The Anti-Leukemic Activity of Natural Compounds

The use of biologically active compounds has become a realistic option for the treatment of malignant tumors due to their cost-effectiveness and safety. In this review, we aimed to highlight the main natural biocompounds that target leukemic cells, assessed by in vitro and in vivo experiments or clinical studies, in order to explore their therapeutic potential in the treatment of leukemia: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL). It provides a basis for researchers and hematologists in improving basic and clinical research on the development of new alternative therapies in the fight against leukemia, a harmful hematological cancer and the leading cause of death among patients.


Introduction
Cancer is one of the leading causes of death worldwide and a major challenge for the public health system [1]. The incidence of cancer is constantly increasing and is estimated to increase by 70% over the next 20 years [2].
Conventional anticancer therapies have limited efficacy and are associated with many side effects, such as hepatotoxicity, myelosuppression, or tumor lysis syndrome [3]. Chemotherapy and radiation therapy are frequently correlated with side effects, such as hair loss, loss of appetite, diarrhea, vomiting, liver damage, and neurological disorders [4]. Therefore, it is necessary to find new therapeutic approaches with high efficacy and fewer side effects. The main treatments used in leukemia are radiotherapy, hyperthermia, and chemotherapy. Conventional drug treatment is associated with cytotoxicity and systemic side effects. Therefore, efforts in cancer treatment are focused on finding strategies that can specifically target tumor cells without affecting normal cells [5]. Understanding the molecular mechanisms involved in hematologic cancers is useful in developing of the new therapeutic strategies that target various molecular abnormalities. Recently, there has been an increase in molecularly targeted therapies approved by the FDA in various types of leukemia, but there are insufficient data on the use of these drugs. Thus, in the case of AML, several agents are available for various clinical stages, but the best response rates were obtained by combining new molecularly-targeted treatments with conventional induction chemotherapy [6]. However, the patients experience short-term nausea/vomiting, diarrhea, hair loss, mouth sores, infection, rash; and for the long-term, organ dysfunction, chemobrain, fatigue, neuropathy, as well as resistance of leukemia cells to chemotherapy drugs [7][8][9], highlighting the need for the development of less toxic and targeted therapies. 50% of cells underwent apoptosis after treatment with 20 mg/mL boswellic acid acetate for 24 h [61].
The main pharmacological effects exerted by natural compounds against acute myeloid leukemia (AML) are summarized in Table 1.
The natural compounds with anti-tumoral activity against acute mieloid leukemia (AML) by in vitro and in vivo experiments or synergic activity with antineoplastig drugs, are summarized in Figure 1.
Fusion of the Abelson gene (ABL1) on chromosome 9 with the cluster breakpoint region (BCR) on chromosome 22 generates the oncoprotein BCR-ABL, an active tyrosine

Resveratrol
In vitro HL-60 cells ↓ CSC-related Shh expression, Gli-1 nuclear translocation, and cell viability in IL-6-treated HL-60 cells -had synergistic effect with Shh inhibitor cyclopamine on inhibiting cell growth [72] Resveratrol In vitro U937 and MV-4-11 cells -interacted synergistically with HDACIs in human myeloid leukemia cells -coadministration with HDACIs led to enhanced DNA damage, mitochondrial injury, and caspase-3, caspase-9, and caspase-8 activation -blocked HDACI-mediated RelA acetylation and NF-κB activation -induced S-phase accumulation and sensitized leukemia cells to HDACIs [73] Pterostilbene In vitro MV4-11 HL-60, U937, and THP-1 AML cells -suppressed cell proliferation in various AML cell lines -induced G0/G1-phase arrest when expressions of cyclin D3 and CDK2/6 were inhibited -induced cell apoptosis occurred through activation of caspases-8/-9/-3, and a MMP-dependent pathway -treatment of HL-60 cells with PTER induced sustained activation of ERK1/2 and JNK1/2, and inhibition of both MAPKs by their specific inhibitors significantly abolished the PTER-induced activation of caspases-8/-9/-3 -PTER-induced cell growth inhibition was only partially reversed by the caspase-3-specific inhibitor, Z-DEVE-FMK -promoted disruption of LMP and release activated cathepsin B -induced HL-60 cell death via MAPKs-mediated mitochondria apoptosis pathway [74] Gambogic acid In vitro U937 and HL-60 cells -had cytotoxic effect on AML cells -inhibited cell growth and promoted differentiation in U937 and HL-60 cells ↑ the expression of p21waf1/cip1 in the two cell lines [75] 3-O-acetyl-11-keto-βboswellic acid (AKBA) In vitro HL-60 cells -inhibited dose-dependent proliferation of HL-60 and apoptosis rate of HL-60 cells -changed the cell cycle by increasing of G(1) phase and decreasing of S phase -anti-proliferation and apoptosis-inducing effects on HL-60 cells [76] Boswellic acid acetate In vitro NB4, SKNO-1, nK562, U937, ML-1, and HL-60 cells -inhibited cell growth and induced cell toxicity of myeloid leukemia cell lines -induced apoptosis through a p53-independent pathway by activation of caspase-8 induced proteolysis of Bid ↓ mitochondrial membrane potential without production of hydrogen peroxide ↑ the levels of DR4 and DR5 mRNA in apoptotic cells [61] Avocatin B In vitro OCI-AML2 cells ↓ human primary AML cell viability without effect on normal peripheral blood stem cells -selectively toxic toward leukemia progenitor and stem cells -induced mitochondria-mediated apoptosis -inhibited fatty acid oxidation and ↓ NADPH levels, resulting in ROS-dependent leukemia cell death [77] Parthenolide In vitro U937 cells -inhibited growth of U937 cells -induced apoptosis in U937 cells ↓ the CD38+ population of U937 cells ↓ osteopontin gene expression in U937 cells [78] Molecules 2021, 26

Parthenolide
In vitro AML cells, bcCML cells, normal bone marrow, and umbilical cord blood cells -induced apoptosis in primary human AML cells and bcCML cells sparing normal hematopoietic cells -targeted preferentially leukemic but not normal progenitor and stem cell activity [43] In vivo Nonobese diabetic/severe NOD/ SCID mice -the molecular mechanism of PTL mediated apoptosis is associated with inhibition of NF-κB, proapoptotic activation of p53, and increased ROS -the activity of PTL triggers LSC-specific apoptosis

Emodin
In vitro AML HL-60/ADR cells -induced growth inhibition and apoptotic effects in resistant HL-60/ADR cells in vitro as well as in the HL-60/H3 xenograft models in vivo ↑ chemosensitivity of AML cells to Ara-C, inhibited leukemic cell growth, and improved survival in mouse xenograft model of AML [37] In vivo BALB/C-nude mice

Emodin
In vitro NB4, MR2 and primary AML cells -inhibited cell proliferation in NB4 cells, MR2 cells, and primary AML cells -enhanced differentiation induction of ATRA in retinoid-responsive NB4 cells as well as in retinoid-resistant MR2 cells -induced cell apoptosis in NB4 cells, MR2 cells, and primary AML cells -the apoptotic induction in AML cells was associated with the activation of caspase cascades involving caspase-9, caspase-3, and PARP cleavage -induced the activation of the caspase-dependent pathway -induced the degradation of RARα protein in NB4 and MR2 cells -inhibited activation of the PI3K/Akt signaling pathway in AML cells -inhibited p-Akt at Ser473 as efficiently as mTOR at Ser2448 -suppressed the phosphoration of mTOR downstream targets, 4E-BP1 and p70S6K [79] Thymoquinone In vitro HL-60 cells ↓ HL-60 cell viability -induced apoptosis in HL-60 cells ↓ the expression of WT1 and BCL2 genes [80] Ajoene In vitro KG1 cells ↓ bcl-2-expression ↑ the inhibitory effect of the two chemotherapeutic drugs, cytarabine and fludarabine, on Bcl-2-expression in KGI cells -the two drugs, cytarabine and fludarabine, ↑ the activated caspase-3 level in KGI myeloid leukemia cells -ajoene enhanced the activation of caspase-3 in both cytarabine-and fludarabine-treated KGI cells [81]

OSU-A9
In vitro HL-60 and THP-1 cells and primary leukemia cells from AML patients -induced cytotoxicity in AML cell lines and primary leukemia cells from AML patients ↓ cyclin A and cyclin B1 in AML cell lines -induced apoptosis, caspase activation, and PARP cleavage in AML cell lines -induced autophagy but not autophagic cell death in AML cell lines -OSU-A9-mediated cytotoxicity and hypophosphorylation of Akt were dependent on the generation of ROS -suppressed the growth of THP-1 xenograft tumors and prolonged the survival of tumor-bearing athymic nude mice [82] In vivo athymic nude mice
Fusion of the Abelson gene (ABL1) on chromosome 9 with the cluster breakpoint region (BCR) on chromosome 22 generates the oncoprotein BCR-ABL, an active tyrosine kinase that induces cytokine-independent cell proliferation, which causes excessive accumulation of myeloid cells in hematopoietic tissues [86]. The Bcr-Abl oncoprotein activates several downstream pathways, responsible for inducing cell proliferation, loss of adhesion, cell differentiation blocking, and inhibition apoptosis [87,88].
The main pharmacological effects exerted by natural compounds against chronic myeloid leukemia (CML) are summarized in Table 2.
The natural compounds with anti-tumoral activity against chronic myeloid leukemia (AML) by in vitro and in vivo experiments, are summarized in Figure 2.

Natural Compounds in Acute Lymphoblastic Leukemia (ALL)
Acute T-cell lymphoblastic leukemia (T-ALL) is an aggressive malignant blood disorder [ 112]. Currently, the T-ALL treatment protocols include high doses of chemotherapeutics, which have significant toxic side effects [ 113,114]. Natural products with various biological activities and specific selectivity have served as important sources of antitumor agents that have been developed for clinical use [ 115].
Anthocyanins, a subclass of flavonoids, are glycosides of anthocyanidins [ 116]. Blueberries are an important source of anthocyanins [ 117]. Anthocyanins showed, antimutagenesis and anti-carcinogenesis activity [ 118,119]. They have been shown to have a strong antitumor effect by inducing a pro-apoptotic mitochondrial-mediated response [ 120].
Anthocyanins from blueberry extract (Antho 50) induced apoptosis in Jurkat cells by decreasing the expression of Polycomb group proteins. This effect was mediated by an increase in intracellular ROS and depolarization of the mitochondrial membrane [ 117]. In another study, two anthocyanins extracted from blackcurrant juice, delphinidin-3-Oglucoside and delphinidin-3-O-rutinoside, induced apoptosis in human Jurkat leukemic cells [ 121]. Additionally, blackcurrant juice and blackcurrant extract inhibited  ↓ the viability of of leukemic cells -induced apoptosis of peripheral blood lymphocytes isolated from human CLL patients via mitochondrial pathway -induced the activation of proapoptotic Bax ↓ the expression of antiapoptotic Bcl-2 protein -released cytochrome c from mitochondria into cytosol -activated caspase-3, subsequently leading to the activation of apoptosis of B-CLL cells [90] Quercetin In vitro K-562 cells -induced apoptosis in K-562 cells -abrogated K-562 cells proliferation ↓ genes expression of HSP70, Bcl-X(L), and FOXM1 -improved Bax, caspase-3, and caspase-8 expression

Natural Compounds in Acute Lymphoblastic Leukemia (ALL)
Acute T-cell lymphoblastic leukemia (T-ALL) is an aggressive malignant blood disorder [112]. Currently, the T-ALL treatment protocols include high doses of chemotherapeutics, which have significant toxic side effects [113,114]. Natural products with various biological activities and specific selectivity have served as important sources of antitumor agents that have been developed for clinical use [115].
Anthocyanins, a subclass of flavonoids, are glycosides of anthocyanidins [116]. Blueberries are an important source of anthocyanins [117]. Anthocyanins showed, anti-mutagenesis and anti-carcinogenesis activity [118,119]. They have been shown to have a strong antitumor effect by inducing a pro-apoptotic mitochondrial-mediated response [120].
Anthocyanins from blueberry extract (Antho 50) induced apoptosis in Jurkat cells by decreasing the expression of Polycomb group proteins. This effect was mediated by an increase in intracellular ROS and depolarization of the mitochondrial membrane [117]. In another study, two anthocyanins extracted from blackcurrant juice, delphinidin-3-Oglucoside and delphinidin-3-O-rutinoside, induced apoptosis in human Jurkat leukemic cells [121]. Additionally, blackcurrant juice and blackcurrant extract inhibited proliferation, induced cell cycle arrest in the G2/M phase, and apoptosis in Jurkat cells. These effects have been associated with increased expression of p73 and caspase 3, Akt and Bad dephosphorylation, and down-regulation of UHRF1 and Bcl-2 [121].
The main pharmacological effects exerted by natural compounds against acute lymphoblastic leukemia (ALL) are summarized in Table 3.
The natural compounds with anti-tumoral activity against acute lymphoblastic leukemia (ALL) by in vitro and in vivo experiments or antagonizing activity against cytotoxicity of antineoplastic drugs, are summarized in Figure 3.

Natural Compounds in Chronic Lymphocytic Leukemia (CLL)
Chronic lymphocytic leukemia (CLL) is the most common type of hematologic cancer in the western countries (22-30%) [ 134,135]. CLL is a monoclonal lymphoproliferative disorder characterized by the proliferation and accumulation of morphologically mature, but immunologically dysfunctional B-cell lymphocytes [ 136]. CLL B cells interact with their microenvironment, and B cell survival is enhanced by contact with bone marrow stromal cells. Therefore, the lifespan of B cells increases, causing their abnormal accumulation [ 137]. The main sites of the disease include peripheral blood, spleen, lymph    Gambogic acid In vitro Jurkat and Molt-4 cells -inhibited proliferation, induced apoptosis, and activated autophagy in T-ALL cell lines -antileukemic effect against peripheral blood lymphocyte cells in patients with ALL -inhibited phospho-GSK3β S9 protein levels to inactivate Wnt signaling -suppressed β-catenin protein levels [112] Gallic acid In vitro Jurkat cells ↓ cell viability [129] Parthenolide In vitro B-and T-ALL cells -effective against bulk B-and T-ALL cells -prevented engraftment of multiple LIC populations in NOD/LtSz-scld IL-2Rγ c -null mice -restoration of normal murine hemopoiesis

Natural Compounds in Chronic Lymphocytic Leukemia (CLL)
Chronic lymphocytic leukemia (CLL) is the most common type of hematologic cancer in the western countries (22-30%) [134,135]. CLL is a monoclonal lymphoproliferative disorder characterized by the proliferation and accumulation of morphologically mature, but immunologically dysfunctional B-cell lymphocytes [136]. CLL B cells interact with their microenvironment, and B cell survival is enhanced by contact with bone marrow stromal cells. Therefore, the lifespan of B cells increases, causing their abnormal accumulation [137]. The main sites of the disease include peripheral blood, spleen, lymph nodes, and bone marrow [136]. It mainly affects adults [138].
Although there are many therapeutic protocols, CLL is still an incurable disease [138]. Current treatment options include conventional chemotherapy, monoclonal antibodies, and hematopoietic transplantation [139]. These standard treatment methods are not sufficient to eliminate all CLL cells and have a number of side effects. Additionally, standard treatment promotes the development of resistance to treatment and most treated patients relapsed. Therefore, it is necessary to develop new therapeutic strategies that could eliminate apoptosis-resistant CLL cells. Recently, there has been a growing interest in the use of agents derived from natural compounds for cancer therapy [140].
Bcl-2 plays a key role in regulating cellular responses to treatment due to its proand anti-apoptotic properties [141]. The anti-apoptotic protein Bcl-2 is overexpressed in several hematological malignancies, including CLL. This overexpression is considered to be responsible for defective apoptosis in CLL [142].
The effects of polyphenols on cell proliferation, gene regulation, and apoptosis have been studied on several cancer cell lines [143]. Alhosin et al. (2015) demonstrated that a standardized blueberry extract containing 50% anthocyanins (Antho 50) had the ability to induce apoptosis in CLL B cells via the Bcl-2/Bad pathway. They evaluated the pro-apoptotic effect of Antho 50 on CLL B cells from 30 patients and on peripheral blood mononuclear cells (PBMCs) from healthy subjects. The main phenolic compounds in cranberry extract responsible for the pro-apoptotic effect in CLL B cells were delphinidin-3-O-glucoside and delphinidin-3-O-rutinoside. Antho 50-induced apoptosis has been associated with caspase-3 activation, down-regulation of UHRF1, dephosphorylation of Akt and Bad, and down-regulation of Bcl-2 [144].
Luteolin significantly induced apoptosis in chronic lymphocytic leukemia (CLL) cell lines by increasing caspase activity and triggering the intrinsic apoptotic pathway [145].
The main pharmacological effects exerted by natural compounds against chronic lymphocytic leukemia (CLL) are summarized in Table 5.
The natural compounds with anti-tumoral activity against chronic lymphocytic leukemia (CLL) by in vitro and in vivo experiments or antagonizing activity against cytotoxicity of antineoplastic drugs, are summarized in Figure 4.  Curcumin Clinical study Twenty-one patients with stage 0/1 CLL ↓ ALC at four patients (20%) ↓ in ALC was accompanied by an ↑ in CD4, CD8, and NK cells [150] Curcumin and rapamycin PBMCs from patients with B-CLL -induced apoptosis in B-CLL cells obtained from patients with CLL ↑ caspase-9, -3, and -7 activity ↓ anti-apoptotic Bcl-2 levels, ↑ the pro-apoptotic protein Bax [151] [156] In vivo BALB/c mice Indole-3-carbinol In vitro PBMCs cells hMSC-TERT cells -induced cytotoxicity in CLL cells but not in normal lymphocytes ↓ XIAP and cIAP1/2 and induced caspase 9-dependent apoptosis of CLL cells -sinergic activity with fludarabine in CLL cells and overcame stroma-mediated drug-resistance -mechanism of cell death involved p53-dependent and independent apoptosis -sinergic activity with F-ara-A in all types of CLL cells and restored F-ara-A sensitivity in fludarabine-resistant CLL cells [157] In vivo C57bl/6 mice

Clinical Trials and Synergic Activity with Conventional Anti-Leukemic Drugs
Several clinical studies are published in database ClinicalTrials.Gov regarding the anti-tumor action of biactive compounds and synergies with anti-neoplastic therapy of leukemias.
The effect of genistein was tested in a phase I/II clinical study in combination with decitabine in pediatric relapsed refractory malignancies. Genistein was administered orally twice daily from day 2 to day 21, followed by a 7-day break (clinical trial number: NCT02499861). The aim of the research includes assessment of a tolerated dose of the combination of intravenous decitabine with oral genistein for children with refractory or recurrent solid malignancies and leukemia. The adverse events of the combination therapy and clinical benefit in phase IIa of the study measured by either volumetric MRI for solid tumor or by bone marrow aspiration or biopsy for leukemia at the end of cycles 2, 4, 6, 9, and 12 were assessed. To date, the results are not yet published in the database ClinicalTrials.Gov.
The efficacy of concomitant administration of curcumin and colecalciferol was investigated in a phase II trial in the treatment of patients with chronic lymphocytic leukemia in stage 0-II, previously untreated and small lymphocytic lymphoma (clinical trial number: NCT02100423).
Given that green tea extract contains ingredients that can slow the growth of certain cancers, its effect was tested in a phase I/II trial in the treatment of patients with chronic lymphocytic leukemia in stage 0, I, or II (clinical trial number: NCT00262743). In the phase I trial, patients were given orally 400 to 2000 mg of green tea extract (Polyphenon E) twice a day for 6 months [ 158]. In the phase II trial, oral administration of 2000 mg of Polyphenon E twice daily for 6 months was well tolerated [ 159]. Most patients experienced a decrease in absolute lymphocyte count (LAC) and lymphadenopathy following treatment with Polyphenon E [ 158,159].

Conclusions
In this review, we presented the natural compounds that have shown an antileukemic activity in experimental studies on different cell lines or primary cultures, preclinical and clinical studies, results that could propose them in subsequent therapeutic

Clinical Trials and Synergic Activity with Conventional Anti-Leukemic Drugs
Several clinical studies are published in database ClinicalTrials.Gov regarding the anti-tumor action of biactive compounds and synergies with anti-neoplastic therapy of leukemias.
The effect of genistein was tested in a phase I/II clinical study in combination with decitabine in pediatric relapsed refractory malignancies. Genistein was administered orally twice daily from day 2 to day 21, followed by a 7-day break (clinical trial number: NCT02499861). The aim of the research includes assessment of a tolerated dose of the combination of intravenous decitabine with oral genistein for children with refractory or recurrent solid malignancies and leukemia. The adverse events of the combination therapy and clinical benefit in phase IIa of the study measured by either volumetric MRI for solid tumor or by bone marrow aspiration or biopsy for leukemia at the end of cycles 2, 4, 6, 9, and 12 were assessed. To date, the results are not yet published in the database ClinicalTrials.Gov.
The efficacy of concomitant administration of curcumin and colecalciferol was investigated in a phase II trial in the treatment of patients with chronic lymphocytic leukemia in stage 0-II, previously untreated and small lymphocytic lymphoma (clinical trial number: NCT02100423).
Given that green tea extract contains ingredients that can slow the growth of certain cancers, its effect was tested in a phase I/II trial in the treatment of patients with chronic lymphocytic leukemia in stage 0, I, or II (clinical trial number: NCT00262743). In the phase I trial, patients were given orally 400 to 2000 mg of green tea extract (Polyphenon E) twice a day for 6 months [158]. In the phase II trial, oral administration of 2000 mg of Polyphenon E twice daily for 6 months was well tolerated [159]. Most patients experienced a decrease in absolute lymphocyte count (LAC) and lymphadenopathy following treatment with Polyphenon E [158,159].

Conclusions
In this review, we presented the natural compounds that have shown an anti-leukemic activity in experimental studies on different cell lines or primary cultures, preclinical and clinical studies, results that could propose them in subsequent therapeutic protocols of different types of leukemia: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL). Mechanistically, they demonstrated the ability to induce cell cycle blockage and apoptosis or autophagy in cancer cells, as well as inhibition of proliferation/migration and tumor progression, antagonizing activity of cytotoxicity exerted by antineoplastic drugs, or exerted synergy with conventional therapy. Although in vitro results are promising, most bioactive compounds have not yet been tested in preclinical or clinical studies. Moreover, some of the compounds are not soluble and therefore have a reduced bioavailability when administered orally (e.g., flavonoids), which reduces their potential. Therefore, special formulations or chemical modification are needed to increase the bioactive potential. Overall, nature provides a wide range of bioactive compounds with anti-leukemic potential, and extensive research is still needed for them to be considered viable therapeutic options for the treatment of various types of leukemia.