Natural Products for Treatment of Chronic Myeloid Leukemia Natural Products for Treatment of Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a hematological malignancy that arises due to reciprocal translocation of 3 ′ sequences from c-Abelson ( abl ) protooncogene on chromosome 9 with 5 ′ sequence of truncated break point cluster region ( bcr ) to chromosome 22. The fusion gene product BCR-ABL, a functional oncoprotein p210, is a constitutively activated tyrosine kinase that activates several cell proliferative signaling pathways. BCR-ABL-specific tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib and ponatinib potently inhibit CML progression. However, drug resistance owing to BCR- ABL mutations and overexpression is still an issue. Natural products are chemical compounds or substances produced by living organisms. They are becoming an impor- tant research area for cancer drug discovery due to their low toxicity and cost-effective-ness. Several lines of evidence show that many NPs such as alkaloids, flavonoids, terpenoids, polyketides, lignans and saponins inhibit CML cell proliferation and induce apoptosis. NPs not only differentiate CML cells into monocyte/erythroid lineage but also can reverse the multi-drug resistance (MDR) in CML cells. In this chapter, we review the anti-CML activity of various NPs.


Chronic myeloid leukemia
Chronic myeloid leukemia (CML) is a hematoproliferative neoplasm that is marked by uncontrolled myeloid cell divisions in the bone marrow [1]. CML arises due to a reciprocal translocation between chromosome 9 and chromosome 22 [(9;22) (q34;q11)], eventually culminating in the genesis of the bcr-abl oncogene. Approximately 90% of CML patients have shortened chromosome called "Philadelphia chromosome" (Ph) [2].
Targeting Abl kinase is clearly a proven successful strategy to combat CML. First generation tyrosine kinase inhibitor (TKI), imatinib, also known as Gleevac or STI571 inhibited BCR-ABL and suppressed CML progression [4]. Second generation TKIs such as nilotinib, dasatinib & bosutinib and third generation TKIs (Ponatinib) that are more potent to inhibit BCR-ABL kinase are currently used to treat CML [5,6]. All these TKIs were approved by the US Food and Drug Administration (FDA). TKIs have changed the clinical course of CML. However, mutations in bcr-abl and multi-drug resistance (MDR) due to efflux of the drug as a result of overexpression of p-glycoprotein (p-gp) make TKIs less effective. Primary or secondary resistance to TKIs therapy still exists; however, there is a constant need for alternative therapeutic strategy ( Figure 1) [7].

Natural products
Natural products (NPs) represent a large family of diverse secondary metabolites with profound biological activities. NPs are produced in several organisms like bacteria, fungi, plants [26]. Combination of imatinib and camptothecin increased Bax, cleavage of PARP-1, DNAdependent protein kinase (DNA-PK) in CML cells [27].
Capsaicin, an active component of capsicum genus, is a homovanillic acid derivative experimentally is shown to exhibit anti-mutagenic activity [28]. Capsaicin treatment of K562 cells decreased microRNA (miRNA) expression such as miR-520a-5p, a putative target of STAT3. Hence, capsaicin induced apoptosis via reducing mRNA involved in JNK/STAT pathway [29]. Capsaicin also stimulated GATA-1 promoter in CML cells which is an essential transcriptional factor for the development of erythroid cells [30].
Sanguinarine, a benzophenanthridine alkaloid, isolated from blood root plant Sanguinaria canadensis, belonging to the Papaveraceae family inhibited CML cell growth in a dose-dependent manner. At 1.5 μg/ml, sanguinarine induced apoptosis in CML cells. At higher concentration (12.5 μg/ml), sanguinarine caused blister formation in CML cells [33].
Staurosporine, an alkaloid isolated from the bacterium Streptomyces staurosporeus, not only inhibited CML cell growth but also induced differentiation of myeloid cell lineage to megakaryocytic lineage resulting in polypoidy formation. Staurosporine treatment resulted in upregulation and activation of JAK/STAT3, p-STAT3 nuclear translocation and downregulation of c-myc [34,35]. Staurosporine also induced differentiation of CML cells into erythroid cells via increased CD61 and CD42b levels [36]. 7-Hydroxy staurosporine (UCN-01), a potent PKC inhibitor is effective in inhibiting CML cell proliferation at a concentration of 3 μM for 24 h [37,38].
Tetrandrine is a bis-benzylisoquinoline alkaloid that is isolated from Chinese herb Stephania tetrandra. Combination of tetrandine and imatinib showed syngerisitic effect significantly inhibited CML cell growth. The combination treatment arrested CML cells at G1/S phase, enhanced caspase 3 mRNA, protein levels and decreased Bcl-2 mRNA, protein levels [39]. Combination of nilotinib and tetrandrine also effectively decreased the IC 50 of daunorubicin (DNR) on K562/A02 to 3.12 ± 0.13 μg/ml. This combinational effect not only increased Bax mRNA and protein levels but also decreased the survivin mRNA and protein levels [40]. Tetrandrine citrate, a novel tetrandrine salt which is highly soluble in water, Inhibited the growth of K562/IR, primary leukemic cells and primitive CD34 (+) leukemic cells with IC 50 ranging from 1.2 to 2.97 μg/ml. Tetrandrine citrate lowered BCR-ABL mRNA and β-catenin protein levels. Nude mice bearing CML tumors when orally administered with tetrandrine citrate ( Tylophora alkaloids (tylophorine, tylophorinine, tylophorindine) Tylophora indica -Nuclear condensation, ↑Caspases activation, release of cyt.C [60] 44 and 53 μM - [61] potently reduce tumor growth in in vivo models (Table 2). Besides, some alkaloids such as capasaicin, staurosporine induces differentiation of CML cells (Table 3).
Oroxylin A, an O-methylated flavone, found in the medicinal plant Scutellaria baicalensis, was tested against MDR K562/ADR cells. Oroxylin A specifically enhanced the sensitivity of K562/ ADR to ADR by selectively inducing apoptosis. The treatment downregulated CXCR4 expression and inhibited PI3K/Akt/NF-κB pathways [75]. NOD/SCID mice-bearing K562 xenograft, treated with oroxylin A (30 mg/kg BW) alone or in combination with imatinib enhanced the sensitivity of imatinib to K562 cells through suppression of STAT3 pathway, decreasing p-gp levels thus reversing MDR in CML cells [76].
Quercetin (Q), a major flavonol, found in the kingdom Plantae, exhibits many biological effects including Antioxidant, anti-inflammatory, anti-cancer and anti-diabetic activities [77]. While evaluating the anti-proliferative effect of pytoestrogens, it was found that Q specifically inhibits K562 and MDR K562/A cell growth [78]. When K562 cells were treated with Q at a ↑upregulation, ↓downregulation, ⊥cell cycle arrest & * -Inhibition rate (IR) at 100 μg/ml. Anti-cancer Drugs -Nature, Synthesis and Cell concentration of 9.2 mg/ml for 72 h, it induced apoptosis and reduced the BCR-ABL levels in CML cells [79]. Combination of Q and ADR was tested on MDR K562/ADR cells. Combined treatment enhanced activation of caspases 3,8 and loss of mitochondrial membrane potential (MMP). Furthermore, it lowered Bcl-2, Bcl-xl and enhanced the p-c-Jun-N terminal kinase and p-p38 mitogen-activated protein kinase (p-p38-MAPK). Q also significantly decreased the p-gp levels [80] and sensitized MDR K562/ADM to DNR and reversed MDR in CML cells [81]. Q inhibited K562 and MDR K562/A in the range of 5-160 μM. Q treatment of K562/ADR cells (5 μM) enhanced accumulation of ADR and, in addition, decreased the expression of MDRcausing proteins like ABC, solute carrier (SLC). Moreover, it reduced Bcl-2, TNF expression reversing MDR in CML cells [82]. Moreover, Q arrested CML cells at G2/M phase [83]. IC 50 of Q on K562 and K562/ADR was found to be 11 ± 2 μM and 5 ± 0.4 μM [84]. It also inhibited the Hsp70 levels in CML cells [85]. Q induced apoptosis via inhibiting the telomerase enzyme by enhancing human telomerase reverse transcriptase (hTERT) enzymes in CML cells [86].
In sum, flavonoids not only inhibit the growth of CML cells ( Table 4) but also induce their differentiation into erythroid or monocyte lineage (Table 3). Flavonoid fractions of plant extracts also inhibit CML cell proliferation and induced apoptosis .

Saponins
Saponins are a diverse group of secondary metabolites widely distributed in the plant kingdom. They produce soap-like foam when shaken in aqueous solutions. Their structure comprise of triterpene or steroid aglycone and one or more sugar chains. They exhibit anti-cancer and anti-cholesterol activities [164,165]. Various saponins inhibited CML cell proliferation (  Tegillarca granosa, when administered in mice-bearing MDR K562/ADM cell tumors inhibited tumor growth and downregulated mdr1 gene, BCR-ABL and sorcin [177]. HSS was also tested against MDR K562/ADR cells, and it induced apoptosis at 20 mg/l [178]. HSS also inhibited K562 cells at G0/G1 and S phase and lowered Bcl-2 and enhanced Bax levels ( Figure 2) ( Table 6) [179].
Since, there is a need for alternative strategy to develop new BCR-ABL inhibitors; NPs (obtaining from living organisms) offers an alternate, effective and inexpensive design for CML therapy. Moreover, they have less (or) no side effects. Studies conducted so far have revealed that many NPs inhibit CML cell proliferation and, in addition, induce cell death through apoptosis. NPs alone or in combination with other TKIs are able to reverse the MDR, thereby increasing the sensitivity of TKIs towards CML. Moreover, many NPs are able to differentiate CML cells into erythroid, monocyte or macrophage lineage. In vivo results have clearly shown that NPs potently suppress tumor growth. In sum, NPs serve as an inexhaustible source which renders an attractive alternate strategy to combat CML.

Conflict of interests
The authors declare that they do not have any competing interests.