Table 3. Concentration-dependent antagonism of AhR, relative
to its activation by B[a]P at 0.75 µM, by the most potent
CYP1A1 and CYP1B1 inhibitors, identified in Sacchrosomes and
recombinant CYP-expressing human cells.
around 14 and 0.8 µM, respectively (shown in Table S5 of
Supporting Information), indicating toxicity of B[a]P is mediated
by expression of the CYP1A1 gene. The CYP1A1 inhibitor 3j
was tested for chemo-preventive activity that would avert
CYP1A1 mediated B[a]P toxicity in HEK293 cells. It was
observed that 3j rescued cells from B[a]P toxicity, which is
shown in Figure 3. Compounds 3s and 3n which were inactive
against CYP1A1, does not rescue cells from B[a]P toxicity.
AhR-ARNT complex in eGFP units at
Entry
-
1 µM
955
600
920
955
5 µM
955
220
770
955
10 µM
955
150
455
955
20 µM
955
120 1.2
285
955
9
9
9
9
9
5
7
9
9
9
5
9
9
3j
3n
3s
5
9
15
M
µ
All values, presented in eGFP units (excitation/emission monitored at
489/509 nm), represent the mean and standard deviations of three
independent experiments.
n
i 10
0
5
C
E
5
0
P
]
CYP family of enzymes is part of a metabolic machinery of
the cell.4, 16, 33 Recent studies have shown a correlation between
CYP1B1 expression in human cells and the metabolism of
anticancer drugs such as paclitaxel, docetaxel, doxorubicin,
mitoxantrone, tamoxifen and cisplatin.16 CYP1B1-mediated
metabolism leads to decrease in the cellular efficacy of cytotoxic
drugs, and eventually cancer cells become resistant to these
drugs. Such issues of drug metabolism were specifically been
addressed by using CYP1B1-specific inhibitors.16, 18 Cui et al.
and Horley et al. have shown improvement in docetaxel and
cisplatin efficacy by potent CYP1B1 inhibitors.16, 18 Therefore,
CYP1B1 inhibitors confirmed in live human cells were tested for
reduction in resistance to cisplatin in adherent HEK293 cells. The
introduction of CYP1B1 gene bearing plasmid in HEK293
reduces the cytotoxicity of cisplatin by increasing the EC50 from
10.5 to 65 µM (shown in Table S4 of supporting information).
CYP1B1 inhibitors 3j and 3n completely restore cytotoxicity of
cisplatin at the tested concentrations, as shown in Figure 2. Due
to inhibition of cisplatin metabolism, the EC50 of cisplatin was
restored to ~10 µM. This demonstrates that CYP1B1 inhibitors
have the ability to re-establish cisplatin’s cytotoxicity in cells
harboring the CYP1B1 gene. The compound 3s, which was
inactive in live cells assays, does not reverse the cisplatin-
resistance.
a
[
B
Control 3j
3n
3s
ANF
Figure 3. Protection from CYP1A1 mediated B[a]P toxicity by CYP1A1
inhibitors. A range of concentrations of B[a]P (0.05 µM – 100 µM) were
used, in the presence of 8 x IC50 value of 3j, whereas compounds 3n, 3s and
ANF (α-naphthoflavone) were used at 20 µM concentrations. IC50 values
were determined in the human cell assay where cells were grown in
suspension. HEK293 cells were transfected with pcDNA3.1/hCYP1A1 (the
plasmid which encodes the human CYP1A1 gene). All values, presented in
µM concentrations, represent the mean and standard deviations of three
independent experiments.
In summary, CYP1B1 structure-guided information, allowed
us to develop SAR for 2-pyrrolyl chalcones as CYP1 family
inhibitors. CYP1B1 selectivity can find potential for reversal of
cisplatin resistance through restoration of cisplatin’s toxicity in
human cancer cells, while CYP1A1 inhibitory activity can find
application in the prevention of cancer. Further studies, based on
these findings, could lead to the discovery of novel modulators
that would allow chemo-prevention and overcome chemo-
resistance in cancer.
Acknowledgement:
BC would like to thank the UK Higher Education Innovation
Fund (HEIF) and Research Business Innovation (RBI) directorate
of De Montfort University for support. PJ acknowledges CSIR
for an award of Senior Research Fellowship.
80
60
40
20
0
References and notes:
1. Guengerich, F. P., Chem. Res. Toxicol. 2008, 21, 70-83.
2. McFadyen, M. C.; Breeman, S.; Payne, S., J. Histochem.
Cytochem. 1999, 47, 1457-1464.
3. Gajjar, K.; Martin-Hirsch, P. L.; Martin, F. L., Cancer Lett. 2012,
324, 13-30.
Control
3j
3n
3s
ANF
Figure 2. Overcoming cisplatin resistance by CYP1B1 inhibitors. A range of
concentrations of cisplatin (0.05 µM – 100 µM) were used, in the presence of
6 x IC50 values of compounds 3j and 3n, whereas compound 3s and ANF (α-
naphthoflavone) were used at 20µM concentrations. IC50 values had been
determined in the human cell assay where cells were grown in suspension.
HEK293 cells were transfected with pcDNA3.1/hCYP1B1 (the plasmid
encoding the human CYP1B1 gene). All values, presented in µM
concentrations, represent the mean and standard deviations of three
independent experiments.
4. Murray, G. I.; Taylor, M. C.; McFadyen, M. C.; McKay, J. A.;
Greenlee, W. F.; Burke, M. D.; Melvin, W. T., Cancer Res. 1997,
57, 3026-3031.
5. Muskhelishvili, L.; Thompson, P. A.; Kusewitt, D. F.; Wang, C.;
Kadlubar, F. F., J. Histochem. Cytochem. 2001, 49, 229–236.
6. Brockdorff, B. L.; Skouv, J.; Reiter, B. E.; Lykkesfeldt, A. E.,
Int. J. Cancer 2000, 88, 902-906.
7. Carnell, D. M.; Smith, R. E.; Daley, F. M.; Barber, P. R.; Hoskin,
P. J.; D., W. G.; Murray, G. I.; Everett, S. A., Int. J. Radiat.
Oncology Biol. Phys. 2004, 58, 500-509.
8. Cavalieri, E. L.; Stack, D. E.; Devanesan, P. D.; Todorovic, R.;
Dwivedy, I.; Higginbotham, S. M.; Johansson, S. L.; Patil, K. D.;
Gross, M. L.; Gooden, J. K.; Ramanathan, R.; Cerny, R. L.;
Rogan, E. G., Proc. Natl. Acad. Sci. USA 1997, 94, 10937-10942.
B[a]P is a pro-carcinogen, which upon metabolism by
CYP1A1 enzyme is converted into benzo[a]pyrene-6,7-
dihydrodiol-9, 10 epoxide which is then able to intercalate DNA,
thus producing toxicity. In the literature various CYP1A1
inhibitors have been reported to protect cells from B[a]P
toxicity.20, 32 The B[a]P EC50 values in untransfected cells and
cells transfected with the CYP1A1 gene-bearing plasmid are
Williams, Ibidapo S.
