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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 important research area for cancer drug discovery due to their low toxicity and cost-effectiveness. 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.
Keywords
chronic myeloid leukemia (CML)
BCR-ABL
TKIs
natural products (NPs)
multi-drug resistance (MDR)
chapter and author info
Authors
Kalubai Vari Khajapeer
Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Pondicherry, India
Rajasekaran Baskaran*
Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Pondicherry, India
*Address all correspondence to: baskaran.rajasekaran@gmail.com
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].
The bcr-abl oncogene encodes a constitutively activated tyrosine kinase, BCR-ABL. The catalytically activated kinase, in turn, activates multiple cell proliferatory signaling pathways such as RAS, a small GTPase, mitogen activated protein kinase (MAPK), signal transducers and activator of transcription (STAT), and phosphoinositide-3-kinase (PI3K) pathways [3].
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].
Figure 1.
Schematic representation of NPs and TKIs on BCR-ABL inhibition and downregulation of downstream signaling pathways (NP—natural products, TKI—tyrosine kinase inhibitor, CML—chronic myeloid leukemia, MDR—multi-drug resistance).
2. 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 and marine animals. NPs are inexpensive and have less (or) no side effects; hence, NPs are currently being explored as an invaluable source for treatment of cancerous and infectious diseases. As of 2013, 1453 new chemical entities (NCEs) have been approved by the US FDA, of which 40% are NPs or NP-inspired (semi-synthetic NP derivatives, synthetic compounds based on NP pharmacophores, or NP mimics) [8, 9]. A number of NPs Such as alkaloids, flavonoids, terpenoids, polyketides, lignans, saponins, peptides and plant extracts exhibited potent anti-CML activity.
2.1. Alkaloids
Alkaloids are naturally occurring organic compounds containing heterocyclic ring with nitrogen atom. Alkaloids, widely distributed in plant kingdom, are bitter secondary metabolites synthesized by plants, microbes and animals. They possess several physiological activities like anti-malarial, anti-asthmatic, anti-cancer, anti-bacterial, antiviral, anti-hyperglycemic and vasodilatory activities [10–13]. Their anti-CML activity is described below.
Berbamine (BBM) is a natural bisbenzylisoquinoline product, isolated from traditional Chinese herbal medicine Berberis amurensis, was tested on imatinib resistant K562 cell line (K562/IR) both in vitro and in vivo. The IC50 value was found to be 17.1 and 11.1 μM at 24 and 48 h. BBM downregulated Bcl-2, Bcl-xL, mdr-1 mRNA, p-gp levels and enhanced Bax & cytochrome C (cyt.C) release. BALB/c or nu/nu mice were injected with K562-r subcutaneously and the tumor-bearing mice, when treated with BBM [60 mg/kg body weight (BW)] intravenously effectively suppressed the xenotransplated tumors in these mice [14]. BBM also induced apoptosis in CML cells via downregulating survivin protein levels [15]. At 8 μg/ml dose of BBM, NFκB nuclear, IKK-α, IKB-α [16], BCR-ABL, p-BCR-ABL level were decreased [17]. Furthermore, BBM-induced differentiation of CML cells into RBC, granulocyte and megakaryocytes [18]. Interestingly, BBM is a heat shock protein 90 (Hsp90) inhibitor [19]. BBM inhibited MDR K562/adriamycin (ADR) [20] and K562/A02 cell lines consequently inducing apoptosis by reducing mdr-1 gene expression and reversing MDR effect [21]. 4-chlorobenzoyl berbamine (BBD9), an analogue of BBM was also tested against K562/IR. BBD9 with IC50 0.5 μg/ml was found to be more effective than BBM (IC50 8 μg/ml), BBD9-lowered BCR-ABL, IKK a, nuclear NF-κB. Furthermore, it increased the cleaved caspases 3,9, Poly(ADP-Ribose) polymerase (PARP) and LC3-phosphatidylethanolamine conjugate (LC3 II) expression levels. In nude mice model bearing K562 tumors, BBD9 was effective in reducing the tumor weight, promoting tumor regression [22]. E6, a derivative of BBM, was tested against MDR K562/doxorubicin (DOX) with 1, 3, 10 and 30 μM concentrations, and it significantly reduced the IC50 of DOX from 79.19 μM to 35.18, 21.86, 6.31 and 1.97 μM. Co-treatment of E6 with DOX arrested K562 cells at G2/M phase [23].
Camptothecin, isolated from Camptotheca acuminate, is documented to display anti-CML activity. Homocampthothecin (hCPT), a synthetic analogue of camptothecin, showed potent activity at IC50 value of 11 nM suggesting its potential use compared to parent compound camptothecin (IC50 57 nM) [24]. BN80927, an analogue of camptothecin, effectively inhibited K562 cell proliferation with IC50 of 8.4 nM [25]. NSC606985, an analogue of camptothecin, inhibited CML cell growth in a dose-dependent manner. The IC50 was found to be 6.25 nM [26]. Combination of imatinib and camptothecin increased Bax, cleavage of PARP-1, DNA-dependent 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].
Homoharringtonine (HHT), isolated from Cephalotaxus harringtona, has been documented to inhibit CML cell proliferation in a dose-dependent manner. The IC50 was found to be 43.89 ng/ml. HHT arrested K562 cells at G0/G1 phase and, in addition, downregulated Bcl-2, NF-κB, p-JAK2, p-STAT5, p-Akt, p-BCR-ABL levels [31, 32].
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 IC50 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 IC50 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 (100 mg/kg BW), reduced the tumor growth [41]. Combination of 5-bromotetrandrine (analogue of tetrandrine) and DNR decreased p-JNK 11,2 and MDR/p-gp levels in ADR resistant K562 cells [42].
Alkaloids from plant and microbial source inhibited CML cell proliferation in micromole (μM)/microgram (μg) concentration (Table 1) (Figure 2) [43–66]. Alkaloids are well documented to potently reduce tumor growth in in vivo models (Table 2). Besides, some alkaloids such as capasaicin, staurosporine induces differentiation of CML cells (Table 3).
Figure 2.
List of anti-CML alkaloids as 1—Ancistrotectorine E, 2—Berbamine, 3—BBD9, 4—Camptothecin, 5—BN80927, 6—NSC606985, 7—Homocamptothecin, 8—Capsaicin, 9—Cathachunine, 10—Cephranathine, 11—Crambescidin 800, 12—Crebanine, 13—Curine, 14—Cyanogramide, 15—9-deacetoxyfumigaclavine C, 16—d-Dicentrine, 17—Evodiamine, 18—Homoharringtonine, 19—Naamidine J, 20—Piperine, 21—Salvicine, 22—Staurosporine, 23—UCN-01, 24—Tetrandrine, 25—5-bromotetrandrine, 26—α-tomatine, 27—tylophorine, 28—tylophorinine, 29—5-chlorosclerotiamide, 30—10-episclerotiamide, 31—Eupolauramine, 32—Sampangine, 33, 34, 35—Arthpyrones A, B and C, 36–38—Auranomides A, B and C and 39—Virosecurinine.
List of some NPs and its differentiation capacity.
2.2. Flavonoids
Flavonoids belong to polyphenolic compounds which are prevalent in plants. They contain two phenyl rings A, B and a heterocyclic ring C (commonly referred as C6-C3-C6 skeleton) and are classified into many major classes like flavones, flavonols, flavanones, flavanonols and isoflavonoids (Figure 3). They exhibit antioxidant, anti-inflammatory, anti-bacterial, antiviral and anti-cancer activities and play a significant role in human health [67–74].
Figure 3.
Anti-CML activity of some NPs which include ? flavonoids: 1—Apigenin, 2—Baicalein, 3—Fistein, 4—Galangin, 5—Genistein, 6—Kaempferol, 7—Myricetin, 8—Naringenin, 9—Nobiletin, 10—Oroxylin A and 11—Tamarixetin. Terpenoids: 1—Gukulenin A, 2–4—Hebeiabinin A, D & E, 5—Parvifolines C, 6—3-hydrogenwadaphnin, 7—Tanshinone I, 8—EM23, 9, 10—Felixins F & G, 11—Kadlongilactone D. Polyketides: 1—Epiaspinonediol, 2—aza-EpoB, 3—dEpoF, 4—Heveadride, 5—Gilvocarin HE, 6—Rhizoxin, 7—Salarin C, 8—Tausalarin C, 9—Trineurone E. Lignans: 1—Arctigenin, 2—Cleistanthin A, 3—Honokiol, 4—6-hydroxyjusticidin C, 5—(+) –lariciresinol 9’-p-coumarate, 6—4-methoxy magndialdehyde. Peptides: 1, 2—chujamide A, B, 3—gombamide A.
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 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 MDR-causing 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]. IC50 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 [87–109].
Terpenoids are naturally occurring products representing the largest secondary metabolites. Approximately 60% of NPs are terpenoids. They are basically made up of five carbon isoprene units (IU). Depending upon the number of isoprene units present, terpenoids has been classified into hemiterpenoids (1 IU), monoterpenoids (2 IU), sesquiterpenoids (3 IU), diterpenoids (4 IU), sesterterpenoids (5 IU), triterpenoids (6 IU), tetraterpenoids (8 IU) and polyterpenoid (n IU). They have been documented to possess antioxidant, anti-inflammatory, anti-helminitic and anti-cancer activities [110–115].
Sesquiterpenoids, diterpenoids, sesterterpenoids and triterpenoidshas been shown to potently inhibit CML cell proliferation and induce apoptosis (Figure 3) (Table 5) [116–144]. Other diterpenoids such as scapaundulin C (from Scapania undulate (L.) Dum.,) [120], parvifoline Z, parvifoline AA (from Isodon parvifolius) [121], labdane-type diterpenes (from Chloranthus henryi Hemsl.) [124] and sesterterpenoid compounds 3, 11 and 12 (from Sarcotragus sp.) [133] and triterpenoid compounds 1, 2, 5, 7 and 9 (from Ganoderma hainanense) [135], (24R/S)-24-hydroxy-3α 10α-epoxy-9-eip-cucurbita-25-ene (1a, b) (from Fructus Viticis Negundo) [136] are also shown to efficiently inhibit CML cell proliferation.
Polyketides represent a large group of natural products that are produced by microorganisms and plants. These are secondary metabolites, derived by the repetitive condensation of acetate units or other short carboxylic acids catalyzed by multi-functional enzymes called polyketide synthases (PKSs) which is similar to fatty acid synthases [145]. Many polyketides suppress CML cell proliferation and induce apoptosis (Figure 3) (Table 6) [146–155].
Anti-CML activities of polyketides, lignans, saponins and peptides.
*LC50—lethal concentation.
2.5. Lignans
Lignans, natural compounds that are exclusively found in plants, are derived from amino acid phenyl alanine. They possess anti-oxidant and anti-cancer activities [156]. Various lignans effectively inhibit CML cell proliferation and induced apoptosis (Figure 3) (Table 6) [157–163].
2.6. 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 (Table 6) [166–174].
2.7. Peptides
Two peptides, chujamides A (1) and B (2), isolated from the marine sponge Suberites waedoensis inhibited K562 cell growth with LC50 values of 37 and 55.6 μM [175]. Another peptide, gombamide A (1), isolated from the marine sponge Clathria gombawuiensis inhibited CML cell proliferation with LC50 of 6.9 μM [176]. Haishengsu (HSS), a protein extract from 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].
2.8. Others natural products
Other natural products such as acetylenic metabolites, betanin, bufadienolide, mamea a/ba, cryptotanshinone, bavachalcone, polyanthumin, cubebin, denbinobin, digallic acid, perforanoid A, β- and α-mangostin, parthenolide, perezone, polyphyllin D, squamocin, toxicarioside H, tripolide, woodfordin I and rhodexin A inhibited CML cell proliferation (Table 7) [180–230]. Moreover, many plant crude extracts enriched with NPs inhibited the CML cell proliferation and induced apoptosis (Table 8) [231–280].
Of the several natural products, Homoharringtonine (alkaloid) (NCT00114959) is currently under phase II study sponsored by Chem Genex pharmaceuticals to reverse the Gleevac resistance in CML patients [281]. 17-AAG (analogue of glendamycin–polyketide) (NCT00100997) is currently under phase I clinical trial sponsored by Jonsson Comprehensive Cancer Center collaborated with National Cancer Institute (NCI). Efforts are underway to determine the side effects and optimal dose of 17-AAG for treating patients with CML in chronic phase who did not respond to imatinib-mesylate [282]. Paclitaxel (diterpenoid) (NCT00003230) is currently under Phase I/II trials to study the effectiveness in treating patients with refractory or recurrent acute leukemia or CML. This work is sponsored by Swiss Group for Clinical Cancer Research [283].
3. Conclusion
CML is a hematological malignancy that arises due to chromosomal translocation resulting in the presence of Ph chromosome. Initially, TKIs were designed to compete with the ATP binding site of BCR-ABL. TKIs effectively inhibited wild-type BCR-ABL; however, mutations in BCR-ABL and overexpression of drug efflux proteins following treatment decreased their potency.
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.
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