some combination therapies.

Figure 1

Effect of doxorubicin and etoposide on (a and b) ATP levels and (c and d) caspase 3 activity in two lymphoid leukaemia (CCRF-CEM and Jurkat), two myeloid leukaemia (THP-1 and KG-1a) cell lines and two non-tumour control cells (CD133⁺ HSC and CD34⁺ HSC). The lowest-significant doses (LSD) which significantly reduced ATP levels and induced apoptosis was determined for each etoposide inhibitor in each cell lines. The '*' indicated LSD in each cell line.

Figure 2

The effect of doxorubicin (DOX) and etoposide (ETP) when used in combination with quercetin (QUE), apigenin (AP), emodin (EMO), rhein (RH) or cis-stilbene (CIS) on ATP levels in two lymphoid leukaemia cell lines (Jurkat and CCRF-CEM), two myeloid leukaemia cell lines (THP-1 and KG-1a) and two non-tumour control cells (CD133+ HSC and CD34+ HSC). This was evaluated by CellTiter-Glo assay. Cells were treated with doxorubicin or etoposide and polyphenols alone and in combination for 24 h using their lowest-significant doses (LSD); together with a vehicle control. All data were normalised to the vehicle control, which was assigned 100% cell viability. The data were expressed as medians and ranges (n=4). The black bars show the vehicle controls and treatments alone; the coloured bars indicate significant additive effects in brown, synergistic effects in yellow, competitive antagonistic effects in purple and antagonistic effects in pink. Statistical significance was set at P⩽0.05 compared with vehicle control, drugs alone and expected values of individual drugs when combined.

Figure 3

The effect of doxorubicin (DOX) and etoposide (ETP) when used in combination with quercetin (QUE), apigenin (AP), emodin (EMO), rhein (RH) or cis-stilbene (CIS) on caspase 3 activity of two lymphoid leukaemia cell lines (Jurkat and CCRF-CEM), two myeloid leukaemia cell lines (THP-1 and KG-1a) and two non-tumour control cells (CD133+ HSC and CD34+ HSC). This was evaluated by NucView caspase 3 activity assay. Cells were treated with doxorubicin or etoposide and polyphenols alone and in combination for 24 h using their lowest-significant doses (LSD), together with a vehicle control. All data were normalised to the vehicle control, which was assigned a 0% apoptotic level. The data were expressed as medians with ranges (n=4). The black bars show the vehicle controls and treatments alone; the coloured bars indicate significant additive effects in brown, synergistic effects in yellow, competitive antagonistic effects in purple and antagonistic effects in pink. Statistical significance was set at P⩽0.05 compared with vehicle control, drugs alone and expected values of individual drugs effects.

Figure 4

The effect of doxorubicin (DOX) and etoposide (ETP) on caspases 8 and 9 activity when used in combination with quercetin (QUE), apigenin (AP), emodin (EMO), rhein (RH) or cis-stilbene (CIS) in lymphoid leukaemia cell lines (Jurkat and CCRF-CEM); and when used in combination with QUE or AP in myeloid leukaemia cell lines (THP-1 and KG-1a). This was evaluated by Caspases-Glo Luminescent 8 and 9 Assays. Cells were treated with DOX or ETP and polyphenols alone and in combination for 24 h using their lowest-significant doses (LSD). Data were normalised to the vehicle control, which was assigned a 0 RLU. The data were expressed as medians with ranges (n=4). The black bars show the vehicle controls and treatments alone; the coloured bars indicate significant additive effects in brown, synergistic effects in yellow, competitive antagonistic effects in purple and antagonistic effects in pink. Statistical significance was set at P⩽0.05 compared with vehicle control, drugs alone and expected values of individual drugs effects.

Figure 5

(a) Basal glutathione (GSH) levels in two lymphoid leukaemia (CCRF-CEM and JURKAT), two myeloid leukaemia (THP-1 and KG-1a) cell lines; and two non-tumour control (CD133⁺HSC and CD34⁺ HSC) cell lines. Untreated cells were evaluated by the GSH-Glo Glutathione Assay. The data are expressed as median with range in triplicate. Statistical significance was set at P⩽0.05. (b) The effect of doxorubicin (DOX) and etoposide (ETP) when used in combination with quercetin (QUE), apigenin (AP), emodin (EMO), rhein, (RH) or cis-stilbene (CIS) was determined on GSH levels in two lymphoid leukaemia cell lines (Jurkat and CCRF-CEM) and two myeloid leukaemia cell lines (THP-1 and KG-1a). GSH levels were evaluated by the GSH-Glo Glutathione Assay. Cells were treated with DOX or ETP and polyphenols alone and in combination for 24 h using their lowest-significant doses (LSD). Data were normalised to the vehicle control, which was assigned 100% of GSH level. The data were expressed as medians and ranges (n=4). The black bars show the vehicle controls and treatments alone; the coloured bars indicate a significant additive effect in brown, a synergistic effect in yellow, a competitive antagonistic effect in purple and an antagonistic effect in pink. Statistical significance was set at P⩽0.05 compared with vehicle control, drugs alone and expected values of individual drugs effects.

Figure 6

The effect of doxorubicin (DOX) and etoposide (ETP) 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) of two lymphoid leukaemia cell lines (Jurkat and CCRF-CEM) and two myeloid leukaemia cell lines (THP-1 and KG-1a). This was evaluated by the immunofluorescent staining using Alexa Fluor 647 Mouse anti-H2AX (pS139). Cells were treated with DOX or ETP and polyphenols alone and in combination for 24 h using their lowest-significant doses (LSD). The data were expressed as medians and ranges (n=4). The black bars show the vehicle controls and treatments alone; the coloured bars indicate a significant additive increase in DNA damage in brown, a synergistic increase in DNA damage in yellow, a competitive antagonistic decrease in DNA damage in purple and an antagonistic decrease in DNA damage in pink. Statistical significance was set at P⩽0.05 compared with vehicle control, drugs alone and expected values of individual drugs effects.