Effect of pomegranate extracts on apoptosis and cell cycle in haematological malignancies.

Leukaemia is a complex form of blood malignancy characterized by the uncontrolled proliferation of haematopoietic cells and progressive accumulation of these cells within the bone marrow (BM) and secondary lymphoid tissues. The exact cause of leukaemia remains unknown. Leukaemia is a major problem worldwide affecting many people each year. However, current treatment options still have several limitations not least, the cytotoxicity of these therapies to normal cells and the fact that certain chemotherapy agents may cause bone marrow toxicity and organ damage. Several epidemiological studies have shown that high intake of fruit and vegetables are associated with low incidence of a number of human cancers. Researchers suggest that pomegranates contain bioactive chemicals with potential for treatment and prevention of cancer. Pomegranate juice (PJ) have been shown to inhibit cellular proliferation and tumor growth and induce cell death via apoptosis in a number of cancer cell lines. However, to date, few studies have investigated the potential of PJ in the treatment of Leukaemia. Here, PJ significantly induced apoptosis in 8 leukaemia cell lines and non tumour control cells although the lymphoid and 2 myeloid cell lines were affected to a greater extent than non-tumour control cells and 2 of the myeloid cell lines. Furthermore, PJ induced cell cycle arrest. These results provide evidence that PJ contain bioactive compounds that could be used in the treatment of Leukaemia.Treatment of four Leukaemia cell lines with five fractions obtained from PJ by solid phase extraction demonstrated that only the acetonitrile fractions decreased adenosine triphosphate (ATP) levels in all Leukaemia cell lines. Acetonitrile fractions also significantly activated caspase-3 and induced nuclear morphology characteristic of apoptosis. S phase arrest was induced by acetonitrile fractions which matched S phase arrest seen previously following whole PJ treatments. The acetonitrile fractions contained higher phenol content than whole PJ whereas only low levels of phenols were seen in any other fraction. Liquid chromatography mass spectrometry (LC-MS)analysis demonstrated that acetonitrile fractions were enriched in ellagitannins, ellagic acid, and hydroxycinnamic acid derivatives but depleted in anthocyanins.The potential protective effect of a number of pure compounds that were either identified in the acetonitrile fraction of PJ were present in the other fractions of PJ or have been identified as PJ components in previous studies were studied on ATP levels in four human leukaemia cell lines. Among the 26 naturally occurring pomegranate compounds investigated, only seven compounds: delphinidin; cyanidin; pelargonidin; EGCG; gallic acid; ellagic acid; quercetin; and punicalagin induced 50% decrease in ATP levels in the studied leukaemia cell lines.To compare the chemoprotective properties of anthocyanins found in PJ and to understand the relationship between anthocyanin chemical structure and chemoprotective activity, the inhibition of cell proliferation and induction of apoptosis in leukaemia cell lines was measured. Anthocyanins activity was found to be dependent on the ortho-hydroxyphenyl structure and the most potent glycosidic form of the anthocyanins with proliferation and apoptosis effects seen in anthocyanins with greatest hydroxyl groups and least glycosidic form, namely delphinidin. Delphinidin induced apoptosis through intrinsic and extrinsic pathways. Suggesting that delphinidin may have potential for use as a chemotherapeutic agent against leukaemia. Furthermore, EGCG, gallic acid, quercetin, and punicalagin induced apoptosis and resulted in cell cycle arrest in the majority of the leukaemia cell lines. Together this study has shown that PJ is a rich source of bioactive compounds which could hold promise for treatment of leukaemia. The most promising compounds found within PJ were delphinidin, gallic acid, and punicalagin. Further investigation into these agents in the treatment and prevention of leukaemia are essential to develop these potential agents as future treatments