Antiproliferative activity of Juglone derivatives on rat glioma

Article abstract of DOI:10.1080/14786419.2016.1214830

Malignant gliomas are aggressive and life-threatening tumours that still show a poor prognosis: the current therapeutic approach based on surgical resection and chemotherapy combined with radiotherapy does not provide a satisfactory chance of long-term su

Full text of DOI:10.1080/14786419.2016.1214830

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Antiproliferative activity of Juglone derivatives on
rat glioma
Valeria Pavan, Giovanni Ribaudo, Maira Zorzan, Marco Redaelli, Raffaele
Pezzani, Carla Mucignat-Caretta & Giuseppe Zagotto
To cite this article: Valeria Pavan, Giovanni Ribaudo, Maira Zorzan, Marco Redaelli, Raffaele
Pezzani, Carla Mucignat-Caretta & Giuseppe Zagotto (2016): Antiproliferative activity of Juglone
derivatives on rat glioma, Natural Product Research, DOI: 10.1080/14786419.2016.1214830
To link to this article: http://dx.doi.org/10.1080/14786419.2016.1214830
View supplementary material
Published online: 28 Jul 2016.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=gnpl20
Download by: [Simon Fraser University]
Date: 29 July 2016, At: 10:30

Natural Product research, 2016
http://dx.doi.org/10.1080/14786419.2016.1214830
Antiproliferative activity of Juglone derivatives on rat glioma
Valeria Pavana, Giovanni Ribaudoa, Maira Zorzanb, Marco Redaellib,d,
Raffaele Pezzanic,d, Carla Mucignat-Carettab and Giuseppe Zagottoa
adepartment of Pharmaceutical and Pharmacological sciences, university of Padova, Padova, Italy;
bdepartment of Molecular Medicine, university of Padova, Padova, Italy; cdepartment of Medical and surgical
sciences-operative unit of endocrinology, university of Padova, Padova, Italy; daIroB, Italian association for
Basic oncological research, Padova, Italy
ABSTRACT
ARTICLE HISTORY
received 16 February 2016
accepted 27 June 2016
Malignant gliomas are aggressive and life-threatening tumours that
still show a poor prognosis: the current therapeutic approach based
on surgical resection and chemotherapy combined with radiotherapy
does not provide a satisfactory chance of long-term survival to
patients. Natural bioactive compounds represent a precious source
of molecules with antiproliferative activity, potentially effective also
against glioma cells. Among these, Juglone is a known allelopathic
compound extracted from the eastern black walnut (Juglans nigra)
whose antimitotic effect has been extensively described in mammalian
cells. We investigated the antiproliferative effect of a synthetic
derivative of this natural compound, 2-(2,4-dihydroxyphenyl)-
8-hydroxy-1,4-naphthoquinone (DiNAF), in rat glioma cells. We
compared this molecule and its effect with the natural reference
compound and with newly synthesised derivatives to build a
preliminar structure–activity relationship. Biological assays and NMR-
based redox experiments confirmed that DiNAF is a promising lead
and supported the hypothesis of a redox mechanism underlying its
cytotoxic activity.
KEYWORDS
Glioma; Juglone; tMZ;
naphthoquinones
1. Introduction
Malignant gliomas are characterised by a high tendency to infiltrate through the brain paren-
chyma and a high resistance to the standard anticancer therapies (Capper & Reifenberger 2015).
CONTACT Giuseppe Zagotto
giuseppe.zagotto@unipd.it
supplemental data for this article can be accessed here at http://dx.doi.org/10.1080/14786419.2016.1214830.
© 2016 Informa uK limited, trading as taylor & Francis Group

2
V. PAVAN et Al.
the current FDA-approved therapeutic approach consists in neurosurgical resection, followed
by chemotherapy with temozolomide (tMZ) in combination with radiotherapy (Stupp et al.
2005), though only the 26% of treated patients reach 2 years of survival (Preusser et al.
2011). Juglone is a natural compound acting as growth-stunting agent (Kiran Aithal
et al. 2009). It can be extracted from Juglans nigra (Juglandaceae family) and it has been also
associated with the induction of oxidative stress in both healthy and cancerous mammalian cells
(Öllinger & Brunmark 1991; Xu et al. 2012). thus, it is known that naphthoquinones may induce
cytotoxicity through two main mechanisms: redox cycling and reactivity towards nucleophiles.
Concerning redox activity, we recently studied, in accordance with the work of other research
groups, the influence of the redox properties of some Juglone derivatives (such as DiNAF, com-
pound 1) and other compounds of the same class on their cytotoxic activity, also towards glioma
cells (Zagotto et al. 2011; Chen et al. 2012; Redaelli et al. 2015; Yang et al. 2014). On the other
hand, for what concerns electrophilic reactivity, it has been observed that naphthoquinones can
react with thiols and amines to give covalent adducts inside the cell, depending on the electro-
philicity of C-3. Moreover, it cannot be ruled out that both the mechanisms contribute to devel-
oping the cytotoxic effect (Klotz 2014). to confirm our observations and to push our search for
novel therapeutic leads against glioma to a further level, we prepared a small set of 1,4-naph-
thoquinone and DiNAF derivatives, with focused modifications on the scaffold to build a prelim-
inary structure–activity relationship. We also extended the investigation of the antiproliferative
effects to the F98 rat glioma cell line, a widely accepted cellular model of glioma (Barth & Kaur
2009), comparing the performance of the synthesised compounds with the effects of Juglone,
tMZ and paclitaxel (PtX).
Although it is generally accepted that naphthoquinones can promote cell death inducing
oxidative stress (Öllinger & Brunmark 1991; Chen et al. 2012; Xu et al. 2012; Yang et al. 2014), we
recently highlighted the relevance of the kinetic aspect of this phenomenon. In fact, we prelim-
inary demonstrated that in solution, even in the presence of an excess of Na2S2O4, 3-(2,4-
dihydroxyphenyl)naphthalene-1,4,5-triol, the non-toxic, reduced form of compound 1, has a
high tendency to quickly reconvert to its oxidised, cytotoxic form (Figure 1), while juglone
promptly undergoes a non-reversible reduction to the non-toxic hydroxy naphthalene under
the same conditions (Redaelli et al. 2015). to better describe this observation, we designed an
NMR-based experiment to study the reduction kinetic of this and similar naphthoquinones in
the presence of 1 equivalent of Na2S2O4.
2. Results and discussion
the new approaches to target glioma cells exploit their action through different mechanisms,
such as the activation/inhibition of specific receptors, by interfering with cellular pathways
Figure 1. redox and toxicity properties of compound 1.

NAtuRAl PRODuCt ReSeARCh
3
or gene expression, or by influencing redox activities (Jung et al. 2012; Phillips et al. 2012;
Yang et al. 2014). With this work, we provide further evidence that the redox reactivity par-
allels with the cytotoxic effect, at least in some of the cases. We prepared a small, focused
set of compounds sharing the 1,4-naphthoquinone scaffold and showing light modifications
in the substituents (Figure 2). the number and the positioning of the hydroxyl groups on
both the naphthoquinone scaffold and on the phenyl ring were modified, and the effect of
the introduction of an electron-withdrawing nitro group was also evaluated.
We investigated the redox properties of some of the compounds by measuring their degree
of reactivity towards Na2S2O4, a reducing agent.We previously demonstrated the high tendency
of the reduced, non-toxic, form of compound 1, 3-(2,4-dihydroxyphenyl)naphthalene-1,4,5-triol,
to be promptly reoxidised to its parent compound in solution and we show here that the opposite
reaction, i.e. the reduction from the naphthoquinone to the hydroxynaphtalene form, is the
slowest when compared to the behaviour of the other tested compounds (for a detailed review
on the electrochemical properties of quinones see Guin 2011).
We designed this NMR-based experiment to highlight the role that the number and position
of electron donor groups (hydroxyls) have in redox cycling, in contrast with the effect of the
deactivating nitro group that we inserted in compound 6. Curiously, most of the compounds,
including Juglone and compound 6, were promptly converted to the reduced form just after the
addition of the Na2S2O4 solution. Compounds 1 and 5 showed otherwise a lower tendency to
be converted to the hydroxy naphthalene form, with 1 demonstrating a t1/2 three times longer
than the one of 5. Compound 5 anyway showed a curious degree of conversion to the reduced
form just after the addition of Na2S2O4 (see Supplementary Material for representative spectra
and reduction profiles). even if a more evidently different trend for the behaviour of the com-
pounds with an electron-rich scaffold and compound 6 may have been expected, this observation
anyway suggests that, among the screened compounds, compound 1 performs peculiarly and
shows the highest tendency to remain in solution in its oxidised, toxic form.
Mtt-based cell viability assay on F98 rat glioma cells reported a significant reduction in
cell viability induced by 5 and 0.5 μM DiNAF (1), compared to the same concentrations of
Juglone, tMZ and PtX (Figure 3(A)).
Morphological analysis of F98 cells treated with DiNAF (1) showed a statistically significant
increase in apoptosis (Figure 3(B)), which was noteworthy compared to the results obtained
for Juglone, tMZ and PtX. the difference was found to be significant at all tested concen-
trations. Comparison between DiNAF (1), Juglone, tMZ and PtX also showed that the necrosis
Figure 2. structures of the studied compounds.

4
V. PAVAN et Al.
Figure 3. a Mtt assay results for F98 cells treated with diNaF (1), Juglone, tMZ and PtX. t-test compares
diNaF (1) with other treatments *p < 0.05, ***p < 0.001. Morphological analysis of F98 cells treated with
diNaF (1), Juglone, tMZ and PtX: percentages of apoptotic (B) and necrotic (c) cells (Mean seM). t-test
compares diNaF (1) with other treatments *p < 0.05, **p < 0.01, ***p < 0.001.

NAtuRAl PRODuCt ReSeARCh
5

6
V. PAVAN et Al.
(Figure 3(C)) induced by DiNAF (1) is significantly reduced at the highest and the lowest
concentrations (50 and 0.05 μM) in comparison with tMZ. In addition, necrosis induced by
DiNAF (1) resulted to be significantly lower for all the tested concentrations in comparison
with PtX and Juglone.
the other synthetic Juglone derivatives sharing the same naphthoquinone scaffold (com-
pounds 2–6) were tested for their cytotoxic effect on F98 cells. A significant reduction in cell
viability compared to control cells was observed only at the highest concentration (50 μM)
of compounds 2, 3 and 5 (Figure 4).
Preliminary in vivo experiment were carried out on three adult Wistar rats, after 3, 7 and
10 day animals did not show any neurological alteration related to DiNAF brain injection.
the biological results, combined with those observed from the NMR experiments, suggest
that a lower tendency of DiNAF (1) to undergo reduction is accompanied by a higher reduc-
tion in glioma cell viability and a more efficient pro-apoptotic effect compared to Juglone
and the other derivatives.
3. Conclusion
We prepared a set of naphthoquinones with the aim of describing their ability of reducing
glioma cells viability. 2-(2,4dihydroxyphenyl)-8-hydroxy-1,4-naphthoquinone (DiNAF, 1)
represents the most promising compound of the series, confirming our preliminary obser-
vation in other cellular models. the biological results supported those observed from NMR
experiments, strongly suggesting the influence of redox kinetics in exploiting the toxic effect.
Moreover, DiNAF (1) showed a significantly increased apoptosis induction in vitro and cell
viability reduction on rat glioma cells when compared with Juglone, tMZ and PtX.
Interestingly, small focused modifications in DiNAF (1) resulted in a decreased cytotoxicity,
confirming the peculiar features of the lead derivative. the results reported in this study
therefore prompted further investigation including other cell lines and using animal models
in order to evaluate the tolerability and the viability of treatment based on DiNAF (1) or on
novel Juglone derivatives, whose testing is currently being carried out.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
the research project was financially supported by university of Padova.
References
Barth RF, Kaur B. 2009. Rat brain tumor models in experimental neuro-oncology: the C6, 9l, t9, RG2,
F98, Bt4C, Rt-2 and CNS-1 gliomas. J Neurooncol. 94:299–312.
Capper D, Reifenberger G. 2015. Classification of gliomas: current progress and perspectives. Nervenarzt.
86:672–683.

NAtuRAl PRODuCt ReSeARCh
7
Chen C, lin M, Ong P, Yang J. 2012. Novel multiple apoptotic mechanism of shikonin in human glioma
cells. Ann Surg Oncol. 19:3097–3106.
Guin PS, Das S, Mandal PC. 2011. electrochemical reduction of quinones in different media: a review.
Int. J. electrochem. Article ID 816202. Available from: http://dx.doi.org/10.4061/2011/816202
Jung J, Jung K, Kim D, Kim h. 2012. Selective inhibition of MMP-9 gene expression by mangiferin in
PMA-stimulated human astroglioma cells: involvement of PI3K/Akt and MAPK signaling pathways.
Pharmacol Res. 66:95–103.
Kiran Aithal B, Sunil Kumar MR, Nageshwar Rao B, udupa N, Satish Rao BS. 2009. Cytotoxic, genotoxic
and oxidative stress induced by 1,4-naphthoquinone in B16F1 melanoma tumor cells. Cell Biol Int.
33:1039–1049.
Klotz lO, hou X, Jacob C. 2014. 1,4-Naphthoquinones: from oxidative damage to cellular and inter-
cellular signaling. Molecules. 19:14902–14918.
Öllinger K, Brunmark A. 1991. effect of hydroxy substituent position on 1,4-naphthoquinone toxicity
to rat hepatocytes. J Biol Chem. 266:21496–21503.
Phillips JJ, huillard e, Robinson Ae, Ward A, lum Dh, Polley M, Rosen SD, Rowitch Dh, Werb Z. 2012.
heparan sulfate sulfatase SulF2 regulates PDGFRα signaling and growth in human and mouse
malignant glioma. J Clin Invest. 122:911–922.
Preusser M, de Ribaupierre S, Wöhrer A, erridge SC, hegi M, Weller M, Stupp R. 2011. Current concepts
and management of glioblastoma. Ann Neurol. 70:9–21.
Redaelli M, Mucignat-Caretta C, Isse AA, Gennaro A, Pezzani R, Pasquale R, Pavan V, Crisma M, Ribaudo G,
Zagotto G. 2015. New naphthoquinone derivatives against glioma cells. eur J Med Chem. 96:458–466.
Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, taphoorn MJB, Belanger K, Brandes AA,
Marosi C, Bogdahn u, et al. 2005. Radiotherapy plus concomitant and adjuvant temozolomide for
glioblastoma. N engl J Med. 352:987–996.
Xu hl, Yu XF, Qu SC, Qu XR, Jiang YF, Sui DY. 2012. Synthesis and photoluminescence properties of
low-t(g), photorefractive polymers of double function. Food Chem toxicol. 50:590–596.
Yang J, li Z, Wu J, lu F, Chen C. 2014. An oxidative stress mechanism of shikonin in human glioma cells.
PloS ONe. 9. Available from: http://dx.doi.org/10.1371/journal.pone.0094180
Zagotto G, Redaelli M, Pasquale R, D'Avella D, Cozza G, Denaro l, Pizzato F, Mucignat-Caretta C. 2011.
8-hydroxynaphthalene-1,4-dione derivative as novel compound for glioma treatment. Bioorg. Med.
Chem. lett. 21:2079–2082.