Synthetic hispidin, a PKC inhibitor, is more cytotoxic toward cancer cells than normal cells in vitro

Article abstract of DOI:10.1023/A:1007321227010

The trypanocidal activity of naturally occurring 6-(3,4-dihydroxystyryl)-4-hydroxy-2-pyrone (hispidin) prompted us to examine its cytotoxic activity toward normal and cancerous cells in culture. Hispidin synthesized in our laboratory to a high degree of purity (checked by ¹H and ¹³C NMR spectroscopy) was shown to be cytotoxic (between 10^-3 mol/L and 10^-7 mol/L) toward normal human MRC-5 fibroblasts, human cancerous keratinocytes (SCL-1 cell line), and human cancerous pancreatic duct cells (Capan-1 cell line). Interestingly, addition of hispidin in three successive doses (between 10^-5 mol/L and 10^-7 mol/L) led to a 100-fold increase in activity with an enhanced activity on cancer cells compared to normal cells (50%). Synthetic hispidin was found to inhibit isoform β of protein kinase C (IC₅₀ of 2 x 10^-6 mol/L), but not E. coli and placental type XV alkaline phosphatases. The enhanced activity of hispidin toward the cancerous cell lines is discussed.

Full text of DOI:10.1023/A:1007321227010

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Cell Biology and Toxicology. 1997; 13: 141^153.
# 1997 Kluwer Academic Publishers. Printed in the Netherlands
Synthetic hispidin, a PKC inhibitor, is more cytotoxic toward cancer cells than
normal cells in vitro
C. Gonindard1, C. Bergonzi2, C. Denier2, C. Sergheraert3, A. Klaebe2, L. Chavant4
and E. Hollande1
1Laboratoire de Biologie Cellulaire, Equipe de Cytophysiologie des Cellules Eucaryotes Humaines,
Universite Paul Sabatier, Toulouse;
2Laboratoire IMRCP, Groupe de Chimie Organique Biologique, Universite Paul Sabatier, Toulouse;
3Institut Pasteur de Lille, Lille;
4Mycologie (4R1), EA 822, Universite Paul Sabatier, Toulouse, France
Received 26 February 1996; accepted 5 July 1996
Keywords: anti-neoplastic drug, cancer, hispidin, keratinocyte, protein kinase C
Abstract
The trypanocidal activity of naturally occurring 6-(3,4-dihydroxystyryl)-4-hydroxy-2-pyrone (hispi-
din) prompted us to examine its cytotoxic activity toward normal and cancerous cells in culture.
1
Hispidin synthesized in our laboratory to a high degree of purity (checked by H and 13C NMR
spectroscopy) was shown to be cytotoxic (between 10^3 mol/L and 10^7 mol/L) toward normal
human MRC-5 ¢broblasts, human cancerous keratinocytes (SCL-1 cell line), and human cancerous
pancreatic duct cells (Capan-1 cell line). Interestingly, addition of hispidin in three successive doses
(between 10^5 mol/L and 10^7 mol/L) led to a 100-fold increase in activity with an enhanced activity
on cancer cells compared to normal cells (50%). Synthetic hispidin was found to inhibit isoform b of
protein kinase C (IC50 of 2610^6 mol/L), but not E. coli and placental type XV alkaline
phosphatases. The enhanced activity of hispidin toward the cancerous cell lines is discussed.
Abbreviations: AP, alkaline phosphatase; IC50, inhibitory concentration 50%; PKC, protein
kinase C
Introduction
synthetic antineoplastic drugs that interfere
with nucleic acids or their metabolism, such
as 5-£uorouracil (5-Fu) (Hartmann and Hei-
delberger, 1961; Chadwich and Rogers, 1972;
Ardalan and Glazer, 1981; Malet-Martino et
al., 1986; Wurzer et al., 1994), daunorubicin
(Tarasiuk et al., 1990), adriamycin (Blum and
Carter, 1974), and cisplatin (Rosenberg et al.,
1969; Roberts and Pascoe, 1972; Cohen et al.,
1979). Other compounds such as colchicine
Considerable e¡ort has been devoted to the
search for antineoplastic drugs that have more
activity toward cancer cells than toward nor-
mal ones. Unfortunately, most of the available
drugs are active against both kinds of cell,
although some of them have been shown to
have a preferential action on highly proliferat-
ing cells. These agents include the natural or

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143
decarboxylation of hispidin (Figure 1).
(3) The Capan-1 cell line, established by
Fogh, derived from a liver metastasis biopsied
from a 40-year-old Caucasian patient with a
pancreatic adenocarcinoma (Fogh et al., 1977).
They are considered to be ductal type cells
(Kyriazis et al., 1982; Hollande et al., 1990).
They were obtained on the 14th passage from
the American Type Culture Collection (ATCC,
Rockville, MD, USA). Every 6 days, cells were
maintained by passage on a 15% FCS supple-
mented RPMI 1640 medium (Gibco).
To study its biological activity, hispidin was
synthesized using a method based on that of
Edwards and Wilson (1961). The purity and
structure of each intermediate was checked by
1H and 13C NMR spectroscopy. All chemicals
were obtained from Aldrich (Saint Quentin
Fallavier, France). 4-Hydroxymethyl-6-methyl-
2-pyrone was condensed on a suitable substi-
tuted benzaldehyde (i.e., 3,4-di(methoxy-
methylenoxy)) in a Knoevenagel type reaction
under the conditions described by Edwards
and Wilson (1961). Spontaneous dehydration
then leads to a styryl-pyrone methylated on
C-4 and methoxymethylated on the other
hydroxyl groups.
On each passage, MRC-5, SCL-1, and Ca-
pan-1 cells were seeded out into 25-cm2 £asks
(Nunc, Roskilde, Denmark) at a concentration
of 2.56105 cells/ml. Culture media contained
100 U/ml penicillin, 10^4 g/ml streptomycin,
and 2.5610^7 g/ml fungizone (Gibco) and
were renewed every 2 days. All cell lines were
maintained at 378C in a 95% CO2 atmosphere.
Cultures were checked every month for con-
tamination with mycoplasma by culture on
speci¢c media during three passages or by a
PCR method (Mycoplasma PCR kits, Strata-
gene, La Jolla, CA, USA).
Hispidin was synthesized in the following
stages:
(a) 4-Hydroxy-6-methyl-2-pyrone: 13.5 g
dehydroacetic acid (0.08 mol) was heated at
1208C for 90 min in 30 ml of concentrated
sulfuric acid. The reaction mixture was then
added to ice-cold water and ¢ltered; a white
cake was obtained. This cake, recrystallized
from water, a¡orded the product as ¢ne beige
needles in a 60% overall yield.
Isolation, puri¢cation, and synthesis of hispidin
1H NMR: (DMSO d6) d(ppm) 2.1 (s, 3H)
CH3; 5.2 (d, 1H, J = 3 Hz); 5.9 (m, 1H).
13C NMR: (DMSO d6), d(ppm) 19.1 (C7);
88.0 (C3); 100.4 (C5); 163.6 (C6); 165.4 (C4);
171.1 (C2).
Preliminary extracts were obtained after
freeze-drying of the fruit bodies and of the
cultured mycelium of Inonitus hispidus. Extrac-
tions were carried out in a Soxhlet apparatus
using methanol as solvent. After concentra-
tion, the extracts were fractionated by thin-
layer chromatography. Tests on Trypanosoma
equiperdum cultures revealed two active spots
(Bergonzi, personal communication). Com-
pounds from these spots were then isolated by
circular chromatography on a Chromatotron
(b) 4-Methoxy-6-methyl-2-pyrone: 1.7 g 4-
hydroxy-6-methyl-2-pyrone (0.0135 mol), 10 g
K2CO3, and 1.3 ml dimethylsulfate (0.0135
mol) were re£uxed for 8 h with anhydrous
acetone. After ¢ltration and evaporation of
solvent, methoxylated pyrone was obtained in
quantitative yield.
1
apparatus, followed by H and 13C NMR
1H NMR: (DMSO d6, d(ppm) 2.2 (dd, 3H)
CH3; 3.8 (s, 3H) OCH3; 5.5 (d, 1H) CH; 6.0
(m, 1H) CH.
spectroscopy. The active compounds were
shown to be hispidin (6-(3,4-dihydroxystyryl)-
4-hydroxy-2-pyrone) and one of its derivatives
(6-(3,4-dihydroxyphenyl)-hex-5-en-2,4-dione),
probably formed by opening of the lactone and

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144
Hispidin had shown NMR characteristics as
follows:
(c) 3,4-Di(methoxymethylenoxy)benzaldehyde:
6 g 3,4-dihydroxybenzaldehyde (0.0434 mol)
was re£uxed with 100 ml triethylamine for 1
h. 14 ml chloromethyl methyl ether (0.1878
mol) was then added dropwise and left stand-
ing for 2 h. The reaction mixture was evapo-
rated to dryness and the residue was dissolved
in dichloromethane and washed with water. A
brown oil was obtained after drying and eva-
poration of the organic phase. This oil was
dissolved in toluene and ¢ltered through silica
gel. After evaporation of the ¢ltrate, a yellow
solid was obtained and recrystallized from
petroleum ether. The protected aldehyde was
obtained in a yield of 77%.
1H NMR: (acetone d6), 200 MHz, d(ppm)
5.3 (d, 1H) CH; 6.0 (d, 1H) CH; 6.6 (d, 1H,
J = 16 Hz) CH; 7.1 (d, 1H, J = 16 Hz) CH; 6.9
(m, 3H) aromatic H; 8.0 (s, 1H) OH; 8.3 (s,
1H) OH; 10.2 (s, 1H) OH.
13C NMR: (acetone d6), 50 MHz, d(ppm)
90.7 (C3); 101.0 (C5); 114.7 (C10); 116.4
(C13); 117.5 (C14); 121.6 (C7); 128.8 (C9);
135.9 (C8); 146.3 (C6); 147.9 (C11); 161.2
(C12); 163.9 (C14), 170.8 (C2).
The purity of the synthetic hispidin was
veri¢ed using the following methods: (i) identi-
1
¢cation of an extra signal on the H and 13C
1H NMR: (CDCl3, d(ppm) 3.5 (s, 6H)
2CH3O; 5.3 (s, 2H) CH2O; 5.3 (s, 2H) CH2O;
7.5 (m, 3H) aromatic H; 9.9 (s, 1H) CH == O.
spectra; (ii) thin-layer chromatography (silica
gel with CHCl3^MeOH 8:2 (v/v) as solvent;
(iii) identi¢cation of the molecular peak
(MH+) on mass spectrometry with chemical
ionization (NH3); (iv) determination of melting
point.
(d) 6-(3,4-Di(methoxymethylenoxy)styryl)-4-
methoxy-2-pyrone: This product was prepared
according to Edwards and Wilson (1961) with
a yield of 64%.
Cytotoxicity tests
1H NMR: (CDCl3), d(ppm) 3.5 (s, 3H)
CH3O; 3.5 (s, 3H) CH3O; 3.8 (s, 3H) CH3O;
5.2 (s, 4H) 2 (O-CH2-O); 5.5 (d, 1H) CH; 5.9
(d, 1H) CH; 6.4 (d, 1H, J = 16 Hz) H^C ==; 7.2
(m, 3H) aromatic H; 7.9 (d, 1H, J = 16 Hz) H^
C==.
Hispidin was tested on the three cell lines
seeded in Nunc culture plates (4 wells) at the
following concentrations: 2.56104 cells/ml
(MRC-5), 105 cells/ml (SCL-1), and 1.256105
cells/ml (Capan-1).
13C NMR: (CDCl3) d(ppm) 51.6 (C19); 51.9
(C16 and C18); 84.2 (C3); 91.0 (C15); 91.2
(C17); 96.5 (C5); 110.7 (C10); 112.1 (C13);
113.0 (C14); 118.4 (C7); 125.5 (C9); 131.0
(C8); 143.2 (C6); 144.2 (C11); 154.5 (C12);
159.7 (C4); 166.8 (C2).
To identify the various growth stages, growth
curves for each cell line were established in the
presence of 1% DMSO. Hispidin was dissolved
in DMSO and added to the cell culture medium
on day 1 (concentrations between 10^3 and 10^7
mol/L). Control cultures were grown on med-
ium containing the same concentration of
DMSO (1%). In the ¢rst set of experiments,
hispidin was added to the culture medium in a
single daily dose on day 1 and the cells were
counted in a Malassez hematimeter every day
for 5 days. In the second set of experiments,
hispidin was added to the cell cultures on days
1, 2, and 3, and cells were counted daily for a
period of 8 days. Each data point is the mean of
10 experimental values.
(e) 6-(3,4-Dihydroxystyryl)-4-hydroxy-2-
pyrone: 4-Demethylation was carried out as
described by Edwards and Wilson (1961). The
protecting phenol acetals were hydrolyzed by
dissolving the residue in methanol and adding
an acid resin (CG120, 24 h). A yellow powder
was obtained after ¢ltration and evaporation
of methanol. The product was puri¢ed by silica
gel chromatography (CHCl3^MeOH 8:2 v/v).