1
Insights into the Biological Evaluation of Pterocarpanquinones and Carbapterocarpans with AntiTumor Activity against MDR Leukemias Vivian M. Rumjanek *a, Raquel C. Maiab, Eduardo J. Salustiano *a,c, Paulo R.R.Costac a
Laboratório de Imunologia Tumoral, Instituto de Bioquímica Médica Leopoldo de Meis (IBqM), Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; b
Laboratório de Hemato-Oncologia Celular e Molecular, Programa de HematoOncologia Molecular, Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil; c
Laboratório de Química Bio-orgânica (LQB), Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Running title: Anti-tumor activity of isoflavonoid derivatives Keywords: pterocarpanquinones, carbapterocarpans, anticancer agents, tumor cells, leukemias, MDR *
Corresponding authors: Prof. Vivian M. Rumjanek and Dr. Eduardo J. Salustiano, Laboratório de Imunologia Tumoral, Instituto de Bioquímica Médica Leopoldo de Meis (IBqM), Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, Prédio CCS, Bloco H, segundo andar sala 03. CEP.21941-902. Rio de Janeiro, Brazil. Phone: 55 21 3838 6780. e-mails:
[email protected] [email protected]
ACKNOWLEDGEMENTS The authors are grateful to Prof. Alcides J. da Silva, Prof. Camila D. Buarque and Prof. Chaquip D. Netto. Financial support: Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), INCT-Controle do Câncer do Conselho Nacional de Pesquisa, Desenvolvimento Científico Tecnológico e Inovação (CNPq), Fundação do Câncer.
DRAFT MANUSCRIPT as of 30-03-2017. Final version will be provided upon request. The published manuscript is available at EurekaSelect via http://www.eurekaselect.com/openurl/content.php?genre=article&doi =10.2174/1871520618666180420165128.
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Insights into the Biological Evaluation of Pterocarpanquinones and Carbapterocarpans with AntiTumor Activity against MDR Leukemias Vivian M. Rumjanek *a, Raquel C. Maiab, Eduardo J. Salustiano a,c, Paulo R.R.Costac a
Laboratório de Imunologia Tumoral, Instituto de Bioquímica Médica Leopoldo de Meis (IBqM), Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; bLaboratório de Hemato-Oncologia Celular e Molecular, Programa de Hemato-Oncologia Molecular, Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil; cLaboratório de Química Bio-orgânica (LQB), Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Abstract: In an attempt to find anticancer agents that could overcome multidrug resistance (MDR), two new classes of modified isoflavonoids were designed and synthesized, and their effectiveness evaluated against a vast array of tumor cell lines. Pterocarpanquinone (LQB-118) and 11a-aza-5-carbapterocarpan (LQB-223) were the most promising. LQB-118 induced cell death, in vitro, in the M range, to a number of human cancer cell lines as well as to fresh tumor cells obtained from patients with acute or chronic myeloid leukemia, independent on whether they exhibit or not the MDR phenotype. Furthermore, leukemic cells were more sensitive to LQB-118 compared to cells from solid tumors. Given to mice, in vivo, LQB-118 affected the growth of melanoma or Ehrlich carcinoma. Conversely, no general toxicity was observed in vivo, by biochemical, hematological, anatomical or histological parameters and toxicity in vitro against normal cells was low. The process involved in tumor cell death seemed to vary according to cell type. Apoptosis was studied by externalization of phosphatidylserine, DNA fragmentation, caspase-3 activation, reduced expression of XIAP and survivin, ER stress, cytosolic calcium increase and mitochondrial membrane depolarization. Autophagy was also evaluated inhibiting caspase-9, with no effect observed in beclin 1, whereas pretreatment with rapamycin increased cytotoxicity induced by LQB-118. In addition, LQB-118 increased ROS, inhibited NFB nuclear translocation and secretion of TNF- modulated microRNAs miR-9 and miR-21 and modified the cell cycle. Despite being less studied, the cytotoxic effect of the 11a-aza-5-carbapterocarpan LQB-223 was present against several tumor cell lines, including those with the MDR phenotype. Keywords: pterocarpanquinones, carbapterocarpans, anticancer agents, tumor cells, leukemias, MDR. Running title: Anti-tumor activity of isoflavonoid derivatives Graphical Abstract:
Cell proliferation Cell cycle Pterocarpanquinone LQB-118
Carbapterocarpan LQB-223
Multiple pathways affected
Apoptosis
3 1. INTRODUCTION Neoplastic diseases are serious public health problems affecting a significant number of the population worldwide [1]. Due to the increase in cancer incidence driven by a growing and ageing population, cancer treatment represents a major challenge for the next years, especially in developing countries [2]. The development of new antineoplastic drugs is of great relevance due to inability of many current drugs to overcome resistance mechanisms, which are the main obstacles for successful treatment [3]. Therefore, it is highly desirable that these new agents may be capable to overcome the phenomenon of multidrug resistance (MDR). There are diverse mechanisms involved in MDR, including evasion from drug-induced apoptosis, increase of efflux transporter proteins activity, decrease in influx protein and change of the target enzymes, such as topoisomerases [4]. Targeted therapy has revolutionized treatment of cancer because the side effects are tolerable and it produces higher response rates compared to conventional chemotherapy. Despite the success of targetedbased cancer therapy, resistance mechanisms have surpassed the advantages of target-drugs. Taking into account that cancer is a complex disease, it is unlikely to obtain a complete therapeutic success by targeting a single target. Thereby, developing new anticancer drugs is necessary. However, given the complexity in developing new drugs this process is time-consuming. It should ideally reach a molecular multi-target network in cancer cells, sparing normal cells. This is a very difficult scenario, and implicates in robust efforts bringing together biology and medicinal chemistry, besides other important areas of knowledge . 1.1. Pterocarpanquinones and carbapterocarpans: Molecular design Wall et al described in 1995 the isolation of pterocarpan 1 from Petalostemon purpureus and reported for this compound (Figure 1) a strong antiproliferative effect toward KB tumor cell line (ED 50=0.9 g.mL-1, 3M) [5]. This compound was active in a standard in vitro DNA strand-scission assay. Two other products, 2 and 3, were also isolated but were less potent (ED50=4.0 g.mL-1 and 5.6 g.mL-1, respectively 12.73 M and 19.7 M) and were inactive in the DNA strand-scission assay, suggesting the catechol group at the A-ring in 1 is important for antitumor activity [5]. Some years later we synthesized 1, in racemic form, and this compound showed strong antiproliferative effect toward leukemia cell lines (ED 50=1.337.65M), including cell lines with MDR phenotype [6]. As catechol groups can be oxidized in vivo to oquinones, which may present toxic effect, the corresponding o-quinone 4 was also prepared and showed toxic effects toward proliferating lymphocytes [6].
Fig.1 - Structures of natural (1-3) and synthetic pterocarpans (4), and the new p-naphthoquinone hybrid compounds (5 and 6). In 2000, we were engaged in a project aiming at the discovery of new antineoplastic compounds and pterocarpan 1 seemed to us an interesting prototype. We decided to substitute the pro-toxic catechol group at the A-ring in 1 for the p-naphthoquinone moiety, less toxic and present in the structure of a series of anticancer and anti-parasitic drugs used in the clinic. This new structural framework design was later
4 named pterocarpanquinone and 5 is shown in Figure 1 as an example [7]. In the racemic form these compounds showed strong antiproliferative effect toward sensitive and MDR leukemias (ED 50=1.67-6.77 M) [8]. We also prepared compounds with new structural patterns at the E-ring, along with a nonsubstituted derivative, LQB-118 (6), which was selected in our studies as the most promising compound. Due to the interesting pharmacological activities of LQB-118 (6), we explored more this new structural prototype. Firstly, we substituted the oxygen atom at the C-ring for a sulfonamide moiety, a privileged structure in Medicinal Chemistry [9]. The intention was to prepare new derivatives with dual behavior, by the cooperative properties of the two pharmacophores, the p-naphthoquinone and sulfonamide moieties. The resulting compound (7) (Figure 2) and LQB-118 (6) shared similar antiproliferative properties [10-11]. To understand if the sulfonamide moiety could be a pharmacophore as well, the p-naphthoquinone moiety was removed from 7. The resulting compound 8 (11a-aza-pterocarpan) and the corresponding 5carba-analogue 9 (LQB-223) were then synthesized. It is important to note that LQB-223 received different denominations in previous works but, from now on, the correct one would be 11a-aza-5-carbapterocarpan. These compounds presented antiproliferative properties but LQB-223 was the most promising. Interestingly, 9 (LQB-223) and 6 (LQB-118) have different potencies, depending on the target cell lines [11-13].
Fig.2 - Structures of the hybrid aza-pterocarpanquinone (7), the aza-pterocarpan derivative (8) and the 5carba-analogue LQB-223 (9). As more studies have been performed with LQB-118 on the various tumor models, the activity of this compound will be described here in greater length.
1.2. Effect of the pterocarpanquinone LQB-118
1.2.1. Effect of LQB-118 on the cell cycle and the induction of cell death A normal development requires the maintenance of tissue homeostasis. This is a consequence of the balance between cell proliferation, resulting from cell cycle progression leading to cell division, and cell death. By using shared factors in both processes, it is possible to control and maintain the balance between them. In cancer, this equilibrium is lost. This might be a result from either unregulated proliferation or failure to respond to death signals. A number of chemotherapeutic drugs tend to affect cell cycle progression and to induce apoptosis. Inhibition induced by LQB-118 in leukemic cells did not seem to be associated with blockade on a particular phase of the cell cycle [14-16]. However, in prostate cancer cells, inhibition of cell proliferation and a blockade at the G2/M phase of the cell cycle were observed after LQB-118 treatment [17]. In addition to this, c-Myc was reduced as well as cyclins D1 and B1, whereas an increase in p21 mRNA was observed [17]. It is possible that c-Myc reduction led to the decrease in cyclin D1 while the increased levels of p21 could negatively affect the activation of cyclins E and B (Figure 3). Experiments treating leukemic cells with LQB-118 indicated that this treatment suppressed FoxM1 expression in the acute myeloid leukemia
5 (AML) cell line U937 (M4/M5 FAB subtype) but not in another AML cell line, HL-60 (M2/M3 FAB subtype) [16]. FoxM1, a forkhead box transcription factor and cell cycle regulator which is implicated on AML progression [18], is known to inhibit p21 and p27 and to positively affect Cdk1 and Cdk2 [19], and this might explain increased levels of p21 (Figure 3). c-Myc
G0
FoxM1
M
G1
Cyclin D
Cyclin D
Cdk6
Cdk4
Cyclin E Cdk2 p21
FoxM1
FOXO3a
Survivin
Bim
Cyclin B
Cdk1
p27
G2
S
Cyclin A
Cdk2 Apoptosis
Fig.3 - Cell cycle and hypothetical sites where LQB-118 might act. Other chemotherapeutic drugs have been evaluated by their capacity of inducing cell cycle arrest and disturbing the balance between growth and death. Using as a model the CML cell line K562, another group described the activities of the new synthetic naphthoquinone derivative CM363 [20]. This compound was effective in inducing growth inhibition at a low concentration and cytotoxicity with higher concentrations. The authors described that arrest at G0/G1 and/or G2/M phases were dose- and timedependent with increased levels of cyclin E and downregulation of cyclin B, cyclin D3, p27, pRb, Wee1 and BUBR1 [20]. CM363 was also more effective on leukemic cells and decreased viability by the mitochondrial apoptotic pathway with cytochrome C release and cleavage of caspase-3 and -9, and PARP [20], similar to LQB-118. The pterocarpanquinone LQB-118 induced cell death, in vitro, to a number of human cancer cell lines [10-11, 14-17, 21] (Supplementary Table 1) as well as to fresh tumor cells obtained from patients with leukemia [14, 22] (Supplementary Table 2). On the other hand, non-tumor cells were not affected by the drug in the concentrations used [10-11, 13] (Supplementary Table 3). The only exception among the tested cells was a human prostate cell line that was equally sensitive as the prostate tumor cell lines to the effect of LQB-118 [17]. In agreement with the results of not affecting non-tumor cells, murine tumor cell lines were sensitive to death induction by LQB-118 [13] whereas normal cells obtained from mice [11, 13, 23], hamsters [24], or normal monkey cell lines [25-26] were resistant. Generally, leukemic cells were more sensitive to LQB-118 compared to cells from solid tumors. This fact suggests that probably more than one death process is involved in the loss of viability induced by LQB-118. Cell lines that originated from CML [15, 22] and AML [14, 16] as well as cells from patients with these diseases [14, 22] presented evidences of apoptosis that varied depending on the cell type under study. Figure 4 depicts the intrinsic apoptotic pathway and possible sites affected by LQB-118. Similarly, prostatic cancer cell lines as well as a normal human prostate cell line externalized phosphatidylserine [17] and the same was true for cancer cell lines from small and non-small-cell lung cancer [27]. Furthermore, the antiapoptotic proteins (IAPs) Bcl-2 [15], XIAP [14, 22] and survivin [14, 16, 22] were studied. Only a tendency for Bcl-2 decrease was found in Jurkat cell line after treatment with LQB-118. However, the expressions of the IAPs XIAP and survivin were reduced in AML cell lines [14, 16] but, once again, with sensitivities varying according to the cell line. In accordance with these results, other authors have shown that sepantronium bromide (YM155), a small molecule with a 1,4-naphthoquinone moiety, acts in a very similar fashion, suppressing both XIAP and survivin while presenting little effect on the expression of Bcl-2 [28].
6 Chemotherapeutic drugs, irradiation
Apoptotic signal Bcl-2 Bcl-XL Cytochrome C
AIF
APAF-1 Apoptosome
PARP Caspase-9
XIAP Caspase-3 Caspase-7
Apoptosis
DNA fragmentation
Fig.4 - Intrinsic Pathway and effect of LQB-118. Another apoptotic pathway that may be induced by LQB-118 is via endoplasmic reticulum (ER) stress (Figure 5). A number of chemotherapeutic drugs activate this pathway; one example is exposure to the drug lapachone [29]. Treatment with lapachone, a naphthoquinone that bear structural and functional similarities with LQB-118, is accompanied by increased intracellular calcium, activation of caspase-12, MAPK phosphorylation and activation of caspase-7 and calpain [29]. Stress
Ca+2
Bax Bak
Bcl-2 Pro-caspase-4 Pro-caspase-12
Ca+2
Caspase-4 Caspase-12
Calpain
Cytochrome C APAF-1 Apoptosome
Apoptosis
Caspase-3 Caspase-7
Fig.5 - ER stress and hypothetical sites where LQB-118 might act.
Caspase-9
7 The possible involvement of this pathway in LQB-118-induced apoptosis was evaluated using two leukemic cell lines, K562 and Jurkat. Different outcomes were observed when LQB-118 was tested on these two cells. Caspase-12 and caspase-9 were activated in both cell types in a dose-response way but only K562 was protected from cell death using a caspase-12 inhibitor [15]. Increased intracellular calcium levels were observed in both cell lines but LQB-118 only induced mitochondrial membrane depolarization in K562 cells, what could be reversed by the calcium chelator BAPTA-AM. This suggests that at least in this cell line cell death could be ER stress-induced [15]. Autophagy may play a dual role in relation to cell viability, as it may lead either to cell survival or to cell death. The autophagy pathway is essential to recycle intracellular constituents and to maintain cellular homeostasis. Failure to activate autophagy in response to stress can lead to cell death. It has been suggested that caspase-9 plays a role in autophagy-mediated cell survival and when inhibited it may augment cell death. When, following LQB-118 treatment, caspase-9 activation was inhibited K562 was not protected from the effect of LQB-118, whereas there was an increased cytotoxicity in Jurkat cells [15]. Beclin 1 regulates autophagy by forming complexes with a variety of cellular proteins involved in the process [30]. In a different model, using the AML cell lines HL-60 and U937, the expression of beclin 1 was analyzed after LQB-118 treatment. No increase in the levels of beclin 1 was observed in either cell line, on the contrary, a slight decrease was observed in HL-60 [16]. Using a mouse melanoma model B16F10, pre-treatment with rapamycin increased the cytotoxicity induced by LQB-118. However, inhibiting the fusion between lysosomes and autophagosomes using chloroquine, did not suppress LQB-118 cell death [13]. Therefore, if LQB-118 plays a role in the autophagy process this is limited to certain tumors. 1.2.2. Mode of Action of LQB-118 It is quite clear that the effect and possible cellular targets of LQB-118 vary among different tumor cells depending on their intrinsic biological characteristic. This suggests that the original drug target is upstream and what is being seen is the reflection of the way the particular cell tries to circumvent the insult produced by the drug. Many possible mechanisms for the mode of action of LQB-118 have been offered. One proposed mechanism for LQB-118 cytotoxic effect is the induction of oxidative stress through the increase of reactive oxygen species (ROS). However, due to increased level of antioxidants, resistant cells have usually lower ROS levels compared to their sensitive counterpart [31]. In our experience, both ROS resistant and ROS sensitive cells died as a result of LQB-118 [10, 15]. Increased ROS levels could be observed after exposure to LQB-118 [15] and when N-acetyl-cysteine (NAC) was added to K562 cells treated with the drug, there was a partial inhibition of cytotoxicity [Salustiano, unpublished data]. When buthionine sulfoximine (BSO) was added, the cytotoxic effect of LQB-118 was magnified in K562 cells and to a lesser extent in Jurkat [32]. These results suggest that ROS may play some role in the cytotoxic effect produced by this pterocarpanquinone. Another possibility is that the mechanism of action of LQB-118 could result from biotransformation of this compound via one- or two-electron reduction. By incorporation of a single electron, xenobiotic quinones can be reduced to highly reactive and pro-oxidant intermediates semiquinones. Since chronic myeloid leukemias such as K562 and its MDR counterpart Lucena-1 are resistant to oxidative damage [33], this mechanism could not by itself explain the cytotoxicity induced by LQB-118. Alternatively, NAD(P)H:quinone oxidoreductase (NQO1), a detoxifying enzyme expressed by most cancer cells, can convert quinones into bioactive hydroquinones by transferring two electrons by using either NADH or NADPH as reducing cofactor [34]. This mechanism was demonstrated to occur in vitro by structural rearrangement of the hydroquinone intermediate and formation of a DNA alkylating agent [10]. This rearrangement involves the participation of both the pterocarpan and quinone moieties of LQB-118 (Figure 6) and results in a Michael acceptor, which form adducts with essential cell nucleophiles like purines and pyrimidines. Thiol groups (R-SH) such as the one present in glutathione are very powerful nucleophiles, and thus susceptible to form adducts with Michael acceptors [35]. Since NAC and BSO, respectively a precursor and inhibitor of the glutathione synthesis pathway, altered the cytotoxic effect of LQB-118 toward leukemias, this mechanism seems feasible. In agreement with this possibility, blocking the activity of NQO1 with the inhibitor dicoumarol reduced the toxic effect on K562 cells [15]. However, Jurkat cells were also susceptible to the effect of LQB-118 and this cell line does not express NOQ1 [15]. This suggests that at least in some cells the mechanism of action of LQB-118 is independent of DNA alkylation.
8
Apoptosis
Fig.6 - LQB-118 bioreductive activation by NQO1. The cellular target of LQB-118 could be a transcription factor playing a central role in different pathways. The transcription factor NFB is important in carcinogenesis [36] (Figure 7). LQB-118 was shown to inhibit NFB activation in vivo [37] and in vitro [21]. The NFB subcellular localization was evaluated after LQB-118 treatment in K562 and Lucena-1 cells and it was observed that the drug inhibited nuclear translocation. As XIAP, survivin and ABCB1 are targets of NFB their decreased expression is consistent with the modulation of NFB [21]. Despite its effects on hematologic malignancies, LQB-118 is capable to induce toxicity to a variety of solid tumors. Molecules containing a naphthoquinone moiety are prime candidates for the therapy of these tumors mainly due to their capacity of being bioactivated on a hypoxic microenvironment [38]. Availability of oxygen and nutrients is limited since they are consumed by the rapidly proliferating cells from the outer layers of the expanding tumor masses, driving cells from the inner layers to undergo a metabolic adaptation to thrive in (or escape from) that hostile condition. These adaptations are mediated by the hypoxia-inducible factor 1 (HIF-1) transcription factor, which regulates the transcription of diverse genes associated to tumor progression and drug resistance such as VEGF, GLUT1, MMP2 and MDR1 [39]. The generation of an alkylating intermediate under hypoxic conditions is a mechanism common to many quinone-based drugs, in a way that they are considered hypoxia-activated prodrugs (HAPs) [38]. In addition, a complex crosstalk exists between HIF-1, NFB and TNF- under normoxic conditions. Van Uden et al. demonstrated that members from the NFB family are able to bind to the promoter region of the HIF1A gene, an effect that also occurs after exposure to TNF-. Considering that LQB-118 is able to counter both NFB translocation to the nucleus and TNF- production, it is possible that this pterocarpanquinone would play a role in reducing the levels of HIF-1 on solid tumors. LQB-118 is capable of entering the cell. This is quite clear when intracellular parasites, inside normal macrophages or fibroblasts, are killed by the drug [23-25, 41]. Conversely, most normal cells are not killed by LQB-118 (Supplementary Table 3). Therefore, another possibility would be that to kill a tumor cell LQB-118 interacts at the cell surface and that this interaction produces an effect that is more determinant to the fate of tumor cells compared to normal cells. Many cancer cells show aberrant or constitutive NFκB activation and it has been suggested that cytokines produced by tumor cells may activate NFκB in an autocrine/paracrine manner via surface receptors [42]. Therefore, one possibility involves the inhibition by LQB-118 of either the binding or the secretion of extracellular factors that activate NFB. In agreement with that is the observation that secretion of TNF- has been inhibited in vitro [10] and in vivo [37] by LQB-118, but it is difficult to decide what is the primary event. MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression by binding to complementary sites on target mRNA transcripts. Aberrant miRNA expression is the rule rather than the exception in cancer. Furthermore, the expression in tumors is usually downregulated [43]. Regulation of NFκB can also occur via miRNAs [44]. Two microRNAs, miR-9 and miR-21, were analyzed using the CML cell lines K562 and Lucena-1. After treatment with LQB-118 these cell lines produced opposite response with respect to the expression of these miRNAs. Downregulation of miR-9 and miR-21 was
9 observed in K562, whereas increased levels of both miRNAs were observed in Lucena-1 cells [21]. When acting as a suppressor, the increase in miR-9 is associated with inhibition of NFκB1, and miR-21 overexpression has been associated with an oncogenic profile. As for now, these conflicting observations related to the effect of LQB-118 are not clearly understood, as both cell lines are equally susceptible to the drug. LQB-118
BCR-ABL
AKT
TKIs
Ras
LQB-118
LQB-118 LQB-118
ERK1/2 IB Proteasome
miR-9
NFB
miR-21 Survivin
XIAP
Pgp
Apoptosis
Fig.7 - NFκB activation pathway and LQB-118. 1.2.3. Effect of LQB-118 on multidrug resistant leukemic cells The phenomenon known as MDR (multidrug resistance) is a frequent cause of failure of chemotherapeutic intervention in cancer patients. Independent of being acquired or inherent, MDR is characterized by cross-resistance to a broad range of anticancer drugs that differ both in structure and in mode of action. The better studied MDR mechanism involves the expression and activity of ABC transporters (ATP-binding-cassette transporter) leading to increased drug efflux, but the resistance process is multifactorial and may involve other mechanisms such as decreased drug uptake, drug detoxification and resistance to apoptotic mechanisms [3]. The effect of LQB-118 was studied on two human CML MDR cell lines, Lucena-1 cells and FEPS, and compared to the effect on their sensitive counterpart K562. These MDR cell lines have been selected for resistance to the vinca alkaloid vincristine (Lucena-1) or the anthracycline daunorubicin (FEPS) and both cell lines overexpress the transporter ABCB1. Additionally, FEPS overexpresses the transporter ABCC1 as well [45-47]. The evaluation of cytotoxicity was performed by the MTT assay and LQB-118 was effective, at similar concentrations, in the three cell lines (Supplementary Table 1) [10, 22, 32]. Cell proliferation was investigated in K562 and Lucena-1 and no difference between the two cell lines were observed, with 3Hthymidine incorporation being inhibited at concentrations of 2.88 M for K562 and 2.48 M for Lucena-1 [Salustiano, unpublished data]. These results indicate that LQB-118 is equally effective against MDR cell lines suggesting that LQB-118 is not a substrate for ABCB1. Furthermore, the cell surface expression of ABCB1 was partially inhibited in Lucena-1 cells cultured with the drug [22]. Apoptosis was observed in the three cell lines and when caspase-3 was measured in K562 and Lucena-1, activation of this caspase was seen as well as downregulation of the anti-apoptotic proteins survivin and XIAP [22]. Similarly, despite not overexpressing ABCB1 [Maia, unpublished data] the AML cell line Kasumi-1 has many characteristics of resistant cells. The efficacy of LQB-118 was tested on Kasumi-1 cells and downregulation of both XIAP and survivin was verified [14]. When other authors studied the relationship between ABCB1 and the IAPs survivin and XIAP in different MDR cell lines [48], it was realized that the expression of ABCB1 seemed to be independent of the IAPs. By silencing the IAPs they observed that downregulation of survivin or XIAP did not affect ABCB1 expression.
10 Even if IAPs expression is independently regulated, experiments of our group indicated that during MDR development in K562, it was possible to observe a concomitant overexpression of ABCB1 and survivin [49]. Another characteristic of MDR cells is the enhanced antioxidant activity of cells overexpressing ABCB1. Increased catalase activity has been described in Lucena-1 cells [46, 50-51] and FEPS [Vidal, unpublished data] compared to the parental non-MDR line K562. It seemed unlikely, therefore, that the mechanism of LQB-118 could involve oxidative stress in these cells. Another group, studying the AML cell line HL-60/AR, resistant to daunorubicin, that does not overexpress ABCB1 but overexpresses ABCC, verified that despite the high levels of anti-oxidant defenses of this cell line, this did not contribute to the mechanism of resistance to daunorubicin [52].
1.2.4. In vitro antineoplastic effect of LQB-118 on tumor cells from CML and AML patients Multidrug resistance mediated by ABCB1 or other resistance mechanisms can become an obstacle in the treatment of patients with hematological malignancies as they rely on systemic chemotherapy. Due to the encouraging results obtained with a number of neoplastic cell lines, the effect of LQB-118 was evaluated on cells from patients with CML and AML [14, 22] (Supplementary Table 2). Leukemic samples from 13 CML patients, exhibiting phenotypes related to drug resistance such as overexpression of ABCB1, ABCC1 and p53, were exposed in vitro to LQB-118 [22]. Twelve out of 13 samples were positive for ABCB1 and 11 samples expressed ABCC1. An elevated proportion (six out of the 13 samples) expressed p53, associated or not to the presence of efflux pumps. LQB-118 induced apoptosis was observed in samples from all patients independent of Sokal score, p53 expression and coexpression of the two ABC transporters [22]. Fresh blasts obtained from 17 AML patients were positive for ABCB1 expression and the great majority presented efflux activity [14]. In addition the patients presented a poor prognosis. Despite that, LQB-118 was effective in inducing above 20% apoptosis in seven out of 14 samples and only two out of 14 did not respond to LQB-118. Survivin was analyzed in only six samples where it presented higher constitutive levels than in normal samples. After LQB-118 treatment, there was a decrease in survivin levels in five out of the six samples. The effect of the drug on XIAP expression was less clear-cut due to the small number of analyzed samples [14]. These results suggest that LQB-118 is effective in inducing apoptosis in patient leukemic cells independent on whether they exhibit or not the MDR phenotype.
1.2.5. Efficacy and lack of toxicity of LQB-118 in vivo Despite encouraging results produced by LQB-118 in vitro, there was a possibility that it had no efficacy in vivo or presented an unacceptable toxicity. Mice and hamsters were given LQB-118 either orally or by the intra-peritoneal route [13, 16, 23-24, 37] (Supplementary Table 4). Different protocols were tested and the concentrations used for mice varied from 50 mg/kg per day, orally, for 23 consecutive days [53] to 4.5 mg/kg per day, intraperitoneally, for 85 days – five days a week [23]. No effect on the biochemical parameters evaluated was observed [23, 53]. These parameters involved alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, total protein, albumin, globulin, alkaline phosphatase, creatinine, urea, uric acid, sodium, potassium, creatine kinase, calcium, iron, glucose, and cholesterol [53]. Moreover, no changes were observed in hematological parameters [53] nor in the anatomical and histological organ evaluation [53]. Similarly, using a lower dose, a single intraperitoneal injection of 3.6 mg/kg, or 0.36 mg/kg intraperitoneally, for 15 days – five days a week, no weight loss of the animals was observed, and when immune system organs and cells were studied no effect on weight, cell numbers or subpopulations were seen [13]. The one exception was a slight difference observed in thymocytes after a single dose [13]. After LQB-118 treatment, no changes in clinical parameters were observed such as modifications in the skin, hair appearance, eyes, mucous membranes, occurrence of secretions or autonomous activity (e.g., lacrimation and abnormal breathing), stool consistency, and urine color [53]. Similarly, normal weight gain, no fur loss nor any differences in mice behavior were observed [13]. Despite the absence of in vivo toxicity, these concentrations were absorbed and reached effective levels in that they were capable of exhibiting anti-leishmanial [23-24, 53], anti-inflammatory [37] and anticancer effects [13].
11 An impairment of tumor growth was observed in animals treated with LQB-118 [13]. Two-week treatment with 0.36 mg/kg per day LQB-118, significantly reduced tumor masses of B16F10 melanoma and Ehrlich carcinoma. Furthermore, treatment with LQB-118 ameliorated the cachexia produced by B16F10 melanoma [13]. This effect is probably a result of the inhibition of TNF- release [10, 37]. However, despite the encouraging results obtained with in vivo administration of LQB-118, with an effective reduction of tumor sizes, this was only followed by a modest increase in survival of the melanoma-bearing mice [13]. Treatment with higher non-toxic concentrations may overcome this problem. 1.3. Biological activity of the 11a-aza-5-carbapterocarpan LQB-223 The cytotoxic effect of LQB-223 was demonstrated against several tumor cell lines [11-12, 54] (Supplementary Table 5). The new carbapterocarpan was equally effective against cell lines overexpressing ABCB1 [12, 54], a result similar to what was observed with the pterocarpanquinone LQB-118. Lack of proliferation, cell cycle arrest in G2/M and induction of apoptosis were consequences of the exposure of LQB-223 to the leukemic MDR cell lines, Lucena-1 and FEPS, as well as their parental counterpart K562 [54]. Furthermore, despite the fact that FEPS is described as a very resistant cell line and overexpresses ABCC1 as well as ABCB1 [47] this phenotype did not protect these cells from the anti-proliferative and cytotoxic activity of the compound [54]. Concerning models of promyelocytic leukemias, LQB-223 was able to induce ROS in HL-60, but not in Kasumi-1 or U-937 strains. The distribution of NRF2 (nuclear factor E2-related factor 2), a protein responsible for the regulation of the transcription of antioxidant proteins, is similar in these cell lines, being constitutively activated and located in the nucleus. After LQB-223 treatment only HL-60 saw a diffuse distribution of this protein over the citosol [Maia, unpublished data], responses that are consistent with the phenotypes of resistance to oxidative stress of these three AML cell strains. Additionally, when HL-60 was exposed to LQB-223, an increase of microRNAs from the miR-29 family, which are related to the response to oxidative stress, could be observed [Maia, unpublished data]. These observations suggest that, although LQB-223 is capable of inducing oxidative stress, it is important to note that Kasumi-1, U-937, K562, Lucena-1 and FEPS cells are resistant to oxidative stress and are still susceptible to LQB-223. LQB-223 compound could also promote changes in cell viability and proliferation in two different breast cancer cell lines: MCF-7 (invasive breast ductal carcinoma, wild-type p53, estrogen and progesterone receptors positive and Her2/neu overexpression negative), and MDA-MB-231 cell line (invasive breast ductal carcinoma, p53 mutant, estrogen, progesterone and Her2 receptors negative). A reduction on the viability and proliferation was also observed in the doxorubicin-resistant cell line MCF-7 DoxR (doxorubicin-resistant cell line derived from parental MCF-7) after LQB-223 treatment. This inhibition was higher compared to that induced by docetaxel and doxorubicin [12]. Therefore, LQB-223 was effective not only towards breast cell lines overexpressing ABCB1, but also regardless of the p53 status. The effect of LQB-223 was also studied on normal cells [11-12, 54]. Normal human peripheral blood mononuclear cells activated or not with PHA [11] and mice splenocytes, activated or not with the mitogen Concanavalin-A, were still viable after exposure to 10 M LQB-223 [54]. However, unlike LQB118, the effect of LQB-223 on non-neoplastic cells varies according to the cell line. For example, while the non-neoplastic breast human cell line HB4a was resistant to the action of LQB-223 [12], this 11a-aza-5carbapterocarpan reduced the viability of the breast non-tumor cell line MCF-10A in lower ED50 than tumor cells MDA-MB-231 and MCF-7 [Salustiano, unpublished data]. Therefore, in vivo studies are necessary to define the degree of toxicity of this compound. CONCLUSIONS Two new classes of compounds, pterocarpanquinones and carbapterocarpans, were synthesized and shown to display antineoplastic activity. LQB-118 was inhibitory to a vast array of tumor cells, with a special impact on leukemic cells. Moreover, leukemic cells presenting a MDR phenotype were equally susceptible to this drug. This effect, added to the lack of toxicity when given in vivo and to normal cells in vitro, suggests that this compound might be considered as a good candidate for a chemotherapy drug. Indeed and considering these positive results, our group has patented its structure and mode of production under patent number US8835489B2 [55]. Moreover, despite the fact that much more information has been gathered about the pterocarpanquinone LQB-118, the carbapterocarpan LQB-223 also presents interesting features and deserves further studies.
12 ACKNOWLEDGEMENTS The authors are grateful to Prof. Alcides J. da Silva, Prof. Camila D. Buarque and Prof. Chaquip D. Netto. Financial support: Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), INCT-Controle do Câncer do Conselho Nacional de Pesquisa, Desenvolvimento Científico Tecnológico e Inovação (CNPq), Fundação do Câncer. REFERENCES [1] Global Burden of Disease Cancer, C., The global burden of cancer 2013. JAMA Oncol 2015, 1 (4), 505-527. [2] Pedersen, J.K.; Engholm, G.; Skytthe, A.; Christensen, K., Cancer and aging: Epidemiology and methodological challenges. Acta Oncol 2016, 55 Suppl 1, 7-12. [3] Gottesman, M.M.; Lavi, O.; Hall, M.D.; Gillet, J.P., Toward a Better Understanding of the Complexity of Cancer Drug Resistance. Annu Rev Pharmacol Toxicol 2016, 56, 85-102. [4] Baguley, B.C., Classical and Targeted Anticancer Drugs: An Appraisal of Mechanisms of Multidrug Resistance. Methods Mol Biol 2016, 1395, 19-37. [5] Chaudhuri, S.K.; Huang, L.; Fullas, F.; Brown, D.M.; Wani, M.C.; Wall, M.E., Isolation and structure identification of an active DNA strand-scission agent, (+)-3,4-di-hydroxy-8,9methylenedioxypterocarpan. J Nat Prod 1995, 58 (12), 1966-1969. [6] Netto, C.D.; Santos, E.S.; Castro, C.P.; da Silva, A.J.; Rumjanek, V.M.; Costa, P.R., (+/-)-3,4Dihydroxy-8,9-methylenedioxypterocarpan and derivatives: cytotoxic effect on human leukemia cell lines. Eur J Med Chem 2009, 44 (2), 920-925. [7] da Silva, A.J.; Buarque, C.D.; Brito, F.V.; Aurelian, L.; Macedo, L.F.; Malkas, L.H.; Hickey, R.J.; Lopes, D.V.; Noel, F.; Murakami, Y.L.; Silva, N.M.; Melo, P.A.; Caruso, R.R.; Castro, N.G.; Costa, P.R., Synthesis and preliminary pharmacological evaluation of new (+/-) 1,4-naphthoquinones structurally related to lapachol. Bioorg Med Chem 2002, 10 (8), 2731-2738. [8] Salustiano, E.J.; Netto, C.D.; Fernandes, R.F.; da Silva, A.J.; Bacelar, T.S.; Castro, C.P.; Buarque, C.D.; Maia, R.C.; Rumjanek, V.M.; Costa, P.R., Comparison of the cytotoxic effect of lapachol, alphalapachone and pentacyclic 1,4-naphthoquinones on human leukemic cells. Invest New Drugs 2010, 28 (2), 139-144. [9] Casini, A.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, L.T., Sulfonamides and sulfonylated derivatives as anticancer agents. Curr Cancer Drug Targets 2002, 2 (1), 55-75. [10] Netto, C.D.; da Silva, A.J.; Salustiano, E.J.; Bacelar, T.S.; Rica, I.G.; Cavalcante, M.C.; Rumjanek, V.M.; Costa, P.R., New pterocarpanquinones: synthesis, antineoplasic activity on cultured human malignant cell lines and TNF-alpha modulation in human PBMC cells. Bioorg Med Chem 2010, 18 (4), 1610-1616. [11] Buarque, C.D.; Militao, G.C.; Lima, D.J.; Costa-Lotufo, L.V.; Pessoa, C.; de Moraes, M.O.; Cunha-Junior, E.F.; Torres-Santos, E.C.; Netto, C.D.; Costa, P.R., Pterocarpanquinones, azapterocarpanquinone and derivatives: synthesis, antineoplasic activity on human malignant cell lines and antileishmanial activity on Leishmania amazonensis. Bioorg Med Chem 2011, 19 (22), 6885-6891. [12] Lemos, L.G.; Nestal de Moraes, G.; Delbue, D.; Vasconcelos Fda, C.; Bernardo, P.S.; Lam, E.W.; Buarque, C.D.; Costa, P.R.; Maia, R.C., 11a-N-Tosyl-5-deoxi-pterocarpan, LQB-223, a novel compound with potent antineoplastic activity toward breast cancer cells with different phenotypes. J Cancer Res Clin Oncol 2016, 142 (10), 2119-2130. [13] Salustiano, E.J.; Dumas, M.L.; Silva-Santos, G.G.; Netto, C.D.; Costa, P.R.; Rumjanek, V.M., In vitro and in vivo antineoplastic and immunological effects of pterocarpanquinone LQB-118. Invest New Drugs 2016, 34 (5), 541-551.
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16 SUPPORTIVE/SUPPLEMENTARY MATERIAL Table 1 - Effect of LQB-118 on human and murine tumour cell lines
Cell line
K562 (leukemia)
Lucena-1 (leukemia, MDR)
FEPS (leukemia, MDR)
HL-60 (leukemia)
Kasumi (leukemia)
U937 (leukemia)
Concentration (M)
References
Toxicity (MTT) Apoptosis Caspase-3 activation Caspase-9 activation Survivin downregulation XIAP downregulation ROS production Cell cycle arrest Mitochondria membrane potential loss Intracellular calcium increase Caspase-12 activation IBincreased expresssion Cytoplasmatic NFB Proteasome inhibition Decreased microRNAs (miR-9, miR-21) Toxicity (MTT) Apoptosis Caspase-3 activation Survivin downregulation XIAP downregulation Pgp expression inhibition IBelevated expresssion Cytoplasmatic NFB Proteasome inhibition Increased microRNAs (miR-9, miR-21)
1.67,20, 24.3
10, 11, 13
Inhibition of TNFproduction
25-100
10
Toxicity (MTT)
4.2
17
-----------------------------
------------------
---------------
Toxicity (MTT)
18.5
11
Viability
18.5
23
Toxicity (MTT)
24.8
13
Toxicity (MTT)
14.2
13
Morphology changes
>10
25
Toxicity (MTT)
12.8
26
Mitochondrial membrane potential loss
25-200
24
The concentration is given as tested concentrations or ED50 values.
19 Table 4 - In vivo effect of LQB-118 Model
Mice (C57BL/6)
Effect Effective against cutaneous leishmaniasis No toxicity (alanine aminotransferase, aspartate aminotransferase, creatinine) Effective against visceral leishmaniasis No toxicity (clinical, biochemical or hematological parameters) No effect on normal bone marrow cell subpopulations Inhibiton of Ehrlich tumour growth No effect on cells of the immune system Inhibiton of B16F10 melanoma growth No weight loss No hair loss No change in behaviour Anti-inflammatory activity in lung inflammation
Golden hamsters
Effective against L.braziliensis No toxicity
Mice (BALB/c) Mice (BALB/c) Mice (Swiss) Mice (Swiss) Mice (Swiss)
Mice (C57BL/6)
Treatment regimen
Ref.
- Local, 15 g/kg/day - Intraperitoneal, 4.5 mg/kg/day - Oral, 4.5 mg/kg/day (five times a week for 85 days)
23
- Oral, 2.5, 5 or 10 mg/kg/day (23 consecutive days) - Oral, 50 mg/kg/day (23 consecutive days) - Intraperitoneal, 3.8 mg/kg - Intraperitoneal, 3.6 mg/kg (Once) - Intraperitoneal, 3.6 mg/kg (Once) - Intraperitoneal, 0.36 mg/kg/day (five times a week for two weeks) - Intraperitoneal, 3.6 mg/kg/day (Once) - Intraperitoneal, 0.36 mg/kg/day (five times a week for two weeks) - Intraperitoneal, 1, 10 or 100 mg/kg (Once, one hour before LPS inhalation) - Local, 26 g/kg/day (three times a week for 56 days) - Oral, 4.3 mg/kg/day (five times a week for 56 days)
53 53 13, 16 13
13
37
24
20 Table 5 - Effect of LQB-223 on tumor cell lines and normal cells Cells HL-60 (human promyelocytic leukemia) K562 (human chronic myelocytic leukemia) Lucena-1(human chronic myelocytic leukemia MDR) FEPS (human chronic myelocytic leukemia MDR) HCT-8 (human colon cancer SF-295 (human glioblastoma) MDA-MB-435 cell line (human melanoma) MDA-MB-231 cell line (human breast)
MCF-7 cell line (human breast)
Effect
Concentration (M)
Refs.
Toxicity (MTT)
1.0
11
Toxicity (MTT) Cell cycle arrest
1.9, 2.9 2.5
11, 54 54
Toxicity (MTT) Cell cycle arrest
2.49 2.5
54
Toxicity (MTT) Cell cycle arrest
2.12 2.5
54
Toxicity (MTT)
3.0
11
Toxicity (MTT)
> 62.5
11
Toxicity (MTT)
0.5
11
Toxicity (MTT) Apoptosis (annexin V) Cell cycle arrest Colony formation Toxicity (MTT) Apoptosis (annexin V) Cell cycle arrest Colony formation
10.0, 15.0, 20.0 20.0 20.0 5.0, 20.0 5.0, 10.0, 15.0, 20.0 5.0, 20.0 5.0, 20.0 5.0, 20.0 5.0, 20.0
MCF-7 DoxR cell line Toxicity (MTT) (human breast MDR) ----------------------------------- ----------------------------------PBMC + PHA Toxicity (MTT) (human peripheral blood) HB4a cell line (human breast) BGM cell line (Green monkey, kidney) Splenocytes + ConA (Swiss mice) J774 (murine macrophages)
12
12
12
----------------------------- ------7.2
11
Toxicity (MTT)
5.0, 20.0
12
Toxicity (MTT)
> 1000 g/mL
26
Cell death (PI-positive cells)
> 10
54
Toxicity (MTT)
23
11
The concentration is given as tested concentrations or ED50 values.
