Lapachol acetylglycosylation enhances its cytotoxic and pro-apoptotic activities in HL60 cells

Article abstract of DOI:10.1016/j.tiv.2020.104772

Lapachol is a plant-derived naphthoquinone that kills several types of cancer cells. Derivatives of this molecule may therefore prove to be useful chemotherapeutic agents. In this study, we explored whether glycosylation increases the cytotoxic potency of lapachol towards HL-60 human leukemia cells. Two beta-glycosides were synthesized and characterized: LA4A (lapachol-β-glucoside) and LA4C (lapachol-N-acetylglucosamine-β-glucoside). The sugar moieties of both novel molecules were per-acetylated to facilitate cellular uptake. The IC₅₀ values (in μM) for LA4A (5.7) and LA4C (5.3) were lower than those for lapachol (25). LA4A and LA4C triggered typical signs of apoptosis, such as the exposure of phosphatidylserine on the outside of cells, chromatin condensation, DNA fragmentation and a decrease of the mitochondrial transmembrane potential (ΔΨm) prior to cell lysis. Moreover, DNA fragmentation triggered by the lapachol-glycosides was reduced by pre-treatment with the caspase inhibitor, z-VAD-fmk. While LA4A and LA4C activated caspases-3, -8 and -9, lapachol failed to activate these apoptotic proteases, even when used at high concentrations. Finally, the toxicity of lapachol and its derivatives was also tested on non-tumor cells. We used human peripheral neurons (PeriTox test) to evaluate the side effect potential of these compounds. LA4C was clearly less toxic than LA4A. We conclude that LA4C had the most favorable profile as drug candidate (high tumor cell toxicity, reduced neurotoxicity). In general, this study shows that the cytotoxicity of lapachol towards HL-60 can be enhanced by glycosylation, and that the therapeutic ratio may be modified by the type of sugar added.

Full text of DOI:10.1016/j.tiv.2020.104772

Journal Pre-proof
Lapachol acetylglycosylation enhances its cytotoxic and pro-
apoptotic activities in HL60 cells
Lucas Bonfim Marques, Flaviano Melo Ottoni, Mauro Cunha
Xavier Pinto, Juliana Martins Ribeiro, Fernanda S. de Sousa,
Ricardo Weinlich, Nathalia Cruz de Victo, Jaffar Kisitu, Anna-
Katharina Holzer, Marcel Leist, Ricardo José Alves, Elaine Maria
de Souza-Fagundes
PII:
S0887-2333(19)30713-1
https://doi.org/10.1016/j.tiv.2020.104772
TIV 104772
DOI:
Reference:
To appear in:
Toxicology in Vitro
Received date:
Revised date:
Accepted date:
13 September 2019
10 January 2020
11 January 2020
Please cite this article as: L.B. Marques, F.M. Ottoni, M.C. Xavier Pinto, et al., Lapachol
acetylglycosylation enhances its cytotoxic and pro-apoptotic activities in HL60 cells,
Toxicology in Vitro(2020), https://doi.org/10.1016/j.tiv.2020.104772
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2020 Published by Elsevier.

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Lapachol acetylglycosylation enhances its cytotoxic and pro-
apoptotic activities in HL60 cells
1
,2
2
3
Lucas Bonfim Marques
, Flaviano Melo Ottoni , Mauro Cunha Xavier Pinto , Juliana
1
1
4
4
Martins Ribeiro , Fernanda S de Sousa , Ricardo Weinlich , Nathalia Cruz de Victo ,
5
5
5
2
Jaffar Kisitu , Anna-Katharina Holzer , Marcel Leist , Ricardo José Alves , Elaine Maria
1
*
de Souza-Fagundes
.
1-
Departamento de Fisiologia e Biofísica, Universidade Federal de Minas Gerais , Belo Horizonte-MG,
Brasil;
2
-
Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte-MG, Brasil;
Departamento de Farmacologia, Universidade Federal de Goiás, Goiânia-GO, Brasil.
Instituto Israelita de Ensino e Pesquisa Albert Einstein, São Paulo-SP, Brasil.
Chair for In Vitro Toxicology and Biomedicine, Inaugurated by the Doerenkamp-Zbinden Foundation,
University of Konstanz, Konstanz, Germany
3-
4-
5-
*
Corresponding author’s address: Elaine Maria de Souza-Fagundes. Av. Antônio Carlos, 6627, Campus
Pampulha. Belo Horizonte - MG, Brazil. ZIP Code: 30123-970. Departamento de Fisiologia e Biofísica,
Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brasil; Laboratório de Biologia Molecular e
Celular, bloco A4, sala 240. Phone +55 (31) 3409-2941. Email: elainefagundes@ufmg.br
Abbreviations: ΔΨm, mitochondria membrane potential; CCCP, carbonyl cyanide 3-chlorophenylhydrazone;
ETO, etoposide; HST, Hoechst 33342; ROS, reactive oxygen species; peripheral blood mononuclear cells; PI,
propidium iodide.
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Abstract
Lapachol is a plant-derived naphthoquinone that kills several types of cancer cells.
Derivatives of this molecule may therefore prove to be useful chemotherapeutic agents. In
this study, we explored whether glycosylation increases the cytotoxic potency of lapachol
towards HL-60 human leukemia cells. Two beta-glycosides were synthesized and
characterized: LA4A (lapachol-ß-glucoside) and LA4C (lapachol-N-acetylglucosamine-β-
glucoside). The sugar moieties of both novel molecules were per-acetylated to facilitate
cellular uptake. The IC50 values (in µM) for LA4A (5.7) and LA4C (5.3) were lower than
those for lapachol (25). LA4A and LA4C triggered typical signs of apoptosis, such as the
exposure of phosphatidylserine on the outside of cells, chromatin condensation, DNA
fragmentation and a decrease of the mitochondrial transmembrane potential (ΔΨm) prior to
cell lysis. Moreover, DNA fragmentation triggered by the lapachol-glycosides was reduced
by pre-treatment with the caspase inhibitor, z-VAD-fmk. While LA4A and LA4C activated
caspases-3 and -9, lapachol failed to activate these apoptotic proteases, even when used at
high concentrations. Finally, the toxicity of lapachol and its derivatives was also tested on
non-tumor cells. We used human peripheral neurons (PeriTox test) to evaluate the side
effect potential of these compounds. LA4C was clearly less toxic than LA4A. We conclude
that LA4C had the most favorable profile as drug candidate (high tumor cell toxicity,
reduced neurotoxicity). In general, this study shows that the cytotoxicity of lapachol
towards HL-60 can be enhanced by glycosylation, and that the therapeutic ratio may be
modified by the type of sugar added.
Keywords: Lapachol; glycosylation; anticancer drugs; apoptosis
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1
. Introduction
Lapachol, also known as 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone, is
a well-studied natural product isolated from Tabebuia impetiginosa. It displays cytotoxic
effects in several cancer cell lines, and chemical modification of this molecule led to
derivatives with enhanced antitumor activity (Linardi et al., 1975); (Hussain and Green,
2
017); (Romão et al., 2018). For instance, lapachol conjugation with polyamines increased
the cytotoxic effect against glioblastoma cells (Romão et al., 2018). A series of 5-hydroxy-
,4-naphthoquinone analogs of lapachol showed anti-proliferative activity against six
1
human solid tumor cell lines (Bonifazi et al., 2010). Another series of naphthoquinone
derivatives inhibited cell proliferation in human leukemia HL-60 cells, and elevated the
levels of intracellular glutathione disulfide (Inagaki et al., 2015).
Recent studies have demonstrated that the lapachol scaffold can interact with
different targets in cancer cells. These include DNA topoisomerases, monoamine oxidases,
Hsp90, and pyruvate kinase M2 (Romão et al., 2018); (Shankar Babu et al., 2018); (Lee et
al., 2006). The interaction of lapachol with these targets can induce apoptotic death in
cancer cells. The human myeloid leukemia cell line HL60 has been used in several studies
to identify the effects of lapachol and its derivatives. This cell lineage has shown to be
sensitive to β-lapachone and other organometallic complexes (Balsano et al., 2013,
Planchon et al., 1995).
Lapachol is considered a good candidate for chemical derivatization in order to
develop lead compounds for cancer treatment (Hussain and Green, 2017). Its low water
solubility and low oral bioavailability may be improved by derivatization (Tabosa et al.,
2
018). Chemical glycosylation is a type of derivatization that affects polarity, local electron
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density, lipophilicity, membrane permeability, affinity to active transport systems as well as
biodegradability (Grynkiewicz et al., 2008), and thus has a large impact on drugability.
Glycosylation has been widely used, not only to increase water solubility and
bioavailability, but also to improve the molecular stability of proteins and pharmaceuticals.
To achieve higher oral bioavailability, the balance between lipophilicity and water
solubility needs to be further modified. Peracetylation of the glycosyl groups has been
found to be a useful additional modification for increasing the hydrophobicity (membrane
permeability) of phenolic and related compounds (Mao et al., 2018), (Ogawa et al., 2003).
To the best of our knowledge, only two papers have addressed the synthesis of
lapachol glucosides (Linardi et al., 1975 and Polonik et al., 1983). Polonik and co-workers
described the synthesis of several 2-hydroxy-3-alkyl-(alkenyl)-1,4-naphtoquinones but no
biological studies were reported. Linardi and co-workers described the synthesis and anti-
cancer evaluation of lapachol β-glucoside (Linardi et al., 1975). The authors demonstrated
that the peracetylated glucoside of lapachol had an augmented anti-tumor activity in a
mouse model of lymphocytic leukemia (using P-388 cells). In the present manuscript, this
glucoside, named here LA4A, was prepared. In addition, the N-acetylglucosamine β-
glucoside derivative, named LA4C was synthesized (Fig. 1). Based on previous studies, we
chose the HL-60 cell line, in order to test the new lapachol derivatives, and to provide
further insight into the potential utility of lapachol as scaffold for new chemotherapeutic
agents. We have showed that the replacement of an acetoxyl (OAc) group at the C2-carbon
of the glucoside moiety by an acetamido group (NHAc) enhanced its apoptosis-inducing
potency in leukemia cells.
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. Materials and methods
.1. Materials
.1.1. Extraction of lapachol and synthesis of LA4A and LA4C
The lapachol was obtained by extraction of the powdered wood of Tabebuia
impetiginosa sawdust with sodium carbonate 1%, according to the literature (Perez-Sacau
et al., 2005). The glycosyl halides (I or II) were synthesized based on previously described
methods (Conchie et al., 1957),(Adiyala et al., 2018), (Horton, 1972). The preparation of
glycosides LA4A and LA4C was performed by the reaction of 3 mmol of the appropriate
glycosyl halide (I and II) with 1 mmol of lapachol using phase transfer catalysis (PTC)
conditions: 5 mL of 10% w/v sodium carbonate (Na2CO3) aqueous solution, 5 mL of
dichloromethane (CH2Cl2) and 0.3 mmol of tetra-n-butyl bromide (TBAB) as phase transfer
catalyst (Starks, 1971). The reaction mixture was stirred at room temperature for 24 h,
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1
monitored by TLC analysis. A solution of HCl 6.0 mol L (50 mL) was added until pH= 4
and the mixture transferred to a separatory funnel. The organic layer was separated and the
aqueous phase was extracted with 3 x 50 mL of dichloromethane. The combined organic
layers were washed with water (3x 25 mL), dried over anhydrous sodium sulfate and
concentrated in vacuo. The crude classical lapachol glycosides LA4A and LA4C were
purified by recrystallization from methanol. Figure 2 illustrates the synthetic procedure for
the glycosides employed in this work. The chemical structures of lapachol, LA4A and
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LA4C were confirmed by spectroscopic methods ( H,
C NMR, IR and Mass
spectrometry). The purity of all synthetized compounds was ≥ 95% and their mass spectra
showed molecular weights compatible with the proposed structures. The spectra of all
synthesized compounds are provided in Supplementary Material.
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Lapachol, LA4A and LA4C were solubilized in dimethyl sulfoxide (DMSO>
9.5%, Sigma-Aldrich, USA) at a concentration of 20 mM followed by 1:10 dilution in
0X PBS at pH 7.2 (Gibco, Life technologies). In all experiments, compounds were
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1
solubilized in medium or PBS before addition to cells and final maximum concentration of
DMSO was 0.5% v/v.
2
.1.2. Leukemia cell lines
Human promyelocytic leukemia cells (HL60) were kindly donated by Dr. Marcel
Leist (University of Konstanz/Germany) and HL60.Bcl-2 (HL60 cells ectopically
expressing Bcl-2) provided by Dr. Gustavo P. Amarantes-Mendes (São Paulo
University/Brazil). This lineage was obtained by transfecting HL-60 cells with pcDNA-
Bcl-2 vector as previously described (Fernandes et al., 2007) (Fernandes et al.,
2
005),(Brumatti et al., 2003). Cells were cultivated in RPMI 1640 (Sigma-Aldrich, St.
Louis, MO), supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (GIBCO
BRL, Grand Island, NY), enriched with 2 mM of L-glutamine (GIBCO UK, Grand Island,
NY) and 10% fetal bovine serum. All cultures were maintained at 37 °C in a humidified
incubator with 5% CO2 and periodically evaluated regarding mycoplasma contamination.
2
2
.2. Methods
.2.1. Cytotoxicity of lapachol and derivatives against HL60 lineage
Cell viability was measured by the rate of the mitochondrial reduction of the yellow
tetrazolium salt
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
Sigma-Aldrich, St. Louis, MO) into insoluble purple formazan crystals (Pinto et al., 2011).
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Briefly, the HL60 cells were seeded in a 96-well plate at 100,000 cells/well in a 100 L
volume. The plates were pre-incubated in a 95% air-humidified atmosphere with a 5% CO2
for 24 h at 37 °C to allow for the adaptation of cells. The compounds were tested over a
concentration range of 1 – 100 µM. The cell cultures were incubated for an additional 48 h
at 37 °C. MTT solution (20 μL at 5 mg/mL) was added after drug incubation to each well.
After 4 h of MTT incubation, the supernatant was removed, and 200 μL of 0.04 M HCl in
isopropyl alcohol was added. The optical densities (OD) were measured with a
spectrophotometer at 570 nm. The results were normalized by solvent control, and half
maximal inhibitory concentration (IC50) and concentration that inhibited 80% of cell
viability in comparison with control (IC80) values were calculated to lapachol and its two
derivatives using GraphPad Prism (GraphPad Software Inc) according to the recommended
protocol for nonlinear regression of a log (inhibitory) versus inverse response curve.
2
.2.2. Analysis of cell viability by Trypan Blue exclusion test by counting in a
Neubauer chamber
5
HL60 cells were seed at 1.0x10 cells/well (96-well plate, SARSTEDT, Germany)
were pre-incubated in a humidified air environment containing 5% CO2 at 37° C (Avelar-
Freitas et al., 2014). After pre-incubation, cells were treated with lapachol, LA4A and
LA4C at 25 µM for 2, 4, 8, 18 and 24h. A volume of 10 µL was placed to round-bottom
tubes and incubated with 190 µL 0.4% TB solution diluted in PBS to perform the TB
exclusion test with counting in a Neubauer chamber.
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2
.2.3. Investigation of DNA condensation by Hoechst staining
To analyze chromatin condensation, cells were treated with compounds at 25 µM
for three hours and stained with Hoechst 33342 (HST) as described by (Pinto et al., 2011).
The cells were examined and photographed by fluorescence microscopy (EVOS® FL
Color, Life technologies) with one filter (DAPI fluorescent filter, excitation 340–380 nm,
barrier filter 430 nm). For each experiment, three microscopic fields were photographed.
The cell nuclei were classified as normal, condensed or fragmented nuclei, depending on
their morphology.
2
.2.4. Evaluation of phosphatidylserine externalization
Phosphatidylserine externalization was analyzed by flow cytometry using the
Annexin V-FITC apoptosis detection kit I (BD Biosciences, San Diego, CA) and
differentiated into viable, early, and late apoptotic (Pinto et al., 2011). HL60 cells were
seed at density of 250,000 cell/well (24-well plate, SARSTEDT, Germany) and treated with
lapachol (at 25 and 100 µM), LA4A and LA4C at 25μM during 2, 4, 8, 16 and 24h. Cells
were washed twice with ice-cold phosphate-buffered saline and resuspended in 1X binding
buffer. From each cell suspension, 100 μL were transferred to a 5-mL tube, double-stained
with Annexin-V-FITC and PI buffer and incubated at room temperature for 15 min in the
dark. Following incubation, FITC and PI fluorescence were detected using FACSCalibur
(BD Biosciences), and the data were analyzed using FlowJo 7.5.5 (TreeStar Inc., CA).
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2
.2.5. Cell cycle analysis by quantitation of DNA content and analysis of DNA
fragmentation by electrophoresis in agarose gel
Cell cycle and DNA fragmentation were analyzed according to Pinto et al. 2011
(
Pinto et al., 2011), (Pinto et al., 2013). Briefly, HL60 cells were seed at density of 250,000
cell/well (24-well plate, SARSTEDT, Germany) and treated with compounds at 25μM for
, 4, 8, 16 and 24 hours. Cells were suspended in 0.3 mL of hypotonic fluorochrome
solution (50 g/mL propidium iodide and 0.1% Triton X-100 in 0.1% sodium citrate). After
hours at 4°C in the dark, the fluorescence of individual nuclei was measured using
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4
FACSCalibur (Becton–Dickinson, Mountain View, CA). A total of 10,000 events were
acquired using Cell Quest and analyzed using FlowJo 7.6.4® (Tree Star, Inc.) to calculate
the number of cells at each cell cycle phase, as well as the number of cells with fragmented
DNA (sub-G0/G1).
Electrophoresis in agarose gel was performed in order to confirm DNA
fragmentation with treated and untreated cells for 24 h. Briefly, DNA profile was evaluated
by 1.5% agarose gel electrophoresis as previously described (Abdel-Salam et al., 2019).
After treatment with compounds, DNA was extracted and the quantification was performed
by measuring absorption at 260 nm in NanoDrop apparatus (Thermo Scientific). The DNA
was submitted to running in TAE-1X buffer at 100V and 500 mA using a 1.5% agarose gel.
Gel was stained with ethidium bromide (0.5 μg/mL) and DNA profile visualized using
ultraviolet light photographed in transluminator. Typical profile of DNA fragmentation by
apoptosis was observed by formation of bands containing multiples of 180-200 bp ("ladder
standard" or ladder).
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2
.2.6. Evaluation of participation of the classical pathway of apoptosis (caspase-
dependent)
.2.6.1 Cell cycle analysis by quantitation of DNA content after pretreatment with zVAD-
FMK
2
HL60 cells were incubated for 40 min with 40 μM of benzyloxycarbonyl-Val-Ala-
Asp-fluoromethylketone (Z-VAD-FMK), a broad-spectrum caspase inhibitor (Biomol,
Research Laboratories, Plymouth Meeting, PA, USA) prior to adding lapachol, LA4A and
LA4C at 25μM. After 24 h of incubation, cell cycle analysis of DNA content was
performed by flow cytometry. The subdiploid DNA content of each treatment with
compounds was compared with DMSO (0.05%) as previously described (Perdigão et al.,
2
018).
2
.2.6.2 Detection of caspase-3 activation
Evaluation of caspase-3 activation in HL60 cells was performed with the aid of the
FITC Rabbit Anti-Active Caspase-3 reagent (BD PharmingenTM). 200,000 HL60 cells /
well were seed (24-well plate, SARSTEDT, Germany). Cells were incubated in the
presence of solvent 0.5% DMSO or lapachol, LA4A and LA4C at 25μM for three and six
hours. Cells were washed twice with 1X PBS and caspase-3 activity evaluated using the kit
FITC Active Caspase-3 Apoptosis Kit (BD Pharmigen), following flow cytometric
analysis. 10,000 events were acquired. Data analysis was performed using FlowJo 7.6.4®
software (Tree Star, Inc.) and graphical display and statistical evaluation by GraphPad
Prism software version 6.0.
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2
.2.6.3 Detection of caspase-8 and 9 activation
The activity of caspases 8 and 9 was measured using the fluorimetric assay kits
Caspase-8 Protease Assay (Invitrogen) and Caspase9/Mch6/Apaf-3 Colorimetric Protease
Assay (Invitrogen) according to the manufacturer’s instructions. 100,000 HL60 cells / well
were seed in 96-well plates (98-well plate, SARSTEDT, Germany followed by 24 h for
stabilization. Cells were treated with lapachol, LA4A and LA4C at 25μM were treated for 3
and 6 h. DMSO 0.5% was used as untreated control. After treatments cells were washed
twice with PBS and suspended in ‘cell lysis buffer’. From the cell lysates, a 25-μl aliquot
containing 100 μg of protein was plated onto a 96-well plate, followed by the addition of 25
μl of 2X reaction buffer containing 10 mM of DTT and 2.5 μl of 1-mM caspase substrate
(final concentration of 50 μM) per sample. After incubation at 37˚C for 1 h, fluorescent
intensity was measured at 405 nm using a microplate reader (Spectramax –Molecular
Devices®).
2
.2.7. Evaluation of mitochondrial depolarization
Mitochondrial transmembrane potential (ΔѰm) was evaluated using MitoProbe ™
DiIC1 (5) Assay Kit for Flow Cytometry (M34151) according to the manufacturer's
instructions. Cells HL60 cells were seeded at density of 200,000 cells/well in 24-well plates
(SARSTEDT, Germany) and treated with lapachol, LA4A and LA4C at 25μM for 6 hours.
DMSO (0.5%) was used as untreated control and CCCP (carbonyl cyanide 3-
chlorophenylhydrazone) as a positive control of mitochondrial membrane potential loss.
After this time, the cells were labeled with DilC1- (1,1 ', 3,3,3', 3'-hexamethylindodicarbo-
cyanine iodide) at 50nM and incubated for 30 minutes, and after, washed with 1X PBS and
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were read on a flow cytometry (FACScan). The experiments were analyzed using the
Software FlowJo (Tree Star, Inc). Three independent experiments were performed in
duplicate.
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.2.8. Evaluation of phosphatidylserine externalization of HL60 cells overexpressing
the antiapoptotic protein Bcl-2 after treatment with lapachol and derivatives
Phosphatidylserine externalization was detected by flow cytometry using the
Annexin V-FITC apoptosis detection kit I (BD Biosciences, San Diego, CA) following the
manufacturer's instructions, as described in item 2.2.3. Briefly, HL60 wild-type and HL60
expressing Bcl-2 cells were seed at density of 250,000 cell/well (24-well plate,
SARSTEDT, Germany) and treated with DMSO 0.5%, Lapachol, LA4A and LA4C at
2
5μM during 4 and 18h. Cells were washed twice with ice-cold phosphate-buffered saline
and resuspended in 1X binding buffer. From each cell suspension, 100μL were transferred
to a 5mL tube, double stained with Annexin-V-FITC and PI buffer and incubated at room
temperature for 15 min in the dark. Following incubation, FITC and PI fluorescence were
detected using FACSCalibur (BD Biosciences), and the data were analyzed using FlowJo
7
.5.5 (TreeStar Inc., CA). Three independent experiments were performed in duplicate.
Bcl-2 protein expression level in HL60 was checked by western-blot according to a
protocol described elsewhere (Fernandes et al., 2007).
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.2.9. Differentiation of immature dorsal root ganglia neurons and their use in the
PeriTox test
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Cytotoxicity against non-tumoral cells was evaluated using the mammalian model
of peripheral neurotoxicity (PeriTox) that is an interesting model to evaluate potential
hazard of compounds. Human induced pluripotent stem cells were cultured according to
standard protocols (Thomson et al, 1998) and differentiated into immature dorsal root
ganglia (iDRG) neurons, as described previously (Chambers et al, 2012). In detail, the
minicircle induced pluripotent stem cells (mc-iPSCs) were obtained from System
Biosciences (model no. SC301A-1; System Biosciences, Mountain View, CA,
http://www.systembio.com). The import of cells and experiments were authorized under
license no. 170-79-1-4-27 (Robert Koch Institute, Berlin, Germany, http://www.rki.de).
Differentiation of the induced pluripotent stem cell line mc-iPSC to iDRG cells was
prepared on the day of differentiation minus 3 (DoD-3) by replating human induced
pluripotent stem cells (hiPSCs) in a single cell suspension onto Matrigel-coated plates (BD
2
Biosciences, Franklin Lakes, NJ, http://www.bdbiosciences.com; 28,000 cells per cm ) in
“conditioned” keratinocyte culture medium (KCM). This KCM was conditioned for 24
hours on mitomycin C-inactivated mouse embryonic fibroblasts. It was freshly
supplemented with 10 ng/ml fibroblast growth factor 2 (R&D Systems, Minneapolis, MN,
http://www.rndsystems.com) and ROCK inhibitor Y-27632 (10 µM; Tocris Bioscience,
Bristol, U.K., http:// www.tocris.com). On DoD0’, neural differentiation was started by
adding neural differentiation medium KSR-S (knockout Dulbecco’s modified Eagle’s
medium [DMEM] with 15% serum replacement, 1 x Glutamax, 1 x nonessential amino
acids, and 50 µM β-mercaptoethanol; all ingredients from Invitrogen, Darmstadt, Germany,
http://www.thermofisher.com) and the combination of five small molecule pathway
inhibitors. In detail, noggin (17.5 ng/ml; R&D Systems) and SB-431642 (10 µM; Tocris
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Bioscience) were added on DoD0’–5’ and CHIR99021 (1.5 µM; Axon Medchem, Vienna,
VA, http://www/axonmedchem.com), SU5402 (5 µM; Tocris Bioscience), and DAPT (γ-
Secretase Inhibitor IX; 5µM; Merck, Darmstadt, Germany, http://www.merck.de) were
added on DoD2’–12’. From DoD4’ onward, the medium was gradually replaced by N2-S
medium (DMEM/F12, with 2 mM Glutamax, 0.1 mg/ml apotransferrin, 1.55 mg/ml
glucose, 25 µg/ml insulin, 100 µM putrescine, 30 nM selenium, and 20 nM progesterone).
On DoD12’, the cells were cryopreserved.
For the PeriTox Test, DoD12’ cells were thawed, and seeded at a density of 0.1 x
6
2
1
0 cells per cm in 75 µl of in differentiation medium (25% KSR-S and 75% N2-S
medium supplemented with CHIR99021 (1.5 µM), SU5402 (5 µM), and DAPT (5 µM))
and plated onto Matrigel-coated 96 well plates (BD Biosciences). Serial dilutions of test
chemicals were prepared in differentiation medium and 25 µl was added to the cells 1 hour
after seeding. All tests were performed 24 hours after treatment.
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.2.9.1. Image acquisition and quantification
Acquisition was performed as described previously by Krug et al. and Stiegler et al.
Briefly, cells were live-stained with Hoechst H-33342 and calcein-AM for image
acquisition. The neurite area was calculated as the total calcein positive area corrected for
the somatic area, i.e. the Hoechst positive area, expanded by a surrounding ring of 3.2 µm
representing the neuronal body. The images were used simultaneously to assess viability.
Double positive cells were counted as viable; Hoechst positive objects without calcein stain
were counted as dead cells. Viability was expressed as % viable cells (treatment) / % viable
cells (control) x 100 %. Three independent experiments were performed in technical
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triplicates. First, data of the technical replicates were expressed as percent of solvent-
treated. After that, technical replicates were averaged to reduce technical variability.
Subsequently, the mean of three biological replicates was determined for neurite area and
viability relative to solvent-control. Fits were calculated using a 4 parameter Hill model
with constraining the baseline to max. 100 % of solvent-treated signal. The equation of the
Hill model was solved for f(x) = 50 % to determine the EC50, i.e. concentration at which
the neurite growth inhibiting/destabilizing effect was at 50% of the solvent control.
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.2.10. Prediction of blood brain barrier permeability
The predictions for blood brain barrier permeability were based on a study by Liu et
al. (2014) which looked at some of the current limitations in developing new
neurotherapeutics for the treatment of Alzheimer disease (Liu et al., 2014). In this study, a
prediction model for blood brain barrier permeability was built by applying a set of
algorithms on four types of fingerprints (i.e MACCs, PubChem, Molecular 2D and Open
babel) of 1593 reported compounds from a CNS library. To make a prediction, the
compound structure was uploaded into the user interface (http://www.cbligand.org/AD/) as
an sdf file generated using ChemAxon version 17.1.23. Predictions were made based on
MACCs fingerprints which offered scores with a threshold of 0.02 above which a
compound qualified as being able to reach significant concentrations in the central nervous
system as described by (de Souza-Fagundes et al., 2018).
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.2.11. Statistical analysis
Statistical analysis concerning the studies with HL60 was conducted using the
Prism statistical package (GraphPad Software, USA). To ascertain significance, we used a
one-way ANOVA with Bonferroni post-test or Student’s unpaired t-test. Statistical
significance was considered at a limit of p<0.05 from three independent experiments
conducted at least in duplicate. Except for the assays using peripheral neurons, as described
above, data are expressed as the means ± SD (standard deviation).
3
3
. Results
.1 Cytotoxic activity of the Lapachol-based glycosides against HL60 cells
The potency among lapachol and its derivatives LA4A and LA4C against HL60
cells was determined through the ability of these cells to metabolize MTT. The assays were
performed 48 hours after compounds treatment. Lapachol displayed an IC50 value of 25.0 ±
4
.7 µM against HL60 cells. The IC50 values to lapachol derivatives decreased around 5-fold
as shown in figure 3. The values were 5.7 ± 1.4 and 5.3 ± 1.5 µM for LA4A and LA4C,
respectively, demonstrating an increase in potency of the derivatives compared with
lapachol.
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3
.2.Cytotoxic effect of lapachol derivatives LA4A and LA4C in HL60 cells and
morphological changes in HL60 cell nucleus are is associated with apoptosis
In order to compare cytotoxicity of lapachol and its two derivatives LA4A and
LA4C against HL60, nuclear morphology and membrane integrity of cells were
investigated. Studies were carried out at same concentration of 25μM for all compounds
during 24h. Assays were performed using the Hoechst-33342 marker, which is permeable
to the plasma membrane and interacts with the DNA allowing its visualization. HL60 cells
exposed to DMSO 0.5% presented preserved morphology of nucleus after 24 hours of
incubation, without sign of DNA fragmentation. However, HL60 cells treated with LA4A
or LA4C at 25μM for 24 hours showed a considerable increase of cells presenting
chromatin condensation, DNA fragmentation and apoptotic bodies (Figure 4A, red arrows).
Treatment with lapachol, at same concentration of 25μM did not induce such alterations at
nuclear level. Kinetics studies were performed to evaluate cell membrane integrity using
trypan blue. Eighteen hours after incubation, a significant increase in dead cells after
treatment with LA4A and LA4C was observed but not with lapachol using the same
concentration. (Figure 4B). Moreover, after 24h the number of dead cells increased after
treatment with lapachol at 25 μM, however without statistical difference. To shed some
light on cell death mechanism induced by lapachol and its derivatives, we used the IC80
values of both derivatives (approximately 25 μM). At the IC80 of lapachol (100 μM), the
compound was less soluble in aqueous solution. The IC80 estimation is an important
parameter to be measured in the early stages of drug development and it is used in studies
to characterize different compounds in different disease models as previously described
(Kano et al., 2007),(Serova et al., 2007). Therefore, the cytotoxicity of lapachol was studied
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at its soluble IC50 of 25 μM. The alteration in the physicochemical characteristics of
lapachol demonstrates that glycolysation, besides increasing its activity, observed by
reducing the IC50 values of its derivatives by about 5-fold, also improved solubility. Further
experiments were performed at concentration of 25 μM for all compounds to identify cell
cytotoxic mechanism. Lapachol derivatives LA4A and LA4C induced DNA fragmentation
in HL60 cells.
Since we have observed changes in nucleus morphology of HL60 cells treated with
lapachol derivatives LA4A and LA4C, we decided to perform a time course experiment to
analyze the morphological alterations related to increase of the subdiploid DNA content
(sub-G0 / G1), that is one characteristic of cells in apoptosis. Cells were treated with
lapachol and derivatives at same concentration (25 μM). As observed in figure 5, HL60
cells treated with the LA4C and LA4A have displayed a significant increase of subdiploid
or sub-G1 DNA content (>60%) comparing with lapachol, in a time-dependent way. This
effect was significant after 4 hours of incubation with LA4C, but only after 8h for LA4A.
At same conditions, only after 24h treatment with lapachol, a significant increase of sub
diploid DNA content was observed in HL60, but less than 20%. In fact, this increase of
DNA fragmentation was confirmed after analysis by electrophoresis in agarose gel.
Etoposide (eto), an anticancer drug that induces DNA fragmentation in HL60 was used as
positive control in our experiments and induced nuclei fragmentation observed by the
presence of smaller fragments (180-200 base pairs) typical of cells in apoptosis (figure 5B).
Interesting, when evaluated at 25μM lapachol presented a fragmentation profile different
from its derivatives and etoposide, demonstrating that lapachol glycosylation altered its
activity improving the pro-apoptotic potential.
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3
.4 Pre-treatment of HL60 cells with ZVAD-FMK reduced the increase of subdiploid
DNA content after treatment with LA4A e LA4C demonstrating activation of the
apoptotic cell death caspase-dependent
Once observed an increase in sub diploid DNA content, we evaluated DNA
fragmentation in HL60 in the presence of the non-specific pan caspase inhibitor zVAD-fmk
(Figure 6A). This experiment clarifies if apoptosis induced by lapachol derivatives LA4A
and LA4C is associated with induction of the classical pathway of apoptosis. HL60 cells
incubated with 25 µM LA4A and LA4C presented an increase in sub-G0/G1 DNA content
(>70%), when compared to DMSO control. On the other hand, the treatment with lapachol
in the same conditions (25 µM), less than 15% of HL60 cells displayed an increase of sub-
G1 DNA content comparing with control. The pre-incubation with zVAD-fmk was able to
significantly reduce the increase of subdiploid DNA content induced by LA4A and LA4C.
This result demonstrated that the DNA fragmentation induced by LA4A and LA4C is
dependent of caspase activity.
In fact, caspase activity was confirmed by evaluation of the executors (caspase 3/7)
and initiators (caspases-8 and 9). Lapachol derivatives LA4A and LA4C, significantly
increased caspase-3 activity after 3 and 6 hours in HL60 cells when compared to DMSO
control (Figure 6B). At 25μM concentration these compounds have demonstrated to be
more potent than lapachol at 25μM to activate caspase-3. Similar result was observed
regarding caspase 9, an important protein in intrinsic apoptosis pathway, which was
strongly activated after 3 and 6 hours in HL60 cells treated with LA4A and LA4C, when
compared with control. In same conditions, lapachol at same concentration did not activate
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caspase-9 (Figure 6C). Figure 6D shows that LA4A and LA4C at 25μM promoted a
significant caspase-8 activation in HL60 cells after 3 and 6 hours, when compare to DMSO
control. Lapachol, at the same concentration of 25 μM did not promote significant caspase
activation in HL60 cells up 6 hours.
3
.5.Lapachol derivatives LA4A and LA4C induced phosphatidylserine exposure in
HL60 cells
We have established that lapachol derivatives LA4A and LA4C, promote
cytotoxicity, morphological changes and caspase activation associated with classical
apoptosis in HL60 cells. To further investigate the apoptotic mechanism involved in the
actions of the compounds, a kinetic of phosphatidylserine exposure was performed using
labelling with Annexin-V (AV) by flow cytometry. HL60 cells were treated for 2, 4, 8, 16
and 24 hours with 25 μM LA4A, LA4C and lapachol and the phosphatidylserine exposure
was evaluated for each time. Representative histograms of labelling with Annexin-V (FL1)
only are displayed in figure 7A after 4h of incubation. No differences were observed during
2
h (data not shown). As observed in Figure 7A, at 25 μM only LA4A and LA4C induced
an increase of fluorescence FL1 (cells labelled only with Annexin-V, AV+, early apoptosis)
up to 20%, when compared with control. This increase was more than 30% for LA4C.
Quantification of double-labelled cells (late apoptosis) displayed in figure 7B, clearly
demonstrated an increase in Annexin-V/PI positive cells, in a time-dependent manner.
LA4C was the most effective in earlier periods with a significant increase in the number of
+
+
AV /PI cells in the first 8 hours of incubation, comparing with lapachol and LA4A (Figure
B), demonstrating a pro-apoptotic effect and different behavior of compound LA4C.
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This profile was also observed during 8h and 16h when compared to untreated
control DMSO (0.5%). Treatment with LA4A started to induce a significant increase in the
+
+
amount of AV /PI cells only after 16 hours of exposure. Incubation with the original
lapachol molecule at 25 μM did not result in any significant change in the amount of
AV+/PI+ HL60 cells when compare to DMSO control, in any time of incubation. Taken
together, these results demonstrated that LA4C at 25 μM is more effective to induce
apoptosis in earlier periods then LA4A and the original molecule lapachol.
3
.6.Lapachol derivatives LA4A and LA4C promoted loss on mitochondrial function
ΔѰm)
Since it was observed activation of caspase-9 by lapachol derivatives LA4A and
(
LA4C, we evaluated the loss of the mitochondrial transmembrane potential (ΔѰm),
important for the intrinsic pathway of apoptosis (Figure 8). HL60 cells were incubated for 6
hours in the presence of negative control (DMSO 0.5%), positive control of mitochondrial
membrane potential loss (CCCP 500 μM, as suggested by supplier), lapachol, LA4A and
LA4C at 25 μM. As expected, the cells incubated with the negative control solution showed
ΔѰm maintenance, while cells exposed to 500μM CCCP showed a ΔѰm loss, which is
characterized by MitoProbe ™ DiIC1(5) increased labeling. DiIC1 is a dye that is
accumulated in the hyperpolarized membranes and translocated to lipid bilayer. LA4A or
LA4C incubation caused ΔѰm loss. However, no loss of mitochondrial membrane was
observed in HL60 cells when treated with lapachol at 25μM. These data indicate that
lapachol derivatives LA4A and LA4C induce cell death through the mitochondrial pathway
of apoptosis, but not lapachol.
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3
.7.Lapachol derivatives LA4A and LA4C effects are reduced by Bcl-2 overexpression
in HL60 cells.
Once LA4A and LA4C are capable to induce ΔѰm loss and caspase-9 activation,
we analyzed the role of Bcl-2, once the mitochondrial pathway of apoptosis is dependent
upon the Bcl-2. To check this idea, we evaluated the effect of lapachol and its two
derivatives on HL60 cells ectopically expressing the antiapoptotic protein Bcl-2 that
confers resistance of these cells to various pro-apoptotic agents that stimulate intrinsic cell
death pathway as previously described (Brumatti et al., 2003), (Zhang et al., 2013). We
have observed that HL60 cells overexpressing Bcl-2 protein were resistant to pro-apoptotic
effects of LA4A and LA4C (Figure 9). It is noted that after 18h, the two derivatives at
25μM significantly reduced the exposure of phosphatidylserine in HL-60.Bcl-2 cells in
comparison to HL60 wild type cells that demonstrated an increase in Annexin-V/PI positive
cells after treatments the derivatives in comparison with DMSO or lapachol at 25μM.
Moreover, after 18h treatment of HL60.Bcl-2 cells with LA4A a total reduction on
phosphatidylserine exposure was observed, comparing with HL60 cells, at levels observed
for lapachol at 25μM was inactive. The expression level of Bcl-2 protein in HL60
overexpressing Bcl2 is displayed in figure 9C. These results support the findings observed
regarding the loss of mitochondrial potential and caspase-9 activation and indicate that
LA4A and LA4C act through mitochondrial pathway of apoptosis and demonstrated that
LA4C was more potent than lapachol and LA4A.
3
.8 Effects of lapachol derivatives LA4A and LA4C on sensory neurons as a non-
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tumoral cell model in the PeriTox
The most common side effect caused by antineoplastic agents, with a prevalence of
9% to over 85%, is chemotherapy-induced peripheral neuropathy. Due to its high
1
prevalence among cancer patients, this problem affects not only patients, but also survivors
for whom treatment possibilities of this syndrome are limited (Zajaczkowska et al., 2019).
Thus, evaluation of the toxicity of substances with anticancer potential against non-tumoral
model that predict neurotoxicity has been in evidence (de Souza-Fagundes et al., 2018).
Therefore, lapachol and its derivatives were investigated about this issue using the
predictive model of the peripheral neurotoxicity (PeriTox). This is a sensitive assay to
assess peripheral neurotoxicity, one of the non-desired effects of a drug (Hoelting et al.,
2
016). This test can identify chemotherapeutic agents that induce peripheral neuropathies
(drug-induced axonopathies) which are an important parameter to be evaluated during drug
development. Thus, using this strategy, lapachol derivatives LA4A and LA4C were
evaluated for the first time using the PeriTox test as previously described (Hoelting et al.,
2
016).
Cell viability and neurite growth of immature dorsal root ganglia-like (iDRG)
neurons were used as readouts to assess neurotoxic effects. As shown in figure 10, lapachol
does not reduce cell viability of iDRG neurons whereas cytotoxicity was observed for both,
LA4A and LA4C in the tested concentration range. However, comparison of LA4A and
LA4C demonstrated that, in terms of determined IC50 values, LA4C was 2.3-fold less toxic
regarding neurite growth and 1.9-fold less cytotoxic in comparison to LA4A effects.
In addition, interestingly studies to predict if lapachol and their derivatives are able
to cross the blood brain barrier (BBB) and induce toxicity to central nervous system
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demonstrated that only lapachol may cross the BBB, but the two derivatives would not,
according to the algorithm we used described by Liu and co-workers (2014) (Liu et al.,
2
014). These data suggested that glycosylation of lapachol reduced the permeability of its
derivatives to BBB and consequently their neurotoxicity hazard (Table 1).
4
. Discussion
One of the obstacles to the usefulness of bioactive compounds, such as lapachol, is
their low oral bioavailability caused by poor hydro/lipofilicity balance. In this sense,
glycosylation has been extensively used as a feasible tool to improve safety, potency and
efficacy of new drugs (Grynkiewicz et al., 2008), (Tabosa et al., 2018). Similarly,
molecular structure changes by acylation enhances biological activity, possibly due
improvement of membrane penetration (Zhang et al., 2013),(La Ferla et al., 2011). Several
studies have suggested that molecular modifications of natural compounds bearing
hydroxyl groups by glycosylation and/or acetylation may improve biological activity by
modifying their physicochemical properties and/or adding new biological functions (Zhang
et al., 2013),(La Ferla et al., 2011). Also, glycosylation may act by modulating
immunogenicity, in vivo circulatory half-life and effector functions (Zhang et al., 2013).
Peracetylated glycosides are considered pro-drugs, since they can be converted into the
deacetylated glycoside by esterases. Moreover, it has been observed that glycosylation
plays an important role in the adsorption and metabolism of plant-based natural products; in
addition, the conjugation effect sugar-aglycone can facilitate the active transport across cell
membrane by targeting glucose transporters (Gurung et al., 2017), (Mao et al., 2018). In
this context, the attachment of a β-O-peracetyl-glycosyl group to lapachol seems to increase
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its permeability through the cell membrane by acetylation promoting a bioavailability
increment. The amido group present in the compound LA4C may be related to an
improvement in the stability of this glycosyl-derivative. Noteworthy, amide bond has been
considered as the most stable functional groups in organic synthesis due the amidic
resonance effect (Shi et al., 2018), suggesting this property may be related to the better
cytotoxic profile of LA4C as compared to LA4A.
The present work describes the cytotoxic effects of lapachol derivatives LA4A and
LA4C and how they activate apoptosis in HL60 cells. We demonstrated an increase of
potency of these two derivatives, as observed by the decrease of their IC50 values (around 5
µM for both) when compared with that of lapachol (25µM).
However, no information regarding the cytotoxic mechanism was investigated about
this series of lapachol derivatives. Therefore, to understand the lapachol derivatives
cytotoxic mechanisms and their structure relationship are fundamental for the development
of new analogs with improved potential anticancer properties (Lee et al., 2006), (Xu et al.,
2
016), (Shankar Babu et al., 2018).
Recent studies with lapachol original molecule have shown a cytotoxic activity in
different cancer cells and apoptosis activation (Lee et al., 2006), (Xu et al., 2016), (Shankar
Babu et al., 2018). Studies with β-lapachone, an ortho-naphthoquinone derived from
lapachol, demonstrated that both compounds promote activation of caspase-3 and caspase-9
in T24 cells and induce activation of the intrinsic apoptotic pathway (Lee et al., 2006). In
the C6 glioma cells, lapachol blocked cell proliferation and promotes apoptosis and DNA
damage, being effective on in vitro and in vivo models, being these effects associated to
topoisomerase I and II inhibition (Xu et al., 2016). Recent studies have also shown that
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lapachol inhibits pyruvate kinase M2 (PKM2) in melanoma cells and disrupt glycolysis in
this cancer cell, which can trigger apoptosis (Shankar Babu et al., 2018).
The pro-apoptotic profile for anticancer drugs is desirable because it can eliminate
tumor cells without promoting inflammation. The classical apoptosis (caspase-dependent)
can be divided into extrinsic and intrinsic pathways. In the extrinsic pathway, endogenous
factors activate death receptors and trigger apoptosis by activating caspase-8, which
directly activates the effector protease caspase 3 (Hengartner, 2000), (Mattson and Chan,
2
003). The intrinsic pathway is initiated by cellular stress that impels the mitochondria to
release cytochrome C in the cytosol which interacts with apoptotic protease activating
factor-1, forming the apoptosome, that will activate caspase-9 (Hengartner, 2000), (Mattson
and Chan, 2003). The caspase effectors are responsible for the morphological and
biochemical changes related to apoptosis, such as externalization of phosphatidylserine
(
PS), chromatin condensation and DNA fragmentation (Fesik, 2005), (Pinto et al., 2011)
Mattson and Chan, 2003).
To understand the mechanism of cell death induced by LA4A and LA4C, we
(
analyzed their effect regarding phosphatidylserine exposure and the mitochondrial
membrane potential (ΔΨm) disruption, hallmarks of classical apoptosis cell death activated
by the intrinsic pathway. After 8 hours incubated with lapachol derivatives LA4A and
LA4C, a significant increase in the number of HL60 cells were labeled with Annexin-V
which is related to early events of apoptosis. After 24 hours of treatment with LA4A and
LA4C, early and late apoptotic events (cells positively labeled with Annexin-V and PI)
were observed. We found that HL60 incubated with lapachol derivatives strongly increased
caspase 9 activity and presented a slight increase in caspase 8. On the other hand, after 6
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hours treatment of HL60 with lapachol derivatives, a reduction in ΔΨm was have observed,
which is a feature of intrinsic pathway.
The consequences of mitochondrial potential alteration, caspase activation and
phosphatidylserine exposure in HL60 cell incubated with LA4A and LA4C were also
analyzed. Interestingly, it was observed that overexpression of Bcl-2 protein in HL60 cells
reduced the apoptosis induced by LA4A and LA4C, comparing with HL60 wild-type, due
to the decrease in the number of cells exposing phosphatidylserine as observed by the
reduced quantity of anexin-V/PI positive cells (figure 9). The protein Bcl-2 belongs to a
family of proteins that regulate apoptosis and tumorigenesis. This family has both pro-
apoptotic members such as BAX and BAK proteins, as well as anti-apoptotic members
such as Bcl-2 and BAD, for example (Czabotar et al., 2014). Expression of the
antiapoptotic Bcl-2 is an important determinant of cell sensitivity to apoptotic stimuli such
as chemotherapy. Overexpression of Bcl-2 protein reduces the susceptibility of cells to pro-
apoptotic stimuli. In fact, the ectopic expression of Bcl-2 in HL60 cells has been described
by (Brumatti et al., 2003). These authors have demonstrated that Bcl-2 overexpression
confers resistance of HL60 cells to various pro-apoptotic agents that stimulate the intrinsic
cell death pathway. In our study, this strategy was used to confirm the involvement of the
mitochondrial pathway in the cytotoxic effect of lapachol and its derivatives and are in
agreement with those authors. As shown in Figure 9, HL60.Bcl2 cells demonstrated a lower
percentage of cells with phosphatidylserine exposure compared to wild type HL60. This
shows a greater resistance to the effect of the compounds compared to the wild-type HL60,
corroborating the findings of mitochondrial potential alteration and caspase-9 activation,
besides the nuclear alterations (figure 4) that were consistent with the death process by
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apoptosis. Taken together, this data indicates that mitochondrial pathway is activated by
LA4C and LA4A. These compounds also promoted caspase-3 activation, which confirmed
the classical apoptotic cell death.
In addition, we also observed that LA4A and LA4C promoted chromatin
condensation and DNA fragmentation in HL60 cells. Pre-incubation of HL60 cells with Z-
VAD-FMK, a pan caspase inhibitor, prevented DNA fragmentation in HL60 cells treated
with lapachol derivatives. Taken together, these results indicate that LA4A and LA4C
apoptotic cell death depend on caspase activation.
Regarding the differences between lapachol and its derivatives, we observed that
LA4C was more potent after 8 hours of incubation than LA4A and lapachol in promoting
cytotoxicity, phosphatidylserine exposure, and DNA fragmentation. After 16 and 24 hours
of incubation, LA4C and LA4A did not present differences between each other, but they
are more potent that lapachol to promote cytotoxicity, phosphatidylserine exposure, and
DNA fragmentation at same micromolar concentration (25 µM). These results indicated
that lapachol chemical modifications were capable of improving the kinetics cytotoxicity
and potency of these molecules. This finding was also observed concerning the peripheral
neurotoxicity, once the predictive PeriTox assay displayed LA4C as less toxic comparing to
LA4A. Moreover, prediction about possibility of them to cross the blood brain barrier also
confirmed that lapachol glycosylation decreased the lipophilic profile, as shown by lower
clog P values of LA4A and LA4C (Table 1), rendering these compounds less prone to cross
the BBB as compared to lapachol.
5. Conclusion
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In conclusion, we observed that attachment of a β-O-peracetylglycosyl group to
lapachol improves the cytotoxic effects of the natural molecule possibly by enhancing
bioavailability. Furthermore, both derivatives, especially LA4C showed lower toxicity
against peripheral neurons comparing with LA4A. These data provide evidential basis that
acetylglycosylation is a promising method of modifying hydroxylated compounds and
generating diverse and potent derivatives of natural products.
6
. Acknowledgements
This work was supported by grants from FAPEMIG (Minas Gerais State Agency
for Research and Development) and CNPq (National Council for Scientific and
Technological Development - Brazil). Alves R.J. and Souza-Fagundes E.M. are CNPq
fellowship recipients. The authors also thank CAPES (Brazil) and the Alexander von
Humboldt Foundation (Germany, Process 99999.008121/2014–01), for financial support.
This work was also supported by the DFG (RTG1331), the Land-BW (INVITE), the BMBF
(e: ToP program) and the EC project EU-ToxRisk (Grant No. EC681002).
7
. Conflict of interest
The authors declare that they do not have any conflicts of interest.
8
. References
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J., Dias, F.F., dos Reis, P.V.M., Pimenta, A.M.C., Leist, M., de Lima, M.E., de Souza-Fagundes, E.M.,
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