Phosphine metal complexes have been recently evaluated in the field of cancer therapy. In this research, the cytotoxic effects of some metal phosphines
Some anticancer agents act through production of ROS (reactive oxygen species) to kill tumor cells. Reported studies have shown that cells with high levels of antioxidant enzymes are resistant to some anticancer agents [
In the last three decades, metal complexes have been of interest to cancer therapy researchers. The international community has widely recognized that while some ruthenium complexes exhibit low toxicity to normal cells, they are easily absorbed by tumor tissue and rapidly excreted from the body [
Antioxidant enzymes in cancer cells, such as GPx, GR, and especially TrxR, are major targets for recent antitumor drug studies. Several different clinical antitumor agents have been reported to inactivate TrxR. However, the relationship between TrxR inactivation and apoptosis has been less fully reported [
The cytotoxic activities of Ru(II), Pd(II), and Pt(II) phosphine complexes on A549 and K562 cell lines, and the inactivation of the GPx, Cat, and TrxR enzymes of these cells via the metal phosphine complexes have been investigated in this study.
All reactions were carried out under purified nitrogen using standard Schlenk techniques. Solvents were purified by standard methods and distilled under nitrogen prior to use. [PdCl2((CH2OH)2PCH2)2NCH3] (C1), [PtCl2((Ph2PCH2)2NCH3) (timin)2] (C3) was prepared according to the procedure described in the literature [
An aqueous solution (10 mL) of [((CH2OH)2PCH2)2NCH3] (2 mmol) was added dropwise to the ruthenium precursor [Ru(COD)Cl2] (0.95 mmol) in toluene (10 mL) at 40°C with constant stirring. The mixture was further stirred for 48 h, and the aqueous layer was separated from the organic layer. The aqueous solution was concentrated to 5 mL in vacuum and evaporated slowly at room temperature to afford the green colored complex C2 at 78% yield.
Anal. Calcd. for [RuCl2(((CH2OH)2PCH2)2NCH3)2] (C2): C, 25.6%; H, 5.8%; N, 4.3%. Found: C, 27.1%; H, 6.5%; N, 4.09%. {1H}NMR (D2O, 25°C):
In order to examine the anticancer activities of metal phosphine complexes, two different human cancer cell lines were used: a K562 cell line provided by Cukurova University’s Hematology Clinic and an A549 cell line supplied by Gaziantep University’s Cell Culture Laboratory. The cancer cells were maintained in the logarithmic phase at 37°C in a 5% carbon dioxide atmosphere using a culture medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin RPMI-1640 (Sigma) (developed by Roswell Park Memorial Institute).
The growth inhibitory effect towards cancer cell lines was evaluated by means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) assay [
Catalase activity was measured as described by Claiborne [
Cell extracts (200–400 mg) were added to 3 mL of a 10 mM H2O2 in 50 mM potassium phosphate buffer (pH 7.8) and the disappearance of H2O2 (extinction coefficient (
After applying the same sonicating procedure to the cells, each 5
Enzyme activity was determined spectrophotometrically by monitoring the NADPH dependent production of 2-nitro-5-thiobenzoate (
To determine whether the differences between the activities of the enzymes of two different cancer cells were significant depending on the IC50 values of the metal complexes, analysis of variance and then Tukey’s test were used [
Specific activities of TrxR, GPx, and Cat enzymes in the given period, after incubation of A549 and K562 cells, with IC50 values of metal complexes.
A549 | K562 | ||||
---|---|---|---|---|---|
U/mg protein | U/mg protein | ||||
TrxR | Control |
0.5080ax ± (0.00458)y |
TrxR | Control |
1.0640a ± (0.00529) |
|
|||||
GPx | Control |
0.1520a ± (0.00265) |
GPx | Control |
0.2367a ± (0.00577) |
|
|||||
Cat | Control |
854.73a ± (0.00008) |
Cat | Control |
354.9300a ± (3.27432) |
Among groups
N,N-bis (hydroxymethyl phosphinomethyl) aminomethyl [((CH2OH)2PCH2)2NCH3] (dppam) was synthesized by using the reported procedure [
Platin, palladium, and ruthenium complexes of phosphines were used to investigate their cytotoxic activity towards two different cell lines, A549 and K562. Cytotoxicity was evaluated by means of the MTT test after 24, 48, 72, and 120 hours of treatment with increasing concentrations of the aforementioned compounds. The IC50 values which were calculated from dose dependent curves can be seen in Figures
Inhibition of the enzymes CAT, TrxR, and GPx by organometallic and other metal compounds in the treatment of cancer has been widely studied. The metal phosphine complexes C1, C2, and C3 have been studied to examine the inhibition of the above-mentioned enzymes (Figure
Molecular structures of the phosphine metal complexes used.
Synthesis of [RuCl2(((CH2OH)2PCH2)2NCH3)2] (C2) complex.
IC50 values of metal complexes at 48, 72, and 120 h for A549 cells.
IC50 values of metal complexes at 24, 48, and 72 h for K562 cells.
Microscope images (20x) of K562 cells with controls (H2O and DMSO) and IC50 values of metal complexes for 48 and 72 hours. (a) Image of K562 cells at 72 h with distilled water as a control; (b) and (c) images of the cells at 72 h with IC50 values of C1 and C2 complexes, respectively; (d) image of the cells at 48 h, incubated with DMSO as a control of C3 complex; (e) image of the cells at 48 h, incubated with IC50 value of C3.
The enzyme activities of the cells incubated with the IC50 values of metal complexes tended to decrease at 72 h when compared with untreated A549 and K562 cell lines. Table
The TrxR activities of A549 and K562 control cells (untreated with any complex) were found to be 0.508 U/mg protein and 1.064 U/mg protein, respectively, and these activities were accepted as 100% activity. The TrxR activity of the K562 cells was higher than that of the A549 cells. The TrxR activity of the A549 cells treated with C1 (4.625 mM, 72 h) decreased by 6.69%. The TrxR activity of the A549 cells treated with C3 (0.158 mM, 48 h) decreased by 41.73% (Figure
Microscope images (20x) of A549 cells with controls (H2O and DMSO) and IC50 values of metal complexes for 48 and 72 hours. (a) image of A549 cells at 72 h with distilled water as a control; (b) and (c) images of the cells at 72 h with IC50 values of C1 and C2 complexes, respectively; (d) image of the cells at 48 h, incubated with DMSO as a control of C3 complex; (e) image of the cells at 48 h, incubated with IC50 value of C3.
Percentages of inhibition of TrxR, GPx, and Cat enzymes of A549 cells in the given period, after incubation, with IC50 values of C1, C2, and C3 complexes.
Percentages of inhibition of TrxR, GPx, and Cat enzymes of K562 cells in the given period, after incubation, with IC50 values of C1, C2, and C3 complexes.
The GPx activity of A549 cells as the control group was found to be 0.152 U/mg protein. After incubation of the A549 cells with C1 (4.625 mM, 72 h), C2 (7.436 mM, 72 h), and C3 (0.158 mM 48 h), their specific activities were calculated as 0.068 U/mg, 0.107 U/mg, and 0.046 U/mg protein, respectively. C3 had the most effective inhibition (69.74%). C1 reduced the TrxR enzyme activity in A549 cells by 55.26%, and C2 reduced it by 29.62% (Figure
A metal complex has different effects on the same enzymes in two different cells, which indicates that metal complexes are cell selective. In a study with gold phosphine complexes concerning cell selectivity, most of the complexes showed good inhibitory effects (more than 80%) on the TrxR of A549 cells, but percentages were different in the other tested cell lines. In the same study, it was observed that enzymes were inhibited disparately by the metal complexes. While a gold complex inhibited TrxR by more than 80%, it inhibited both GPx and GR (glutathione reductase) enzymes of the same cell (A549) by under 50% [
The catalase activities of both types of cells were found to be at higher levels than those of the other two enzymes’ activities. The catalase activity of healthy human cells is already higher than GPx and TrxR activity. The catalase-specific activities of the A549 and K562 control cells (untreated with any complex) were found to be 854.73 U/mg protein and 354.93 U/mg protein, respectively, and these activities were accepted as 100% activity. Some of the tested compounds showed good catalase inhibitor properties. After incubation of the A549 cells with C1 (4.625 mM, 72 h), C2 (7.436 mM, 72 h), and C3 (0.158 mM, 48 h), Cat was inhibited at 60.39%, 37.94%, and 70.47%, respectively (Figure
The statistical findings of this study showed that while there was no significant difference between C2 and C3 in terms of TrxR inhibition, both differed significantly from the control in the A549 cell line. There was a remarkable difference in TrxR inhibition in the K562 cell line between all three complexes and the control group. Statistically, C1 inhibited the TrxR enzyme of K562 cells far more than C2 did but only slightly more than C3 did (Table
There were significant differences between all three tested compounds and the control group in terms of GPx inhibition in the A549 cell line (Table
The most significant differences of Cat inhibition in A549 cells were between C3 and the control group. The differences between C1 and the control were greater than those found between C3 and the control, while those between C2 and the control were the smallest. There was significant disparity between all three of the compounds tested and the control group with regard to Cat inhibition, with the greatest difference being between C3 and the control and the least between C1 and the control (Table
In this study, three original metal phosphine complexes were found to have variable cytotoxic activities at the mM level in A549 and K562 cells. In addition to the cytotoxic activities, measurement of the activities of the enzymes TrxR, GPx, and Cat was attempted by use of metal complexes as inhibitors in both cells. Phosphines are widely used as a ligand group in the treatment of cancer via the inhibition of antioxidant enzymes [
Since high levels of the enzyme TrxR in many human cancer cell lines prevent anticancer agents from inducing apoptosis [
Most of the electrophilic compounds (like metal complexes) interact selectively and irreversibly with the SH/Se-group at the active site of the enzyme, thus becoming inhibitors of TrxR [
A sequential thiol-exchange mechanism, in which thiolates act as soft ligands forming covalent bonds with the soft metal ions, is suggested to explain the reactivity and cellular distribution of the tested phosphine compounds which were used for inactivation of the TrxR and GPx enzymes in A549 and K562 cells. For instance, Becker at al. suggest that selenocysteine residue is a suitable site for platination in hTrxR and that the mechanism involves a selenolate-thiolate exchange with the ligand of the Pt(II) compounds. This inactivation via metal complexes may also cause an inhibition of DNA synthesis [
Our studies have contributed to understanding of metal phosphines’ new role in cancer cells. The results have indicated that tested metal phosphine compounds were effectivein terms of cell death on K562 and A549 cell lines
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors are grateful to pediatric immunology Professor Dr. Mustafa Yılmaz for sharing his academic experiences. In addition, the authors are thankful to Çukurova University’s Hematology Laboratory for allowing them to use their facilities and Gaziantep University’s Cell Laboratory for providing cell lines.