**5.1 Flavonoids**

A number of phenolic compounds belonging to the flavonoid class have been found to have an effect on leukaemia cell lines. Quercetin is a flavonol that has been reported to inhibit the proliferation of HL-60 cells and induce their apoptosis by the

#### **Figure 3.**

*The cell cycle – a process inhibited by phenolics. G1 = Gap 1, S = Synthesis phase, G2 = Gap 2, M = Mitosis, G0 = resting phase.*

*Phenolic Compounds - An Emerging Group of Natural Compounds against Leukaemia… DOI: http://dx.doi.org/10.5772/intechopen.98935*

**Figure 4.**

*Some phenolics found to possess anti-leukaemic activity. A = Gallic acid, B = Mangostin, C = Quercetin, D = Resveratrol, E = Syringaresinol and F = Tannic acid.*

activation of caspase-3, the downregulation of Bcl-2 protein and the upregulation of the Bax protein. Its effects have been found to be both dose and time dependent. The action of quercetin on the mitochondrial pathway of apoptosis also involves the inhibition of COX-2 [81]. It has also been suggested that its antiproliferative effect may be due to its capacity to inhibit both cytosolic protein kinase C as well as tyrosine protein kinase [82]. Quercetin has been found to arrest the cell cycle of both HL-60 cells and U937 cells, with treatment resulting in an increase in the number of cells in G2M phase. For U937 cells, this effect was coupled to a decrease in cyclins D, E, E2F1 and E2F2 [83]. Furthermore, the treatment of K562 cells with quercetin results in a number of morphological changes which include nuclear fragmentation as well as nuclear chromatin condensation. It has been found to inhibit the synthesis of heat shock protein 70, which is known to be involved in regulating the processes of both cell proliferation and differentiation [142, 143].

Within the same class of flavonols, both galangin and kaempferol have been found to inhibit the growth of HL-60 cells in a dose dependent manner. For kaempferol, this was attributed to both apoptotic and non-apoptotic effects but for galangin, the increased level of caspase-3 is suggestive of apoptosis [144]. These effects were also observed for two major flavones apigenin and luteolin. For the former, treatment resulted in an increase in both caspase-3 and caspase-9 proteases as well as cytochrome c [145–147]. Furthermore, the treatment of U937 cells with apigenin resulted in the cleavage of Poly (ADP-ribose) polymerase (PARP) as well as in the activation of caspase-3, caspase-7 and caspase-9. As for quercetin, down-regulation of Bcl-2 also occurs [148].

It has been shown that, similarly to quercetin, the flavone chrysin induces both U937 cell proliferation decline and DNA fragmentation. Its apoptotic effect on this cell line has been found to involve activation of caspase-3 as well as the inactivation of Akt (protein kinase B) [149, 150]. A methylated form of chrysin, termed

5,7-dimethoxyflavone was found to inhibit the growth of YCUB leukaemia cell lines in a dose and time dependent manner. Though this effect was seen on both YCUB-2 and YCUB-5 cells, for the former, an accumulation of reactive oxygen species was observed, but this was absent in the latter, suggesting a potentially different mechanism of action. Moreover, when 5,7-dimethoxyflavone was tested in combination with anticancer drugs such as cytarabine, an antagonistic effect was observed, suggesting the use of the compound as a single agent [151].

As a flavanol, epigallocatechin-3-gallate (EGCG) has been found to induce apoptosis in both acute and chronic myeloid leukaemia. For the former, a decline in death associated protein kinase 2 is observed, and an increase in neutrophil differentiation results on treatment of acute promyelocytic leukaemia with both ATRA and EGCG [152]. For the latter, the use of both EGCG and ponatinib results in a synergistic apoptotic effect which involves the downregulation of the CyclinD1 gene and the upregulation of TGF-β2 gene [153]. It has been reported that epigallocatechin-3-gallate causes the downregulation of the 67LR gene, and the induction of apoptosis is selective to cancer cells [154].

The anthocyanin delphinidin-3-sambubioside induces apoptosis in HL-60 cells through activation of three caspases which are caspase-3, caspase-8 and caspase-9, and causes DNA fragmentation [155].

Finally, the flavonoid curcumin and the metabolite tetrahydrocurcumin have both been found to induce apoptosis and autophagy respectively both in HL-60 cells as well as in HL-60 cells resistant to cytarabine [156]. This finding has very promising applications to overcome the issues with drug resistance. Furthermore, the combination of two flavonoids curcumin and quercetin induces mitochondrial apoptosis in CML. Since used in combination, any toxic effects on normal cells are unlikely since the treatment dose is lowered [157].

#### **5.2 Phenolic acids and their derivatives**

For hydroxybenzoic acids, gallic acid has been found to possess cytotoxic activity on HL-60 cells. Furthermore, gallic acid inhibits ribonucleotide reductase and arrests the cell cycle at the G0/G1 phase [89, 90]. The apoptosis of HL-60 cells by derivatives of gallic acid has also been investigated, and it has been concluded that apoptosis is greater in the presence of a long hydrophobic chain [158]. One of the derivatives of gallic acid, ellagic acid has been found to accumulate HL-60 cells in the S phase as well as induce their apoptosis with an increase in caspase-3 expression and PARP cleavage. Moreover, ellagic acid also enhances the differentiation effect of ATRA on HL-60 cells, and thus may be useful in overcoming ATRA resistance [159]. Ellagic acid has also been found to induce apoptosis in B-lymphocytes obtained from untreated CLL patients. This apoptotic effect involved the formation of reactive oxygen species, activation of caspase-3 and release of cytochrome c. Interestingly, this effect was selective to cancerous B-lymphocytes, and no toxic effect was seen for B-lymphocytes obtained from healthy donors [160].

With respect to hydroxycinnamic acids, caffeic acid phenethyl ester (CAPE) and cinnamic acid were found to induce apoptosis in HL-60 cells and K562 cells respectively, where for CAPE, protein, DNA and RNA synthesis in HL-60 cells were found to be inhibited [93, 161]. CAPE treatment resulted in the stimulation of Bax, downregulation of Bcl-2 as well as activation of caspase-3, signifying an apoptotic mechanism [162]. Apoptosis of U937 cells following CAPE treatment has also been recorded, with this effect being accompanied by an increase in cytochrome c [163]. For the cinnamic

acid, a dose dependent arrest in the G0/G1 phase has been observed. Cinnamic acid has also been found to induce differentiation in K562 cells [93].

#### **5.3 Xanthones and stilbenes**

For xanthones, the effect of α-mangostin on HL-60 cells was investigated and its apoptotic effect was found to be caspase-3 dependent [94]. Apart from α-mangostin, β-mangostin also inhibits the growth of HL-60 cells, arrests them at the G0/G1 phase and induces intrinsic apoptosis through the activation of caspases-3, 7 and 9 and Bax, as well as the down-regulation of Bcl-2. Like quercetin, β-mangostin inhibits heat shock protein 70 [95].

With respect to stilbenes, studies have mainly focused on resveratrol, which has been found to be a differentiation inducing agent, as well as an inducer of apoptosis. This has been observed on NB4 cells, which are a type of APL. Like the xanthone α-mangostin, treatment with resveratrol results in an increase in caspase-3 activity. Nonetheless, for both α-mangostin and resveratrol, treatment on HL-60 and NB4 cells respectively does not have an effect on the Bcl-2 protein levels. Hence this is suggestive of an alternative apoptosis pathway. Differentiation of NB4 cells with resveratrol is completely effective when the cells are treated with both ATRA and resveratrol [96]. Furthermore, synthesized resveratrol analogues also arrest the cell cycle of HL-60 cells but do so at all three phases, G0/G1, S and G2/M, contrasting with resveratrol which has been found to be phase specific [164]. It is relevant to highlight that though resveratrol is effective, it is limited by its poor bioavailability [165, 166]. Another two stilbenes namely piceatannol and sophorastilbene A both possess dose dependent cytotoxic activity on HL-60 cells with caspases 3, 8 and 9 being activated, with no changes in Bcl-2 protein expression being recorded [167].
