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.
Highlights
IntroductionDespite considerable improvements in tolerance and efficacy of chemotherapeutic agents, the mortality of leukaemia is still high.[1]Topoisomerase II inhibitors such as doxorubicin and etoposide are the most common chemotherapeutic agents used for leukaemia treatment.[1,2] these agents are commonly associated with severe side effects; and drug resistance is common.[1,2,3,4,5,6] As such combination treatments are under investigation as they could enhance the efficacy of standard chemotherapy agents, and decrease development of drug resistance, toxicity and side effects.[1,2,3,5,7]One strategy could be combination of chemotherapy agents with bioactive compounds such as polyphenols
Doxorubicin and etoposide induced a dose-dependant decrease in ATP levels and increase in caspase 3 activity in all cell lines effects on non-tumour control cells were reduced compared with leukaemia cells (Figure 1)
The lowest-significant dose (LSD) for doxorubicin and etoposide alone, which reduced ATP levels compared with the vehicle control at 24 h, was dependant on all cell types
Summary
Despite considerable improvements in tolerance and efficacy of chemotherapeutic agents, the mortality of leukaemia is still high.[1]Topoisomerase II inhibitors such as doxorubicin and etoposide are the most common chemotherapeutic agents used for leukaemia treatment.[1,2] these agents are commonly associated with severe side effects; and drug resistance is common.[1,2,3,4,5,6] As such combination treatments are under investigation as they could enhance the efficacy of standard chemotherapy agents, and decrease development of drug resistance, toxicity and side effects.[1,2,3,5,7]One strategy could be combination of chemotherapy agents with bioactive compounds such as polyphenols. Despite considerable improvements in tolerance and efficacy of chemotherapeutic agents, the mortality of leukaemia is still high.[1]. We have previously shown that a number of polyphenols reduced cell ATP levels, caused cell cycle arrest and induce apoptosis, in lymphoid leukaemia cells lines, whilst having limited effects on normal haematopoietic cells.[8] Within our previous study, eight polyphenols selected as representatives of the major polyphenol classes were investigated, and the most potent polyphenols identified were quercetin, apigenin, emodin, rhein and cis-stilbene.[8]
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