Lapachol: an overview Hidayat Hussain,*a Karsten Krohn,a Viqar Uddin Ahmad,b Ghulam Abbas Miana,c and Ivan Robert Greend
aDepartment of Chemistry, University of Paderborn, Warburger Straße 100, 33098 Paderborn, Germany bH.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan cRiphah Institute of Pharmaceutical Sciences, Riphah Internation University, Islamabad, Pakistan dDepartment of Chemistry, University of the Western Cape, P/Bag X17, Bellville,7530, South Africa
Abstract Lapachol is a naphthoquinone that was first isolated by E. Paterno from Tabebuia avellanedae (Bignoniaceae) in 1882. A wide spectrum of therapeutic activities have been attributed to lapachol or its derivatives viz., anti-abscess, anti-ulcer, antileishmanial, anticarcinomic, antiedemic, anti-inflammatory, antimalarial, antiseptic, antitumor, antiviral, bactericidal, fungicidal, insectifugal, pesticidal, protisticidal, respiradepressant, schistosomicidal, termiticidal, and viricidal. Originally isolated from species of the Bignoniaceae family, lapachol can also be found in other families such as Verbenaceae, Proteaceae, Leguminosae, Sapotaceae, Scrophulariaceae, and Malvaceae. The interesting and most usefull knowledge on lapachol, which is reviewed in this paper, can be used as a starting point in future research endeavors.
Antitumor activity In a 1968 study, lapachol demonstrated highly significant activity against cancerous tumors in rats.39 Then in 1974, the NCI reported that Phase I clinical trials failed to produce a therapeutic effect with lapachol without side effects and discontinued further cancer research.40 In a small study in 1980 with nine patients with various cancers (liver, kidney, breast, prostate and cervix), pure lapachol demonstrated an ability to shrink tumors and reduce feeling of pain caused by these tumors and achieved complete remissions in three of the patients.41 It is believed that the antitumor activity of lapachol may be due to its interaction with nucleic acids. Additionally it hasbeen proposed that interaction of the naphthoquinone moiety between base pairs of the DNA helix occurs with subsequent inhibition of DNA replication and RNA synthesis.42 Although lapachol has some beneficial effects, it is by no means a perfect anticancer drug. Despite the lack of significant toxicity even in large oral dose levels, sufficiently high blood levels were not attained to show a therapeutic effect.43 This led to the termination of further clinical development of lapachol. Because of its antitumor activity, it is an ideal candidate for systematic modification to develop an understanding of its structure-activity relationships and thus eventually to develop analogs with improved activity. Hartwell and Abbott reported44 that out of 68 synthetic analogs only one, 2-(3,7-dimethyl- 2,6-octadienyl)-3-hydroxy-1,4-naphthoquinone (3) (Figure 4), was active against the Walker 256 tumor cell line. One year later a study of Herman43 indicated a high activity against the Walker 256 tumor cell line for 2-(3’,3’-dibromo-2-propenyl)-3-hydroxy-1,4-naphthoquinone (4). In 1975, Linardi et al.44 developed a lapachol analog, 2-(3’-methyl-2-buteny1)-3-(tetraacetyl-β-Dglucopyranosyloxy)-1,4-naphthoquinone (5), which was effective in increasing the life span by over 80 % in mice inoculated with leukemic cells.Recently, Vargas et al.45 synthesized two lapachol derivatives 6 and 7 (Figure 5) and these compounds were active against lung, breast, melanoma, ovarian, prostate, and renal cancer.
Anti-metastatic activity
Metastasis is the major process responsible for the death in cancer patients. Balassiano et al.46 analyzed the effects of lapachol on a human cancer cell line and evaluated the potential of thissubstance as an anti-metastatic drug using an in vivo assay. The results of this study indicatedthat lapachol, in the maximal non-toxic concentration for HeLa cells of 400 μg/ml(corresponding to 1012 molecules of the drug/cell), induces alterations in the protein profile and inhibits cellular invasiveness, thus representing an important anti-metastatic activity.
Anti-angiogenic activity
Neovascularization is an essential process in tumor development and thus it is conceivable thatanti-angiogenic treatment may block tumor growth.77 In angiogenesis, nitric oxide (NO) is animportant factor which mediates vascular endothelial cell growth and migration. β-Lapachonehas been demonstrated to possess anti-cancer and anti-viral effects. Whether β-lapachone caninduce endothelial cell death or has an anti-angiogenic effect is still uncertain.77 Kung et al.77recently investigated the in vitro effect of β-lapachone on endothelial cells, including the humanvascular endothelial cell line, EAhy926, and human umbilical vascular endothelial cells(HUVEC). Kung et al. demonstrated that NO can attenuate the apoptotic effect of β-lapachoneon human endothelial cells and suggest that β-lapachone may thus have potential as an antiangiogenicdrug.77
Anti-tumor activity by activation of the Mre11-Tel1p G1/S checkpoint
β-Lapachone is an anticancer agent that selectively induces cell death in several human cancercell lines.79 However the precise mechanism of β-lapachone cytotoxicity is not yet fullyunderstood. Menacho-Marquez and Mauricio79 reported that β-lapachone treatment delayed cellcycle progression at the G1/S transition, incremented phosphorylation of the Rad53p checkpointkinase and decreased cell survival in the budding yeast, Saccharomyces cerevisiae.79 Furthermore, β-lapachone induced phosphorylation of histone H2A at serine 129. Thesecheckpoint responses were regulated by Mec1p and Tel1p kinases.79 Mec1p was required forRad53p/histone H2A phosphorylation and cell survival following β-lapachone treatment inasynchronous cultures, but not for the G1 delay. The major and vital conclusion of all thosefindings indicated that β-lapachone activates a Mre11p-Tel1p checkpoint pathway in buddingyeast. Given the conservative nature of the Mre11p-Tel1p pathway, these results suggest thatactivation of the Mre11-Tel1p checkpoint could be of significance for β-lapachone anti-tumoractivity.
Use of β-lapachone in pancreatic cancers
Erik Bey et al.80 discovered that β-lapachone, currently in phase II clinical trials for use inpancreatic cancers, is also effective against NSCLC (Non-Small Cell Lung Cancer). The authorsfound that NSCLC cells over express endogenous NAD(P)H:quinone oxidoreductase 1 (NQO1),similar to pancreatic cancers. In NQO1-positive cells, β-lapachone induced PARP-1-mediatedcell death.80 Typically, PARP-1 facilitates DNA repair by resealing single strand breaks. When NQO1 is confronted with massive DNA damage, such as after β-lapachone treatment, it triggersμ-calpain cell death mechanisms. β-Lapachone was most effective when delivered in short, 2- to4-h pulses. Downstream, the chemotherapeutic agent killed NSCLC cells independent of cellcycle or p53 status and in the absence of proapoptotic factors, according to the authors.80Ough et al.81 also found that cytotoxic in vitro effects of β-lapachone were inhibited with coadministrationof dicumarol, a specific inhibitor of NQO1. In pre-established human pancreatictumor xeno grafts in nude mice, β-lapachone demonstrated greater tumor growth inhibition whengiven intratumorally compared to when complexed with cyclodextrin to increase itsbioavailability.81 In another study on β-lapachone, Park et al.82,83 strongly suggested that NQO1 activity intumors may be further and selectively elevated using either local radiotherapy or hyperthermia,both established cancer treatment modalities, to improve the cytotoxicity of β-lapachone againsthuman cancer cells.82,83,84 7.2.5 Use of β-lapachone in treatment of neuroendocrine tumorsLarsson et al.85 studied in vitro drug sensitivity screening using the fluorometric microculture cytotoxicity assay in one human pancreatic carcinoid and two human bronchial carcinoid celllines. The aim of this study was to investigate drug sensitivity in neuroendocrine tumor cell lines.In addition, a normal human retinal pigment epithelial cell line was used for comparison. A totalof 18 drugs, including β-lapachone, with different mechanisms of action were tested. Thesestudies indicated that some of the tested compounds viz., β-lapachone, could possibly be used inclinical trials and demonstrate a therapeutic effect in patients suffering with neuroendocrinetumors.85
Selective necrotic cell death by DNA damaging activity
Most efforts thus far have been devoted to develop apoptosis inducers for cancer treatment.However, apoptotic pathway deficiencies are a hallmark of cancer cells.86 Sun et al.86 proposedthat one way to bypass defective apoptotic pathways in cancer cells is to induce necrotic celldeath. They showed that selective induction of necrotic cell death can be achieved by activationof the DNA damage response pathways. While β-lapachone induces apoptosis through E2F1 checkpoint pathways, necrotic cell death can be selectively induced by β-lapachone in a varietyof cancer cells. Sun et al.86 also found that β-lapachone, unlike DNA damaging chemotherapeuticagents, transiently activates PARP1, a main regulator of the DNA damage response pathway,both in vitro and in vivo. All these data suggested that selective necrotic cell death can beinduced through activation of DNA damage response pathways, supporting the idea of selective necrotic cell death as a therapeutic strategy to eliminate cancer cells.867.2.7 Use of β-lapachone in apoptosis inductionWoo et al.87 studied the effects of β-lapachone on the growth of the human hepatoma cell lineHepG2. The results showed that β-lapachone inhibits the viability of HepG2 by inducing apoptosis, as evidenced by the formation of apoptotic bodies and DNA fragmentation.87 Reversetranscription-polymerase chain reactions and immunoblotting results indicated that treatment ofcells with β-lapachone resulted in down-regulation of anti-apoptotic Bcl-2 and Bcl-XL and upregulationof pro-apoptotic Bax expression. However, β-lapachone treatment did not affect theinhibitor of apoptosis proteins family and the Fas/FasL system. Taken together, this study indicated that β-lapachone may have potential as a chemopreventive agent for liver cancer.87 given intratumorally compared to when complexed with cyclodextrin to increase its bioavailability.81 In another study on β-lapachone, Park et al.82,83 strongly suggested that NQO1 activity in tumors may be further and selectively elevated using either local radiotherapy or hyperthermia,both established cancer treatment modalities, to improve the cytotoxicity of β-lapachone againsthuman cancer cells.82,83,84
Use of β-lapachone in treatment of neuroendocrine tumors
Larsson et al.85 studied in vitro drug sensitivity screening using the fluorometric microculturecytotoxicity assay in one human pancreatic carcinoid and two human bronchial carcinoid celllines. The aim of this study was to investigate drug sensitivity in neuroendocrine tumor cell lines.In addition, a normal human retinal pigment epithelial cell line was used for comparison. A totalof 18 drugs, including β-lapachone, with different mechanisms of action were tested. These studies indicated that some of the tested compounds viz., β-lapachone, could possibly be used inclinical trials and demonstrate a therapeutic effect in patients suffering with neuroendocrinetumors.85
Selective necrotic cell death by DNA damaging activity
Most efforts thus far have been devoted to develop apoptosis inducers for cancer treatment.However, apoptotic pathway deficiencies are a hallmark of cancer cells.86 Sun et al.86 proposedthat one way to bypass defective apoptotic pathways in cancer cells is to induce necrotic celldeath. They showed that selective induction of necrotic cell death can be achieved by activationof the DNA damage response pathways. While β-lapachone induces apoptosis through E2F1 checkpoint pathways, necrotic cell death can be selectively induced by β-lapachone in a varietyof cancer cells. Sun et al.86 also found that β-lapachone, unlike DNA damaging chemotherapeuticagents, transiently activates PARP1, a main regulator of the DNA damage response pathway,both in vitro and in vivo. All these data suggested that selective necrotic cell death can beinduced through activation of DNA damage response pathways, supporting the idea of selective necrotic cell death as a therapeutic strategy to eliminate cancer cells.86
Use of β-lapachone in apoptosis induction
Woo et al.87 studied the effects of β-lapachone on the growth of the human hepatoma cell lineHepG2. The results showed that β-lapachone inhibits the viability of HepG2 by inducingapoptosis, as evidenced by the formation of apoptotic bodies and DNA fragmentation.87 Reversetranscription-polymerase chain reactions and immunoblotting results indicated that treatment ofcells with β-lapachone resulted in down-regulation of anti-apoptotic Bcl-2 and Bcl-XL and upregulationof pro-apoptotic Bax expression. However, β-lapachone treatment did not affect theinhibitor of apoptosis proteins family and the Fas/FasL system. Taken together, this studyindicated that β-lapachone may have potential as a chemopreventive agent for liver cancer.87