S. Das et al. / European Journal of Medicinal Chemistry 51 (2012) 193e199
199
5.2.5. Cell cycle analysis
References
Cell Cycle analysis was carried out by modifying a literature
[
[
1] U. Das, R.K. Sharma, J.R. Dimmock, Curr. Med. Chem. 16 (2009) 2001e2020.
2] H.N. Pati, U. Das, R.K. Sharma, J.R. Dimmock, Mini-Rev. Med. Chem. 7 (2007)
31e139.
procedure [42]. HCT-116 cells were maintained in McCoys’5A
Modified media (ATCC) supplemented with 10% Foetal Bovine
Serum (Fischer Scientific) and 1% antibiotic-antimycotic solution
1
[3] B. Mutus, J.D. Wagner, C.J. Talpas, J.R. Dimmock, O.A. Phillips, R.S. Reid, Anal.
6
2
Biochem. 177 (1989) 237e243.
(
Sigma). Cells (1 ꢃ 10 cells) were seeded in 75 cm flasks and
[
[
4] G. Baluja, A.M. Municio, S. Vega, Chem. Ind. (1964) 2053e2054.
5] A.B. Okey, P.A. Harper, in: H. Kalant, D.M. Grant, J. Mitchell (Eds.), Principles of
Medical Pharmacology, seventh ed. Elsevier, Toronto, 2007, p. 902.
ꢂ
incubated for 24 h in a humidified incubator at 37 C with 5% CO
2
.
Each flask containing cells was treated with the samples and
ꢂ
[6] M. Galanski, B.K. Keppler, Anti-Cancer Agents Med. Chem. 7 (2007) 55e73.
[
[
untreated control for 48 h in a humidified incubator at 37 C with
7] S. Frantz, Nature 437 (2005) 942e943.
8] L.M. Espinoza-Fonseca, Bioorg. Med. Chem. 14 (2006) 896e897.
5
% CO2. Floating cells were collected and adherent cells were har-
vested with trypsin-EDTA (0.2%) and pooled. The samples were
washed with cold PBS, fixed in 70% ethanol and left on ice for 2 h.
Further samples were washed after 2 h with PBS and resuspended
[9] J.R. Dimmock, P.L. Carter, P.D. Ralph, J. Chem. Soc. Sect. B (1968) 698e703.
[10] J.R. Dimmock, M.L.C. Wong, Can. J. Pharm. Sci. 11 (1976) 35e53.
[
[
11] G. Chen, D.J. Waxman, Biochem. Pharmacol. 47 (1994) 1079e1087.
12] K. Tsutsui, C. Komuro, K. Ono, T. Nishidia, Y. Shibamoto, M. Takahashi, M. Abe,
Int. J. Radiat. Oncol. Biol. Phys. 12 (1986) 1183e1186.
in PBS containing RNase (300
for 20 min in the dark with propidium iodide (20
300 g/ml). The samples were analyzed by a FACScalibur (BD) flow
mg/ml). The samples were incubated
mg/ml) and RNase
[13] S. Das, U. Das, A. Varela-Ramírez, C. Lema, R.J. Aguilera, J. Balzarini, E. De
Clercq, S.G. Dimmock, D.K.J. Gorecki, J.R. Dimmock, ChemMedChem. 6 (2011)
(
m
1892e1899.
cytotmeter. The data were analyzed using Modfit LT free trial
version 3.3 available from Variety software house. Cells were gated
to include G0/G1, S-phase, and G2/M populations.
[
[
14] U. Das, H.N. Pati, H. Sakagami, K. Hashimoto, M. Kawase, J. Balzarini, E. De
Clercq, J.R. Dimmock, J. Med. Chem. 54 (2011) 3445e3449.
15] S. Das, U. Das, P. Selvakumar, R.K. Sharma, J. Balzarini, E. De Clercq, J. Molnár,
J. Serby, Z. Baráth, G. Schatte, B. Bandy, D.K.J. Gorecki, J.R. Dimmock, Chem-
MedChem. 4 (2009) 1831e1840.
5
.2.6. Multidrug resistance reversal assay
The ability of 1aej to reverse MDR in murine L-5178Y cells
[16] U. Das, J. Molnár, Z. Baráth, Z. Bata, J.R. Dimmock, Bioorg. Med. Chem. Lett. 18
2008) 3484e3487.
(
[
[
17] M.R. Boyd, K.D. Paull, Drug Dev. Res. 34 (1995) 91e109.
18] M.R. Grever, S.A. Schepartz, B.A. Chabner, Semin. Oncol. 19 (1992) 622e638.
transfected with the mdr1 gene followed a published procedure
34]. In brief, solutions of the compounds in dimethylsulfoxide
were added to MDR and parenteral cells at room temperature. After
0 min, rhodamine 123 was added and the cells were incubated at
[
[19] H. Gunji, S. Kharbanda, D. Kufe, Cancer Res. 51 (1991) 741e743.
[20] J.F. Haince, D. McDonald, A. Rodrigue, U. Dery, J.Y. Masson, M.J. Hendzel,
G.G. Poirier, J. Biol. Chem. 283 (2008) 1197e1208.
1
[21] M. Malanga, F.R. Althaus, Biochem. Cell Biol. 83 (2005) 354e364.
ꢂ
37 C for 20 min. In these experiments, the FAR value of 1% dime-
[22] Y. Drew, H. Calvert, N.Y. Ann, Acad. Sci. 1138 (2008) 136e145.
[23] A. Peralta-Leal, M.I. Rodriguez, F.J. Oliver, Clin. Transl. Oncol. 10 (2008)
thylsulfoxide is 0.82.
3
18e323.
24] E.T. Sakamoto-Hojo, A.S. Balajee, Anticancer Agents Med. Chem. 8 (2008)
02e416.
[
4
5.3. Determination of log P values
[
[
25] D.W. Nicholson, N.A. Thornberry, Trends Biochem. Sci. 22 (1997) 299e306.
26] R.U. Janicke, M.L. Sprengart, M.R. Wati, A.G. Porter, J. Biol. Chem. 273 (1998)
9357e9360.
The logP values of 1aej which were determined using a soft-
[
[
27] S.A. Susin, E. Douglas, L. Ravagnan, et al., J. Exp. Med. 192 (2000) 571e579.
28] D.A. Le, Y. Wu, Z. Huang, K. Matsushita, N. Plesnila, J.C. Augustinack,
B.T. Hyman, J. Juan, K. Kuida, R.A. Flavell, M.A. Moskowitz, Proc. Natl. Acad. Sci.
USA 99 (2002) 15188e15193.
ware programme [43] are as follows, namely 1a : 5.40, 1b : 5.34, 1c :
.29, 1d : 5.71, 1e : 6.96, 1f : 5.67, 1g : 5.57, 1h : 6.08, 1i: 7.08 and 1j :
.08. Linear, semilogarithmic and logarithmic plots were made
5
7
using a commercial statistical package [44].
[29] D. D’Amours, S. Desnoyers, I. D’Silva, G.G. Poirier, Biochem. J. 342 (1999)
249e268.
[
[
[
30] K.K. Wang, Trends Neurosci. 23 (2000) 20e26.
31] B.B. Aggarwal, A. Kumar, A.C. Bharati, Anticancer Res. 23 (2003) 363e398.
32] L. Moragoda, R. Jaszewski, AP.N. Majumdar, Anticancer Res. 21 (2001)
Acknowledgements
8
73e878.
The authors thank the following agencies for financial support
of this study, namely the Canadian Institutes of Health Research
and the Saskatchewan Health Research Foundation for a grant to
J.R. Dimmock and U. Das, the Ministry of Education, Science, Sports
and Culture for a grant-in-aid to H. Sakagami, and the Szeged
Foundation of Cancer Research to J. Molnár. The measurement of
the FAR values was undertaken by Imre Ocsovszki which is grate-
fully acknowledged. The National Cancer Institute (USA) kindly
provided the data presented in Table 2 and S.G. Dimmock,
Department of Finance, Nanyang Technological University,
Singapore, is thanked for discussions on the PSE expression. D.
Michel kindly assisted with the flow cytometry experiment. Ms. B.
McCullough and Ms. C. Sutton typed various drafts of this report
who are gratefully acknowledged.
[33] B.C. Baguley, Mol. Biotechnol. 46 (2010) 308e316.
[
34] M. Kawase, H. Sakagami, N. Motohashi, H. Hauer, S.S. Chatterjee, G. Spengler,
A.V. Vigyikanne, A. Molnár, J. Molnár, In Vivo 19 (2005) 705e712.
35] X. Shen, G. Chen, G. Zhu, W.-F. Fong, Bioorg. Med. Chem. 14 (2006)
7138e7145.
[
[36] J.R. Dimmock, U. Das, H.I. Gul, M. Kawase, H. Sakagami, Z. Baráth, I. Ocsovsky,
J. Molnár, Bioorg. Med. Chem. Lett. 15 (2005) 1633e1636.
[
[
[
37] J.R. Dimmock, M.P. Padmanilayam, R.N. Puthucode, A.J. Nazarali,
N.L. Motaganahalli, G.A. Zello, J.W. Quail, E.O. Oloo, H.B. Kraatz, J.A. Prisciak,
T.M. Allen, C.L. Santos, J. Balzarini, E. De Clercq, E.K. Manavathu, J. Med. Chem.
4
4 (2001) 586e593.
38] N. Motohashi, W. Wakabayashi, T. Kurihara, H. Fukushima, T. Yamada,
M. Kawase, Y. Sohara, S. Tani, Y. Shirataki, H. Sakagami, K. Satoh,
H. Nakashima, A. Molnár, G. Spengler, N. Gyémént, K. Ugocsai, J. Molnár,
Phytother. Res. 18 (2004) 212e223.
39] F. Yanagisawa-Shiota, H. Sakagami, N. Kuribayashi, M. Iida, T. Sakagami,
M. Takeda, Anticancer Res. 15 (1995) 259e266.
[40] R. Takeuchu, H. Hoshijima, N. Onuki, H. Nagasaka, S.A. Chowdhury, M. Kawase,
H. Sakagami, Anticancer Res. 25 (2005) 4037e4041.
[41] Distributed by Cell Signaling Technology, Inc.
Appendix. Supplementary material
[42] A. Lakshmikuttyamma, E. Pastural, N. Takahashi, K. Sawada, D.P. Sheridan,
[
[
44] Statistical Package for Social Sciences, SPSS for Windows, Release 17.0, SPSS
Inc, Chicago, 2008.