**1. Introduction**

Leukaemia is a malignancy which is characterised by an uncontrolled increase in immature blood cells, termed blasts, in the bone marrow [1]. As a result, these cells permeate the bone marrow and prevent haematopoiesis from occurring normally. Such blasts eventually penetrate into the bloodstream and spread into organs [2]. The earliest observations and descriptions of cases of leukaemia were recorded by Alfred Velpeau, Alfred Donné and John Hughes Bennett [3–5]. Rudolf Virchow is credited with coining the term 'leukaemia' in 1847, from the two Greek words 'leukos' and 'helma', which mean 'white blood' [6].

Broadly, leukaemia can be classified as either acute or chronic. In acute leukaemia, the proliferating cells are very immature, while in chronic leukaemia, these cells have a more mature phenotype [7]. Furthermore, both types are subdivided into myeloid, lymphoid and mixed lineages [8]. On one hand, in acute myeloid leukaemia (AML), these blasts are termed myeloblasts while they are lymphoblasts in acute lymphoblastic leukaemia (ALL). On the other hand, the mature cells are granulocytes or neutrophils in chronic myeloid leukaemia (CML) and are lymphocytes in chronic lymphatic leukaemia (CLL). In general, both chronic leukaemias and AML are more common in adults while ALL is generally prevalent in children [9–12].

Acute and chronic leukaemias differ in terms of onset time. In acute leukaemia, cell proliferation occurs rapidly in days, while in chronic leukaemia, the process is slower and takes months or years [13, 14]. As a result, in acute leukaemia, lack of treatment results in death within a time frame of weeks or months while in chronic leukaemia, this may be either months or years. The signs and symptoms of both types of leukaemia also vary. In acute leukaemia, the rapid proliferation of white blood cells causes bone discomfort, aches as well as swelling in the lymph nodes. The initial symptoms include anaemia, fatigue, fever and swelling in the liver and the spleen [15]. Patients with chronic leukaemia may also show similar symptoms but if anaemia is evident, it is milder than in acute leukaemia. Moreover, most patients diagnosed with chronic leukaemia do not show symptoms at the time of diagnosis [16].

In the following sections, current treatments for different leukaemia subtypes are discussed, as well as their drawbacks. Such disadvantages pave the way for the need for alternative therapies, whereby studies show that phenolic compounds are very promising candidates in this regard.

### **2. Leukaemia prevalence, current treatments and challenges**

Leukaemia is the most common cancer in children under 15 years of age and accounts for 32% of cancers in children of this age. For patients under 20 years of age, leukaemia accounts for 25% of cancers. The most common childhood cancer, ALL, constitutes 23% of childhood cancers and between 75% to 80% of childhood leukaemia cases. AML follows ALL and encompasses between 15% to 20% of childhood leukaemia [17, 18]. Leukaemia is also the most common blood cancer in people older than 55.

Though the treatment offered to a patient diagnosed with leukaemia depends on the leukaemia type, the primary options for treatment of leukaemia remain chemotherapy and radiotherapy. Chemotherapy drugs for AML include cytarabine, daunorubicin, doxorubicin and idarubicin [19]. Where possible, a bone marrow or stem cell transplant is also used following remission. In the latter, though the procedure may result in complications, recovery rates are good [15]. For AML, the intensive chemotherapy treatment administered to the patient as an induction treatment and as consolidation treatment. In the former, the aim is to achieve remission, while the purpose of the latter is relapse prevention [20, 21]. The use of induction and consolidation therapy together with an autologous stem cell transplant results in both a high relapse risk and a high mortality, while the use of consolidation therapy together with allotransplation results in a lower relapse risk but a higher mortality due to risks associated with graft versus host [22].

Although chemotherapy is widely used to treat a variety of cancers, it is broadly cytotoxic to normal tissues. Chemotherapy needs to be administered in more than one cycle since both the proliferating and resting phase cells possess the genetic abnormality. As a result, one chemotherapy cycle alone is not enough to kill all the

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

leukaemic cells [23]. Chemotherapy drugs are classified into five major classes based on their structure and mechanistic action. These are: alkylating agents, topoisomerase inhibitors, antitumour antibiotics, antimetabolites and microtubule inhibitors. Alkylating agents such as cisplatin act by damaging DNA and inhibiting transcription and protein synthesis [24]. Topoisomerase inhibitors like etoposide inhibit DNA topoisomerase from releasing supercoils during DNA replication [25]. Standard chemotherapy drugs such as daunorubicin and doxorubicin fall under the class of antitumour antibiotics which inhibit enzymes involved in DNA replication [26], while cytarabine is an antimetabolite which disrupts the S phase of the cell cycle [27]. Finally, microtubule inhibitors such as paclitaxel interfere with the M phase of the cell cycle, which results in the inhibition of mitosis [28].

For AML patients younger than sixty years of age, chemotherapy results in remission rates of between 50% to 75%, with most suffering a relapse. The incidence in AML is bimodal, with remission rates being lower for older patients and relapse rates being higher [21]. This relapse is a result of haematopoietic stem cells which survive the chemotherapeutic drug treatment and regenerate. Currently five year survival rates are estimated to be around 30% for AML [29, 30]. Moreover, standard chemotherapy for AML may result in side effects including myelosuppression, tumour lysis syndrome and hepatotoxicity [31].

While chemotherapy remains the standard treatment for AML, the use of other drugs has greatly improved survival rates for about 30% of AML cases. Such patients present with FLT3 mutations, with FLT3 being a tyrosine kinase vital for the differentiation of progenitor cells into both myeloid and lymphoid lineages. The first drug approved as an FLT3 inhibitor was Midostaurin, which since 2017 has been used a treatment for FLT3 mutant AML in combination with standard chemotherapy [32, 33]. In 2018, the second FLT3 inhibitor Gliteritinib was approved as a treatment for patients who were found to be resistant to other treatments [34]. Patients with FLT3 mutations are likely to relapse as elimination of cells harbouring the FLT3 mutation is very problematic. Moreover, some patients also become resistant to FLT3 inhibitors after treatment [35].

In AML subtype APL, treatment involves the use of all *trans* retinoic acid (ATRA) and arsenic trioxide (ATO) as induction therapy, combined with mild chemotherapy. This treatment, termed differentiation therapy, has converted the prognosis of APL from poor to favourable. Through differentiation therapy, blasts differentiate, resulting in a decline in proliferative capacity, followed by apoptosis or terminal differentiation initiation. This method contrasts highly with chemotherapy which is generally nonspecific and is often accompanied by highly toxic side effects [36]. Moreover, it is also advantageous in that while it causes terminal differentiation, it does not result in bone marrow hypoplasia, and unlike chemotherapy, the proliferating cells are not killed but their maturity is induced, leading to death [37–40].

More than 98% of APL patients possess the characteristic translocation t(15;17), which results in the fusion between two genes - the PML gene and the RARα. As a result, the fusion protein PML-RARα is formed. PML-RARα is conformationally changed by ATRA at concentrations between 10−7 and 10−6 M, resulting in co-repressor dissociation and co-activator activation, leading to a relaxation in chromatin, the activation of transcription of genes involved in differentiation, resulting in the terminal differentiation of promyelocytes to granulocytes [41, 42].

Three decades ago, APL was fatal as a result of coagulation disorders, and via anthracycline based chemotherapy, the prognosis was still poor for approximately 70% of patients. Differentiation therapy using ATRA and ATO has resulted in complete remission (CR) for around 85% of patients, and 70% of patients being cured. The use

of ATRA as a differentiating agent to differentiate promyelocytes into granulocytes was first discovered by Breitman *et al* in 1980. A problem that has been encountered with the use of ATRA is ATRA resistance. This has improved through the use of ATO combined with ATRA, yet drug resistance to ATRA and ATO remains an issue [43].

Moreover, though ATRA has been pivotal in the treatment of APL, this treatment may result in another complication known as retinoic acid syndrome or differentiation syndrome (DS). It has been found to occur in around 2% to 27% of children with APL who are treated with ATRA, and in up to 50% of patients. This may result in pulmonary haemorrhage, renal failure, as well as heart failure and for this reason is termed life threatening. Differentiation syndrome typically occurs around a week or two following the start of ATRA and/or ATO therapy [44]. If DS is severe and has resulted in pulmonary or renal dysfunction, the use of ATRA is ceased [45–48]. Compared to patients who do not develop this complication, patients with DS have a lower overall free survival and event free survival [49]. Though the exact mechanism of DS is not fully known, the main key player is thought to be an excessive inflammatory response. This response stems from leukaemic cells during their differentiation process, and is due to a higher level of chemokine production and adhesion molecules on APL cells. Inflammation leads to capillary leak syndrome and blast cells infiltrating organs such as the lungs, and organ failure [50]. Treatment for DS is required early in the diagnosis, and the corticosteroid dexamethasone is administered intravenously. Corticosteroids decrease chemokine production and stop lung infiltration [51].

Reported benefits of other agents of differentiation include histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors. A key DNMT inhibitor is 5-aza-cytidine while examples of HDAC inhibitors include sodium butyrate and valproic acid [52]. Moreover, for HDAC inhibitors, the combination of both valproic acid and ATRA has been found to be beneficial for older patients with AML [53, 54]. On one hand, HDAC inhibitors act by remodeling chromatin by subduing the activity of HDACs leading to histone acetylation. This results in the expression of genes involved in the processes of differentiation as well as apoptosis. On the other hand, the effect of DNMT inhibitors is DNA hypomethylation, which leads to the re-activation of tumour-suppressor genes silenced by methylation. The use of such inhibitors stems from the fact that the differentiation block of leukaemic cells may be a result of epigenetic changes including histone acetylation and DNA hypermethylation, which may be reversed through the action of these inhibitors [55].

In contrast to other leukaemias, in CML, the genetic abnormality, termed the Philadelphia chromosome is a result of the bcr-abl protein, which was identified by Nowell and Hungerford in 1960 [56]. This oncogenic protein leads to an upregulation of tyrosine kinase and inactivation of phosphoinositide-3-kinase resulting in the proliferation of myelocytes. Imatinib is a tyrosine kinase inhibitor which acts by binding to the bcr-abl protein. This inhibition allows the cells to differentiate into mature granulocytes and subsequently die by apoptosis [57–59]. Following imatinib administration, the cytogenic response to the treatment can be at one of three levels – cytogenic response, major cytogenic response and complete cytogenic response. In 80% of the patients, it is the latter that results, and following imatinib administration, most remain stable. However in some patients, mutations in the bcr-abl tyrosine kinase domain result in lack of inhibition by Imatinib. This leads patients to rely on chemotherapy and stem cell transplantation [60, 61]. A number of unfavorable effects following Imatinib treatment have been reported and include episodic bone pain, fluid retention, lethargy and weight gain. These usually occur within the first two years of treatment, and through continued treatment, they may also be reversed [62].

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

For ALL, 80% of cases occur in children, and like AML, its distribution is bimodal. Though the outcomes for children have greatly improved, the same cannot be said for elderly patients, with remission rates lying between 30 and 40% for this age group [63, 64]. Treatment involves the use of chemotherapy as induction treatment, consolidation therapy and also maintenance. For the former, an anthracycline, vincristine as well as corticosteroids [65] or the Hyper-CVAD chemotherapy regimen are used [66]. For ALL patients who are Ph-positive, survival rates have improved through the use of second generation tyrosine kinase inhibitors coupled to Hyper-CVAD [67]. Recently, great advancements have been made for relapsed or refractory ALL patients through CAR-T cell therapy [68]. Between 70-90% of these patients respond well to this treatment, however it is associated with challenges such as antigen escape, toxicity and tumour infiltration [69].

Contrastingly, many patients with CLL have indolent disease and are asymptomatic. For patients with active CLL, treatment involves the use of chemoimmunotherapy such as a combination of fludarabine, cyclophosphamide and rituximab (FCR) or bendamustine and rituximab (BR) [70, 71]. For patients with high risk ALL, other targeted treatment agents include venetoclax, ibutrinib and idelasilib [72–74]. Though toxicity and resistance remain challenges, these may potentially be alleviated by combination therapy.

### **3. Methods**

This chapter discusses studies that have been published to date, that assess the antileukaemic effect of phenolic compounds. These studies are grouped into the following three categories: *in vitro*, *in vivo* and clinical trials, as outlined in the following flow chart (**Figure 1**). Both *in vitro* and *in vivo* fall under the term 'preclinical trials', which are vital prior to moving to clinical trials and aim to determine the usefulness of a drug as therapy, as well as whether treatment is accompanied by any toxicity effect. Coming from the Latin "in glass", *in vitro* refers to experimental work carried out in a laboratory, as opposed to "within the living" for *in vivo*, where experimental work is performed using living organisms. With regards leukaemia, *in vitro* studies include experimental work performed using leukaemia cell lines while *in vivo* studies utilize animal models such as mice injected with leukaemic cells. Lastly, clinical trials are performed using human subjects, and are used to confirm *in vitro* and *in vivo* results, as well as determine drug efficacy and safety, amongst other parameters.

#### **Figure 1.**

*A flow chart outlining the different types of studies recording the effects of phenolics on leukaemia.*

### **4. Phenolic compounds: chemicals with a wide spectrum of bioactivity**

Due to the challenges posed by the current treatments, therapies that may improve patient survival are needed. Novel treatments that are more specific and generally

less toxic than conventional chemotherapy, are highly in demand. Due to their health benefits, the interest in natural products, specifically phenolic compounds, has greatly increased, making phenolics the subject of a number of research efforts over the past decade. Even more so, toxicity studies have shown that phenolics are safe and less toxic than a number of other synthetic and semi-synthetic compounds [75].

In plants, phenolic compounds are secondary metabolites consisting of an aromatic ring with one or more hydroxyl groups, which are involved in defending the plant against stress caused by drought, low or high temperatures, pathogens, restricted soil fertility and ultraviolet radiation [76, 77]. There is a wide range of such compounds and to date around 8000 of them have been identified and grouped into the following classes: phenolic acids (hydroxycinnamic and hydroxybenzoic acids), lignans, stilbenes, coumarins, xanthones and flavonoids [78–80]. Examples of each class of phenolic compounds that have been tested on leukaemia are presented in **Table 1**.

Such phenolics are distributed to varying degrees in particular parts of plants. Caffeic acid, a major phenolic acid is widely present in fruits, tannins are high in fruit pods, wood as well as bark, and flowers are rich in flavonoids [105, 106]. These compounds have been used by man for many years in the field of traditional medicine [76]. Several studies have been carried out which demonstrate the beneficial health effects of phenols. These compounds have been found to inhibit the oxidation of low density lipoprotein (LDL) *in vivo* where LDL oxidation is associated with the formation of atherosclerotic plaques, which play a role in coronary heart disease [107]. Even more so, the phenolic compound hydroxytyrosol has been found to decrease the risk of atherosclerosis and coronary heart disease [108].


Phenolic compounds such as hydroxytyrosol, hydroxytyrosol acetate and oleuropein have also been found to hinder platelet aggregation, in so doing, decreasing the

#### **Table 1.**

*The major classes of phenolics and respective examples found to have an effect on leukaemia.*

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

synthesis of eicosanoids such as thromboxane and thus preventing thrombosis [109, 110]. Another antiatherogenic property of phenols is their ability to reduce endothelial activation by decreasing the mRNA levels of vascular adhesion molecule-1, hence resulting in a decline in its expression. Due to this, adhesion of monocytes to endothelial cells decreases, hence preventing endothelial malfunction [111].

The antioxidant capacity of phenolic compounds has also been widely investigated. Antioxidants are vital in protecting the plant from oxidative stress [112]. Compounds possessing an *ortho-*diphenolic structure are known to display antioxidant behaviour. Examples of such compounds include the phenols hydroxytyrosol and oleuropein, whose scavenging capabilities were compared to those of 2,2-diphenyl-1-picrylhydrazyl (DPPH) [113].

Additionally to antioxidant behaviour, hydroxytyrosol and oleuropein have been found to possess antimicrobial activity against a variety of American type culture collection (ATCC) bacterial strains and clinical bacterial strains [114]. Moreover, such compounds are also anti-inflammatory agents. This is because they have been found to reduce both the release of arachidonic acid as well as production of arachidonic acid metabolites which play central roles in inflammation [115]. Also crucial to inflammation are the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). It has been reported that such enzymes are inhibited by the phenolic compound oleocanthal, in a mechanism like that of ibupforen [116].

## **5. Phenolic compounds and leukaemia:** *in vitro* **studies**

Various *in vitro* studies indicate that phenols possess anticancer properties [117–120]. Phenols are commonly found in foods including nuts, fruits, vegetables and oil. Studies have shown that diets rich in phenols help prevent a variety of cancers [121–124].

Structure-activity-relationship studies have shown that the anticancer properties of these compounds vary as a result of the functional groups present in the structure, where both the hydroxylic groups present as well as the aromatic ring play an important role. With regards to hydroxylic groups, the more the number of such groups, the higher the anticancer properties. Moreover, the presence of a side chain consisting of an unsaturated fatty acid makes the phenolic compound more effective (**Figure 2**) [125, 126].

In general, phenols act by inhibiting the cell cycle, leading to apoptosis (**Figure 3**) [127–129]. In addition, phenols appear to subdue the expression of chemokines as well

#### **Figure 2.**

*The aromatic ring, the number and position of OH groups, and the presence of the unsaturated fatty acid side chain (R) influence activity.*

as cytokines and angiogenesis is stopped. Both of these are vital for tumour development regulation [130–132].

Though a number of *in vitro* studies have focused on the effect of phenols on carcinomas, gliomas, melanomas, lung cancer and breast cancer, other studies have reported the inhibitory effects of phenolic compounds on leukaemia cell lines, with most studies focusing on HL-60, U937 and K562 cells.

The HL-60 cell line was isolated in 1977 and is classified as acute myeloblastic leukaemia with maturation (M2 category in the French-American-British classification) [133, 134]. In this suspension culture, a vast majority of the cells are promyelocytes which can be induced to differentiate into monocytes or granulocytes respectively by a number of compounds such as Phorbol 12-myristate 13-acetate (PMA), sodium butyrate, dimethyl sulfoxide (DMSO) as well as all-*trans* retinoic acid (ATRA) [135]. The U937 cell line was isolated in 1974 from a patient with histiocytic lymphoma and is classified under the M4 category in the French-American-British classification. The cells are promonocytes and can be driven towards monocytic differentiation by PMA. For this reason, the cell line is used as a model for both monocyte and macrophage differentiation [136]. K562 is an example of erythroleukaemia [137]. It was isolated from a patient diagnosed with CML "in blast crisis", which is the final phase of the disease [138, 139]. The K562 cell line is positive for the Philadelphia chromosome, which is present in the vast majority of patients (>95%) diagnosed with CML. The cell line is termed to be proerythroblastic and studies have shown that it can be induced to monocytic, megakaryocytic and erythroid differentiation using chemicals such as proanthocyanidins for the former lineage, PMA for both the megakaryocytic and monocytic lineage and 5-azacytidine, butyric acid and hemin for the latter lineage [140, 141]. The *in vitro* effects of phenolics on the above-mentioned cell lines will be discussed below, and the structures of some of these phenolics are shown in **Figure 4**.
