2082
G. Zagotto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2079–2082
4. Seger, R.; Krebs, E. G. FASEB J. 1995, 9, 726.
showed a remarkable increase of the in vitro apoptotic induction
on human glioma cells when compared with TMZ and PTX. Aiming
for the discovery of novel therapeutic compounds which could sig-
nificantly improve contemporary glioma chemotherapy, these pre-
liminary results prompted the preparation of novel derivatives,
whose testing is currently being carried out under the same condi-
tions. In addition, future cytotoxicity assays on different cell lines
will be considered, thus giving significant and comprehensive data
to better understand the cytotoxic effect of such compounds.
5. (a) Mucignat-Caretta, C.; Denaro, L.; Redaelli, M.; Davella, D.; Caretta, A. BMC
Cancer 2010, 10, 141; (b) Mucignat-Caretta, C.; Cavaggioni, A.; Redaelli, M.;
Malatesta, M.; Zancanaro, C.; Caretta, A. Neurooncology 2008, 10, 958; (c)
Redaelli, M.; Mucignat-Caretta, C.; Cavaggioni, A.; Caretta, A.; D’Avella, D.;
Denaro, L.; Cavirani, S.; Donofrio, G. Virol. J. 2010, 7, 298.
6. Chen, T. C.; Hinton, D. R.; Zidovetzki, R.; Hofman, F. M. Lab. Invest. 1998, 78, 165.
7. See Supplementary data for the detailed procedure of C1 synthesis and the cell
cytotoxicity of the 30 screened compounds. Some kinase inhibitory properties
are also reported.
8. Zonta, N.; Cozza, G.; Gianoncelli, A.; Korb, O.; Exner, T. E.; Meggio, F.; Zagotto,
G.; Moro, S. Lett. Drug Des. Disc. 2009, 6, 327.
9. Högberg, T.; Glimelius, B.; Nygren, P. Acta Oncol. 2001, 40, 340.
10. Cells were maintained in a monolayer using complete growth medium (CGM)
in combination with 90% Dulbecco Modified Eagle’s Medium (DMEM), 10% FBS,
Acknowledgments
100 I.U./ml penicillin, 10
Plasmocin (InVivogen, Milan, Italy). Cells were incubated at 37 °C in
humidified environment with 95% air and 5% CO2, up to 80–90% confluence
(4–6 days). For cytotoxicity assay, cells were plated on 24-wells plates and
grown on 9 mm sterile cover glass. After 48 h, addition of the following species
lg/ml streptomycin, 10 lg/ml tetracycline, 25 lg/ml
The research project was financially supported by University of
Padova, Progetto di Ateneo 2008.
The authors thank Dr. Alfonso Venzo for technical support, CNR-
ISTM, Padova.
a
was carried out whilst maintaining the CGM medium: C1 (50
l
M,
5
l
l
M,
M,
0.5
lM or 0.05
lM), PTX (50
lM, 5
lM, 0.5
lM or 0.05
l
M) and TMZ (50
5
l
M, 0.5
l
M or 0.05 lM). Cells were incubated for 24 h, and then fixed with
Supplementary data
methanol and stained with Wright’s stain; a total of 2400 cells were counted
from each slide. The percentage of apoptotic and necrotic cells was then
calculated, following an established procedure;5b t-test was used to estimate
the amount of apoptotic and necrotic cells (in percentage) between TMZ and
treated cells. The primary cell culture was prepared from glioblastoma biopsy
(patient: male 70 years). The biopsy (3 mm3) was shaken for 5 min in 3 ml of
0.25% Trypsin, 0.02% EDTA solution. The suspension was inactivated with CGM
and centrifuged at 37 °C for 10 min at 1350 rpm. The supernatant was
discharged, the pellet was re-suspended carefully in 10 ml of CGM and
plated in a cell culture flask. The culture was monitored for three weeks
changing the medium every 3 days.5c Cells were maintained as described
above and the cytotoxicity assay has been carried out as described for the
Supplementary data associated with this article can be found, in
References and notes
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GLI36 cell lines using two different concentrations of C1, PTX and TMZ (50
or 0.05 M).
lM
l