New Transplatinum Complexes Containing Phosphane Groups
FULL PAPER
cillin (100 units/mL) and streptomycin (100 µg/mL) at 37 °C in an
trophotometry at 260 nm in a Shimadzu UV-240 spectrophoto-
atmosphere of 95% air and 5% CO2. All cultures were passaged meter, and platinum bound to DNA was determined by total
twice weekly, showing a doubling time between 16Ϫ24 hours.
reflection X-ray fluorescence (TXRF). Experiments were carried
out in triplicate.
Cytotoxicity Assays: Cell survival was evaluated by use of a system
based on the tetrazolium compound MTT, which is reduced by
living cells to yield a soluble formazan product that can be detected
colorimetrically.[28] Cells were platted in 96-well sterile plates at a
density of 104 cells/well in 100 µL of medium and were incubated
for 3Ϫ4 h. The compounds were added to final concentrations
from 0 to 200 µM in a volume of 100 µL/well. Twenty-four hours
later, 50 µL of a MTT solution (1/5 in culture medium) freshly
diluted to a concentration of 1 mg/ml were pipetted into each well,
and the plate was incubated for 5 h at 37 °C in a humidified 5%
CO2 atmosphere. After the periods specified, the cell viability was
evaluated by measurement of the absorbance at 520 nm, by use
of a Whittaker Microplate Reader 2001. IC50 values (compound
concentration that produces 50% of cell growth inhibition) were
calculated from curves constructed by plotting (%) cell survival ver-
sus drug concentration. The therapeutic index (T.I.) of every com-
pound was calculated as the ratio of the IC50 in normal cells versus
the IC50 in transformed cells. In control experiments it was ob-
served that 10% acetone did not have any effect on cell growth.
This was the highest percentage of acetone present in the cell cul-
tures after addition of 200 µM of the compounds. All experiments
were made in quadruplicate.
TXRF Measurements: The analysis by TXRF was performed with
a Seifert Extra-II spectrometer (Seifert, Ahrensburg, Germany).
TXRF determinations were carried out by a previously reported
procedure.[29] Briefly, a 100 µL sample of the DNA precipitated
from the solutions of pBR322 incubated with the platinum com-
plexes was introduced into a 2 mL test tube. This solution was
standardised with 100 ng/mL vanadium [Merck (Darmstadt, Ger-
many) ICP vanadium standard solution]. Afterwards, the sample
was introduced into a high-purity nitrogen flow concentrator at a
temperature of 70 °C until the volume had been reduced five times.
An aliquot of 5 µL was then taken, deposited on a clean quartz
reflector and dried on a ceramic plate at a temperature of 50 °C.
The entire process was carried out in a laminate flow chamber
(Model A-100). The samples were analysed by following the X-ray
molybdenum line under working conditions of 50 kV and 20 mA
with a live-time of 1000 s and a dead time of 35%. Spectra were
recorded between 0 and 20 keV. The following 15 elements were
analysed simultaneously: P, S, K, Ca, V, Fe, Cu, Zn, As, Br, Rb,
Sr, Ni, Mn and Pt, in order to obtain a correct deconvolution of
profiles associated with the general spectrum. The Pt line was used
for Pt quantification. The analytical sensitivity of the TXRF meas-
urements was 0.3 to 22.4 ng Pt in a solution volume of 100 µL,
with repeatability between 2 and 8% (the number of determinations
was n ϭ 3).
DNA Fragmentation Assay: Pam212-ras cells (5 ϫ 105 cells/mL)
were platted in 100 mm sterile dishes. The cells were treated with
the compounds for 24 h to a final concentration of the IC50 under
the conditions described above.[28] The fraction of detached cells
was collected by centrifugation of the culture media and washed
twice with PBS. The cell pellet was disrupted with 700 µL of lysis
buffer (150 m Tris, tris(hydroxymethyl)aminomethane, pH 8.0;
100 m NaCl; 100 m EDTA, ethylenediamine tetracetate). The
fraction of non-detached cells was also washed twice with BPS and
lysed by addition of 700 µL of lysis buffer to the plate. Both cell
fractions were joined and the whole cell lysate was treated with
proteinase K (500 µg/mL) for 2 h at 55 °C. Afterwards, samples
were exposed to RNase A (50 µg/mL) for 16 h at 37 °C. The DNA
was first extracted with phenol, followed by phenol/chloroform/
isoamyl alcohol (25:24:1) and a chloroform/isoamyl alcohol (24:1)
phase. Subsequently, the DNA was precipitated overnight at Ϫ20
°C in 2.5 volumes of cold 100% ethanol/150 m potassium acetate.
After centrifugation at 12,000 rpm for 15 min to recover the precip-
itated DNA, the supernatant was discarded and the pellet was
washed with 70% ethanol. Samples were dried on a SAVANT Speed
Vac Concentrator and then resuspended in distilled water. The
Gel Electrophoresis of Drug:pBR322 Complexes: pBR322 DNA ali-
quots (50 µg/mL) were incubated with the platinum compounds in
a buffer solution containing 50 m NaCl, 10 m Tris.HCl (pH 7.4)
and EDTA (0.1 m⁾ at several ri values (0.5 and 0.1). Incubations
were performed in the dark at 37 °C. 20 µL aliquots of the
drug:DNA complexes containing 1 µg of DNA were subjected to
1.5% agarose gel electrophoresis for 16 h at 25 V in TAE buffer
(Tris-acetate 40 m, EDTA 2 m pH 8.0). The DNA was stained
in the same buffer containing ethidium bromide (0.5 µg/mL). The
gels were photographed with a MP-4 Polaroid camera with a 665
Polaroid film and an orange filter.
Acknowledgments
This work was supported by the Spanish CICYT/ SAF 00/0029.
Sponsorship by COST Actions D20/001/00 and D20/003/00 is also
acknowledged. Johnson Matthey PLC is also acknowledged for
their generous gift of K2PtCl4.
DNA concentration was calculated by determining the OD260
.
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Ramos-Lima, Francisco J.
