SUPPLEMENTARY FIGURE LEGENDS
Figure S1.Analysis of the COX-dependency of Celecoxib toxicity. (A) Comparison, on a logarithmic scale
of concentrations, of COX inhibition vs. toxicity of Celecoxib (MDA-MB-231 cells, 0.5% serum). COX
activity was measured as PGE2 release in the medium in 3h (163±33 pg/106 cells in control cells) by
Radioimmunoassay (RIA), as described [1]. Toxicity is reported as decrease of crystal violet staining at 30h;
(B-C) exogenous PGE2 (1 µM) does not rescue MDA-MB-231 cells from death or growth inhibition induced
by 50 µM Celecoxib in 0.5% serum or 10% serum, respectively. Lower/higher concentrations of PGE 2 or
other prostanoids tested were also ineffective (not shown); (D) The selective COX-2 inhibitor Rofecoxib,
tested up to 200 µM in low serum, is totally devoid of toxicity, despite full COX inhibition is reached in the
sub-micromolar range (not shown), in agreement with previous studies [2, 3].
Overall, growth inhibition and death occurred at concentrations of Celecoxib significantly higher
than COX-inhibition, were not prevented by exogenous PGE2, and were independent of the levels of COX
expression and prostanoid production, which is quite different between the three cell lines tested. In fact
MDA-MB-231 express COX-2 and produce relevant levels of PGE2, while HCT116 and MCF7 are negative
or almost negative for COX-2 and don’t produce measurable levels of PGE2 in basal conditions ([3-5] and
unpublished data). Altogether, these data confirmed that Celecoxib toxicity is COX-independent [3, 6, 7].
Figure S2. Sporadic crystals of Celecoxib observed by normal light microscopy in cell cultures (HCT116).
Top: 30 µM Celecoxib, low serum, 24h. Middle: 50 µM Celecoxib, low serum, 24h. Bottom: 100 µM
Celecoxib, 10% serum, 40h. Within 40h, the absolute limit of detection of well visible crystals was 100, 30,
and 25 µM in high, low and serum-free medium, respectively (see also Fig. S3E).
Figure S3. Analysis of the solubility of Celecoxib, supporting data. (A) The solubility in water, measured by
light scattering [8], is in the expected range ([9]and Table S1) and validates the method successively used on
solutions previously not tested; (B) Approximate quantification of the average number of fluorescent
precipitates observed in squares 100x100 µm in 0.5% (2-3h after Celecoxib dilution) and 10% serum (4-6h).
Inset: magnification of the same graph. Since size and brightness were not considered, these values are
1
biased in favour of small precipitates, albeit very small or non-compact precipitates were missed. An
indicative correction based on size x brightness of the precipitates is shown in (C); (D) Spectrophotometric
(turbidometric) analysis of precipitation in 0.5% serum [8] indicates at 25µM Celecoxib the onset of
turbidity; (E) Correlation between different techniques of solubility analysis (low serum): light scattering,
turbidometric, fluorescent microscopy, and crystals count by normal light microscopy (rough and quick
quantification of crystals in 12 well plates, sum of 6 independent wells). At 25 µM the appearance of visible
precipitates (fluorescent microscopy) corresponds to the onset of turbidity and to a change of the slope of the
light scattering curve, and immediately precedes the appearance of sporadic ramificated crystals (30 µM).
Overall, the different methods defined a coherent picture: a limit of solubility detectable by sensitive
techniques (light scattering, dissolution studies), and a limit of gross/visible precipitation around 20µM in
serum-free medium, 25µM in low serum, 50µM in 10% serum.
Notably, while small precipitates were more reproducibly observed and causally implicated in cell
death, ramificated/well visible crystal appeared numerous only at > 50 µM Celecoxib in low serum, >100
µM in high serum. At lower concentrations they only appeared sporadically and were simple indicators of
the occurrence of gross precipitation, not cause of cell death. Moreover their formation was affected by many
factors, which included the type of plastic used, technical details in the dilution of Celecoxib from DMSO
stocks, temperature of storage before use, and shacking of the medium during all these steps and in cell
culture. In high serum crystals were generally less ramificated and smaller, and influenced by the batch of
serum used.
Figure S4. Fluorescent analysis of Celecoxib precipitates, supporting data in RGB color scale. (A)
Fluorescent visualization of DiA stained precipitates (50 µM Celecoxib, low serum) on the plane of the
coverslip after 2-3h incubation vs. a plane in suspension (middle), and after 16h incubation (left). Seeding
onto glass coverslips only improves the visualisation of Celecoxib precipitates but the total number of
precipitates in suspension is much higher. The precipitates in suspension drop during the time while they
seed or aggregate in bigger crystal (Fig. S6B), with parallel decrease of toxicity (Fig S7B); (B) With 25 µM
Celecoxib (low serum), precipitates in suspension are almost undetectable (<1/100 µm squares). At 16h more
precipitates are detected on the coverslip, which well fits with delayed cell death (Fig 1E). At < 25 µM
2
precipitates are not well visible not only because rare and small, but probably also because the inclusion of
serum proteins or serum coating reduces/prevents clustering, adhesion to the glass, or efficient stimulation of
DiA fluorescence; (C) 12.5 µM Celecoxib, plane on the coverslip. (D) Approximate quantification (noncorrected for brightness) of what shown in the images A and B. Right graph: precipitates of 50µM celecoxib
on the coverslip vs. suspension at 2-3h, and in suspension at 16h. Left: precipitates of 25 µM Celecoxib in
suspension vs. coverslip at 2h and 16h. (E) Higher magnification of the confocal image in Fig 2A, which
probably shows a growing crystal. Very small precipitates, visible as clouds, surround the crystal,
contributing to its growth; (F) Fluorescent visualisation of precipitates in high serum (50 μM Celecoxib at
5h). Inset: crystal formed by 100 μM Celecoxib (30h)
Figure S5. Correlation on a logarithmic scale of concentration indicates overlapping between precipitation
and overall toxicity of Celecoxib (growth inhibition and death), while COX-inhibition (see also Fig. S1A) is
quite distant
Figure S6. Analysis of the interaction cells-precipitates, supporting images (in RGB color scale). (A)
Control CFSE-stained cells (0.5% serum ) (up) and CFSE stained cells treated 45’ with 100 µM
Celecoxib+DiA after 0.45 µm filtration (down). Only a few visible precipitates escape the filtration or are
formed afterwards, and the absence of diffused red staining indicates that most of DiA is precipitate-bound
and retained by the filters (no direct DiA-staining of cellular structures); (B) Higher magnification of Fig.
3B, in RGB, which shows the internalisation of Celecoxib precipitates (white arrows); (C) Fig. 3A in RGB,
cells treated with Celecoxib 100µM + DiA for 20’; (D) Confocal section (medium-apical) showing the
dramatic situation of cells exposed to Celecoxib 100µM for 45’; (E) Fig. 3C in RGB, confocal section of a
cell treated with 150 µM celecoxib for 20’, which shows the cell-damaging capacity of Celecoxib
precipitates
Figure S7.Analysis of the causal relationship precipitation-toxicity, supporting data (MDAMB-231 cells). (A) Impact of filtration (0.2 µm) on cell growth and viability of confluent MDAMB-231 cells (confluent cells, here ≥70000/well in 96 well plates, have less membrane exposition
3
resulting in lower toxicity). Cell death is prevented up to 150 µM Celecoxib, but some growth
inhibition is still present. To exclude binding of soluble Celecoxib to the filter, the first 6 ml of
filtered solutions were discarded, since fluorimetric analysis [10] of serum-free soluble Celecoxib
(5 µM) indicated 20% binding in the first 2 ml filtered fraction, 6% in the second, <2% in the third
one; (B) Celecoxib toxicity (0.5% serum) is gradually lost after incubation in a polypropilene tube,
as precipitates adhere to the plastic. Moreover, incubation with a monolayer of cells, which
efficiently bind precipitates, followed by centrifugation of cellular debris, completely removes the
toxicity. If Celecoxib adhering to the plastic or the cells was recovered by ethanol extraction, almost
100% of the original toxicity was recovered, thus excluding inactivation by chemical modifications
(not shown); (C) Time course of cell viability at different pH values, used as reference for the graph
in Fig. 4B. pH 9.2 was a good compromise between increased Celecoxib solubility and sufficiently
long cell-survival ([9] and Table S1); (D) The toxicity of 25 µM Celecoxib increases at acidic pH,
(slightly increased precipitation or cooperative toxicity low pH-Celecoxib); (E) Time course of celldeath following 10’ contact with 100 µM Celecoxib in the presence of 20% DMSO or Methanol,
which prevent Celecoxib -induced cell death, or 20% Ethanol, excessively toxic for this assay; (F)
Effect of NaN3 and NH4Cl on cell death induced by 50 µM Celecoxib (high-density cells). NH4Cl
(10mM), used as inhibitor of lysosomal acidification [11], was totally ineffective. NaN3 (0.1-0.2%),
used as inhibitor of endocytosis [12, 13], was only weakly active on Celecoxib ≤50 µM; (G)
Evidences that cellular accumulation is not cause of toxicity. Cells (HCT116) were repeatedly
exposed to high volumes of Celecoxib-containing medium (4 times within 16 h, at the density of
1x104 cells/ml, and recovering the floating cells by centrifugation), condition that favours cellular
accumulation of the drug (unpublished observations), while preventing delayed precipitation and
loss of Celecoib on the walls (Fig. S7). If compared with the standard protocol (7.5 to 10x104
cells/ml, single treatment) the effect of low-density precipitates (25 µM Celecoxib) is potentiated,
while only minimal cell death occurs with 12.5 µM Celecoxib (negligible concentration of toxic
precipitates).
4
Figure S8. Analysis of delayed precipitation and toxicity of Celecoxib (low serum). Since toxicity with
low concentration Celecoxib was reported by some authors (e.g. [14, 15]) the causal involvement of delayed
precipitation phenomena in cell death was investigated. (A) Normal light microscopy phase contrast, 20X
objective) showing small late Celecoxib crystals (Celecoxib 12.5µM, incubation 5 days) vs. serum
precipitates. The morphological difference allows discriminating these precipitates; (B) Delayed
precipitation with low concentration Celecoxib is prevented by short incubation with cells (4h), indicating
that membrane binding can deplete supersaturated Celecoxib and small precipitates. The images are
indicative of the size/complexity of sporadic crystals observed in culture plates after 5 days with Celecoxib
down to 12.5 µM, with or without cells. Crystal formation at 12.5 µM Celecoxib is prevented by incubation
with >10000 cells/ml, while >20000cells/ml are necessary to prevent further precipitation of 25 µM
Celecoxib; (C) Analysis of cell-death in low serum (HCT116, left, MDA-MB-231, right) at 96h after
treatment with Celecoxib 12.5 and 25 µM at different cell densities (10000 to 320000 cells/ml). Additional
delayed toxicity only occurs at cell densities unable to prevent delayed precipitation, and fits with the
formation of visible crystals.
Notably, late crystals and toxicity were never observed at 6 µM, soluble according to light
scattering, unless relevant evaporation of the medium occurred, or the dissolution of Celecoxib
from DMSO was not accurate. However, this never occurred in controlled conditions. The high
volumes of medium used in the present study (200ul in 96 wells, etc.), were also selected to
minimize this type of experimental errors.
REFERENCES
[1]
D'Orazi G., Sciulli M.G., Di Stefano V., Riccioni S., Frattini M., Falcioni R., Bertario L., Sacchi A.,
Patrignani P., Homeodomain-interacting protein kinase-2 restrains cytosolic phospholipase A2dependent prostaglandin E2 generation in human colorectal cancer cells, Clin Cancer Res 12 (2006)
735-741.
5
[2]
Kazanov D., Dvory-Sobol H., Pick M., Liberman E., Strier L., Choen-Noyman E., Deutsch V.,
Kunik T., Arber N., Celecoxib but not rofecoxib inhibits the growth of transformed cells in vitro,
Clin Cancer Res 10 (2004) 267-271.
[3]
Schiffmann S., Maier T.J., Wobst I., Janssen A., Corban-Wilhelm H., Angioni C., Geisslinger G.,
Grosch S., The anti-proliferative potency of celecoxib is not a class effect of coxibs, Biochem
Pharmacol 76 (2008) 179-187.
[4]
Liu X.H., Rose D.P., Differential expression and regulation of cyclooxygenase-1 and -2 in two
human breast cancer cell lines, Cancer Res 56 (1996) 5125-5127.
[5]
Liu Q., Chan S.T., Mahendran R., Nitric oxide induces cyclooxygenase expression and inhibits cell
growth in colon cancer cell lines, Carcinogenesis 24 (2003) 637-642.
[6]
Chuang H.C., Kardosh A., Gaffney K.J., Petasis N.A., Schonthal A.H., COX-2 inhibition is neither
necessary nor sufficient for celecoxib to suppress tumor cell proliferation and focus formation in
vitro, Mol Cancer 7 (2008) 38.
[7]
Schonthal A.H., Direct non-cyclooxygenase-2 targets of celecoxib and their potential relevance for
cancer therapy, Br J Cancer 97 (2007) 1465-1468.
[8]
Pan L., Ho Q., Tsutsui K., Takahashi L., Comparison of chromatographic and spectroscopic methods
used to rank compounds for aqueous solubility, J Pharm Sci 90 (2001) 521-529.
[9]
Seedher N., Bhatia S., Solubility enhancement of Cox-2 inhibitors using various solvent systems,
AAPS PharmSciTech 4 (2003) E33.
[10]
Zarghi A., Shafaati A., Foroutan S.M., Khoddam A., Simple and rapid high-performance liquid
chromatographic method for determination of celecoxib in plasma using UV detection: application
in pharmacokinetic studies, J Chromatogr B Analyt Technol Biomed Life Sci 835 (2006) 100-104.
[11]
Myers J.T., Swanson J.A., Calcium spikes in activated macrophages during Fcgamma receptormediated phagocytosis, J Leukoc Biol 72 (2002) 677-684.
[12]
Dar A., Goichberg P., Shinder V., Kalinkovich A., Kollet O., Netzer N., Margalit R., Zsak M.,
Nagler A., Hardan I., Resnick I., Rot A., Lapidot T., Chemokine receptor CXCR4-dependent
internalization and resecretion of functional chemokine SDF-1 by bone marrow endothelial and
stromal cells, Nat Immunol 6 (2005) 1038-1046.
6
[13]
Miliukiene V., Biziuleviciene G., Pilinkiene A., Quantitative evaluation of macrophage
phagocytosing capacity by a fluorometric assay, Acta Biol Hung 54 (2003) 347-355.
[14]
Bocca C., Bozzo F., Bassignana A., Miglietta A., Antiproliferative effects of COX-2 inhibitor
celecoxib on human breast cancer cell lines, Mol Cell Biochem 350 (2010) 59-70.
[15]
Tuynman J.B., Vermeulen L., Boon E.M., Kemper K., Zwinderman A.H., Peppelenbosch M.P.,
Richel D.J., Cyclooxygenase-2 inhibition inhibits c-Met kinase activity and Wnt activity in colon
cancer, Cancer Res 68 (2008) 1213-1220.
7