Use of silver(I) complexes as anticancer agents
10583109 ยท 2020-03-10
Assignee
Inventors
Cpc classification
C07F9/5442
CHEMISTRY; METALLURGY
A61K49/0004
HUMAN NECESSITIES
A61K31/095
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
Abstract
The present invention relates to the use of silver(I) monophosphine complexes as Active Pharmaceutical Ingredients (API's), including anticancer agents, for the treatment, diagnosis and/or prevention of cancer. The present invention also relates to pharmaceutical compositions containing such complexes and further extends to a method of treating or diagnosing a subject/patient suffering from cancer.
Claims
1. A compound having the structure of Complex (5), represented by the following formula: ##STR00006## wherein n is an integer from 1 to 100.
2. A pharmaceutically acceptable salt, polymorph or derivative of the compound of claim 1.
3. A pharmaceutical composition comprising the compound of claim 1.
4. The pharmaceutical composition according to claim 3, wherein the compound is a pharmaceutically acceptable salt, polymorph or derivative thereof.
5. A method for treating or diagnosing cancer, comprising administering to a patient suffering from cancer an effective amount of a compound represented by Complex (5), represented by the following formula: ##STR00007## wherein n is an integer from 1 to 100.
6. The method according to claim 5, wherein the treating comprises selectively inhibiting the activity of cancer cells.
7. The method according to claim 5, wherein the cancer is selected from the group consisting of breast cancer, esophageal cancer, lung cancer, colon cancer, ovarian cancer, leukemia, renal cancer, melanoma cancer, prostate cancer, CNS cancer, carcinoma, lymphoma, blastoma, sarcoma, leukemia, gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, and penile carcinoma.
8. The method according to claim 5, wherein the treating comprises inhibiting metastasis of cancer.
9. The method according to claim 5, wherein the treating comprises reducing cell growth of cancer.
Description
BRIEF DESCRIPTION OF THE FIGURES
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(39) The presently disclosed subject matter will now be described more fully hereinafter with reference to the accompanying Examples, in which representative embodiments are shown. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.
EXAMPLES
A: Silver(I) Thiocyanate Phosphine Complexes of the Invention and the Toxicity Evaluation Thereof on MCF-7 Breast Cancer Cells and SNO-Oesophageal Cancer Cells
1. General
(40) The silver(I) thiocyanate salt, the various phosphine ligands and acetonitrile were purchased from Sigma-Aldrich and used as received. 31P NMR spectra of the silver(I) phosphine complexes were recorded in deuterated chloroform on Bruker AC 300 spectrometer at 121.4 MHz. Melting points were determined on a Mettler Toledo DSC822e device. The fast atomic bombardment (FAB) mass spectra were recorded on a Micromass VG70SEQ instrument at the University of the Witwatersrand, RSA.
(41) For the biological studies, MCF-7 breast cancer cells were obtained from an immortal cell line from human adenocarcinoma from (primary site) mammary gland and (metastatic site) pleural effusion (ATCC no HTB-22).
(42) SNO-oesophageal cancer cells were a gift from the University of Pretoria, RSA. Dulbecco's modified Eagles media (DMEM), Foetal Bovine Serum (FBS), and the antibiotic supplements were all purchased from Highveld Biological (Kelvin, RSA). Cell culture graded dimetyl sulpoxide (DMSO) was obtained from AppliChem (Darmstad, Germany). Cisplatin (CDDP) was purchased from Molekula (Dorset, UK) while hydrogen peroxide (H.sub.2O.sub.2) was purchased from Minema (Gauteng, RSA). AlamarBlue dye and the Annexin-V FITC assay kit were obtained from Serotec (Oxford, UK). All reagents were used as supplied.
2. Synthesis of Complexes
Preparation of Silver(I) Thiocyanate Phosphine Complexes of the Invention
Example 1: [AgSCN(PPh3)2]2 Complex (1)
(43) Solid AgSCN (0.0517 g, 0.31 mmol) was added to the solution of triphenyl phosphine (PPh.sub.3) (0.2481 g, 0.95 mmol) in ethanol (40 cm.sup.3) and DMSO (30 cm.sup.3). The reaction mixture was heated under reflux for 5.5 h at 87 C. The solution was filtered hot and the ethanol was evaporated. The solution was placed in a freezer which resulted in a frozen mixture and upon melting small white crystals were isolated and recrystallized from DCM. Yield: 0.0892 g, 30%; mp 170-173 C. IR: .sub.max/in cm.sup.1: 2058.32 ((SCN), s); 1477.36, 1432.25 ((CC aromatic), s); 1307.63 (w); 1155.06 (w); 1090.38, 1059.97 (asymm, m); 1026.02, 997.43 (m); 744.26 ((aromatic, CH bend), s); 692.39 ((aromatic, CH bend, meta), s). .sup.1H NMR: (400 MHz, CDCl.sub.3) (ppm) 7.31 (m, 45H, H-aromatic). .sup.13C {H} NMR: (100 MHz, CDCl.sub.3) (ppm) 128.80 (d, .sub.1=128.756, .sub.2=128.844, .sup.1J(PC)=6.6 Hz, meta C); 129.81 (para C); 133.77 (d, .sub.1=133.692, .sub.2=133.862, .sup.1J(PC)=12.7 Hz, ipso C). .sup.31P NMR: in CDCl.sub.3 (ppm) 4.58. Elemental Analysis: Anal. Calcd for AgSCN:PPh.sub.3 (1:3): C, 69.33; H, 4.76; N, 1.47; S, 3.37. Found: C, 69.36; H, 5.31; N, 1.48; S, 3.42.
Example 2: [AgSCN{P(4-MeC6H4)3}2]2 Complex (2)
(44) In a 100 ml round bottomed flask, 100 mg of AgSCN (0.603 mmol, 165.95 g/mol) and 0.367 mg (1.206 mmol, 304.37 g/mol) of [P(p-MeC.sub.6H.sub.4).sub.3] were dissolved in 30 ml of pyridine. The reaction mixture was stirred under reflux (115 C.) overnight. After filtration, the pyridine solution was allowed to stand at room temperature and a solid crashed out. The latter was collected and recrystallized in acetonitrile. Yield: 0.314 g; mp (DSC): 181 C.; .sup.1H NMR: (CDCl.sub.3, , 300 MHz): 2.47 (s, 3H); 7.22 (d, 2H); 7.35 (d, 211). .sup.13C NMR: (CDCl.sub.3, , 75.5 MHz): 21.76; 129.00; 129.39; 134.22; 140.69. .sup.31P NMR: (CDCl.sub.3, , 121.4 MHz): 7.48; MS (M/z): 717.3=Ag[P(p-CH.sub.3C.sub.6H.sub.4).sub.3].sub.2; 411.1=AgP(p-CH.sub.3C.sub.6H.sub.4).sub.3 EA: % H: 5.46 (Calculated); 5.29 (Found); % N: 1.81 (calculated); 1.55 (Found); % C: 62.70 (Calculated); 63.40 (Found).
Example 3: [AgSCN{P(4-FC6H4)3}2]2 Complex (3)
(45) To a suspension of AgSCN (1 mmol) in acetonitrile (10 cm.sup.3) was added a solution of P(4-FC.sub.6H.sub.4).sub.3 (1-4 mmol) in acetonitrile (20 cm.sup.3). The mixture was heated under reflux until all solids dissolved. The mixture was cooled to room-temperature and the product allowed to crystallize out. The solids were isolated by filtration and dried in vacuo to give the respective product. mp 210-215 C.; Solid IR ( in cm.sup.1): 3029 ((CH), w); 2360 ((CH), w); 2081 ((SCN), m); 1586, 1558, 1541 ((CC aromatic), asymm, s); 1492 (asymm, s) 1394 ((CC aromatic), m); 1299, 1273, 1224, 1158 ((OCH.sub.3), s); 1093 (s), 1043 (m) 1012 (s); 824 ((aromatic, CH para), asymm, s); 707 ((aromatic, CH mono), asymm, s). .sup.1H NMR: (400 MHz, CDCl.sub.3): (ppm) 6.940 (t); 7.280 (t). .sup.31P NMR: (161 MHz, CDCl.sub.3): (ppm) 2.82. .sup.13C{H} NMR: (100 MHz, CDCl.sub.3): (ppm) 116.36 (q); 128.22 (q); 135.73 (q); 169.05 (d).
Example 4: [AgSCN{P(4-ClC6H4)3}2]2 Complex (4)
(46) A 0.025 g of AgSCN was weighed and added to a solution of 100 mg g, 0.0003M tris parachlorophenyl phosphine ligand [P(p-ClC.sub.6H.sub.4).sub.3] in 40 ml acetonitrile. The solution was then refluxed for 1 hour, hot filtered on a whatman 3 filter paper. The solution was evaporated on a hot plate until the crystals started precipitating out. The complex was then allowed to stand for 24 hrs and the concentrated solvent decanted off. The sample was washed with two portions of fresh 2 ml acetonitrile solution. The product was obtained by drying under high vacuum and the product characterized as follows. IR: 3055w, 2075s(SCN), 1574m, 1560m, 1477s, 1385m, 1298w, 1180w, 1079s, 1011s, 813s, 744s, 704w, 630w. .sup.1H NMR: (400 MHz, CDCl.sub.3) =7.53 (s), 7.46-7.42, 7.28-7.21, 7.06-7.01. .sup.13C NMR: (101 MHz, CDCl.sub.3) (ppm) 137.01 (s.sub.1 d.sub.4) 134.85 (d.sub.1, J=0.18 Hz) 130.85. .sup.31P NMR: (162 MHz, CDCl.sub.3) (PPM) 2.45 (S). (d.sub.2, J=0.189 Hz) 129.36 (d.sub.3, J=0.097 Hz). Elemental Analysis: C(49.53) H(2.70) N(1.556) S(3.57) %. found C(49.42), H(2.65) N(1.75).
Example 5: [AgSCN{P(4-MeOC6H4)3}2]n Complex (5)
(47) In a 100 ml round bottomed flask, 100 mg of AgSCN (0.603 mmol, 165.95 g/mol) and 0.4250 mg (1.206 mmol, 352.37 g/mol) of [P(p-MeOC.sub.6H.sub.4).sub.3] were dissolved in 30 ml of pyridine. The reaction mixture was stirred under reflux (115 C.) overnight. After filtration, the pyridine solution was allowed to stand at room temperature and a solid crashed out. The latter was collected and recrystallized in acetonitrile. Yield: 0.350 g; mp (DSC): 183 C.; .sup.1H NMR: (CDCl.sub.3, , 300 MHz): 3.90 (s, 3H); 6.93 (d, 2H); 7.40 (d, 2H). .sup.13C NMR: (CDCl.sub.3, , 75.5 MHz): 55.62; 114.80; 114.94; 135.72; 161.50. .sup.31P NMR: (CDCl.sub.3, , 121.4 MHz): 5.63; MS (M/z): 813.2=Ag[P(p-OCH.sub.3C.sub.6H.sub.4).sub.3].sub.2; 459.1=AgP(p-OCH.sub.3C.sub.6H.sub.4).sub.3; EA: % H: 4.86 (Calculated); 5.013 (Found); % N: 1.60 (Calculated); 1.40 (Found); % C: 59.36 (Calculated); 61.10 (Found).
(48) For ease of reference, the chemical structures of complexes (1)-(5) are provided herein below:
(49) ##STR00001## ##STR00002##
3. Cell Culturing and Treatment
(50) For the initial dose-responsive studies, complexes (1)-(5) were selected together with two cancer cell lines, namely, the MCF-7 breast cancer cells (as set forth in Item 4.1 below) and the SNO-oesophageal cancer cells (as set forth in Item 4.2 below).
(51) MCF-7 breast cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM), 13.53 g/L with sodium bicarbonate (NaHCO.sub.3), 3.7 g/L supplemented with 10% foetal bovine serum (FBS), 1% gentamycin and 5% Penicillin/Streptomycin/Fungizone in 75 cm.sup.3 culture flasks. Cells were subcultured twice a week and incubated at 37 C. with 5% CO.sub.2 in a humidified atmosphere. Hanks balanced salt solution (HBSS), supplemented DMEM media and trypsin versene were warmed to 37 C. prior to subculturing. Cells were washed twice with 10 ml Hanks balanced salt solution for 1 minute and were then incubated for 4.5 to 5 minutes at 37 C. with 6 ml trypsin versene to remove the cells from the culture flask. To inactivate the trypsin versene, 10 ml supplemented DMEM was added and the cells were pelleted by centrifugation at 1028 g for 4 minutes with an Allegra 25R Centrifuge. The pellet was resuspended in 1 ml supplemented DMEM and 500 l of the cell suspension was then added to 25 ml of supplemented DMEM media in 75 cm.sup.3 culture flasks.
(52) The SNO-oesophageal cancer cells were cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% Foetal bovine serum (FBS), 0.8% Penicillin/Streptomycin/Fungizone and 0.2% Gentamycin. The SNO-oesophageal cancer cells were subcultured every 48 hours and incubated at 37 C. under a 5% CO.sub.2 humidified atmosphere. After 48 hours the cells were trypsinized and plated (610.sup.5 cells) in 3.5 cm culture dishes and left to cultivate for 24 hours.
(53) MCF-7 breast cancer cells, in terms of a first study, and SNO-oesophageal cancer cells, in terms of a second study, were treated with 10 M silverthiocyanide complexes (1)-(5), as well as their ligands (Ligand-1 to Ligand-5) over a 24 hour incubation period. These complexes were dissolved in 0.1% Dimethyl sulfoxide (DMSO). The DMSO alone (vehicle control) decreased the viability with approximately 2.2%, signifying its minimal influence in cancer cell growth. For comparative purposes, 100 M Cisplatin (CDDP) and 25% H.sub.2O.sub.2 was included which respectively serves as the apoptotic and necrotic controls. Cisplatin was prepared in 0.9% NaCl, whereas H.sub.2O.sub.2 was prepared in the supplemented DMEM media right before treatment. An untreated negative control (UT) was exposed to similar conditions as the treated cells and monitored during the experimental study.
(54) Several assays were used to evaluate the effect of complexes (1)-(5) on cell death with respect to the MCF-7 breast cancer cells and the SNO-oesophageal cancer cells. These included cell viability assays such as the AlamarBlue viability and proliferation assay and the Trypan blue exclusion assay, microscopy to evaluate cell morphology and flow cytometric analysis of apoptosis and/or necrosis.
4. Cell Viability and Morphological Studies
(55) 4.1 MCF-7 Breast Cancer Cell Assays
(56) The AlamarBlue Viability and Proliferation Assay of MCF-7 Breast Cancer Cells
(57) The AlamarBlue viability and proliferation assay of complexes (1)-(5) for the MCF-7 breast cancer cells is depicted in
(58) The dose dependant studies of complex (1) are shown in
(59)
(60) The Trypan Blue Exclusion Assay of MCF-7 Breast Cancer Cells
(61) The Trypan blue exclusion assay of complexes (1)-(5) for the MCF-7 breast cancer cells is depicted in
(62) The percentage viability of MCF-7 cells determined by the Trypan blue dye exclusion assay is represented in
(63) The dose dependant studies of complex (1) are depicted in
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(65) In addition hereto, the percentage viability of differentially treated MCF-7 cells as determined by the AlamarBlue viability and proliferation assay is depicted in
(66) Morphological Features of the MCF-7 Breast Cancer Cells Following Differential Treatments as Evaluated by Inverse Light Microscopy
(67) The morphological features of MCF-7 cells following differential treatments were evaluated by inverse light microscopy as depicted in
(68) Flow Cytometric Analysis of Apoptosis and/or Necrosis for MCF-7 Breast Cancer Cells
(69) Flow cytometric analysis was conducted in order to determine PS externalization and membrane integrity in differentially treated MCF-7 cells (
(70) Pie charts representing flow cytometric analysis to determine PS externalization and membrane integrity in differentially treated MCF-7 cells are depicted in
(71)
(72) Discussion
(73) The effects of the silver complexes of the present invention on the viability and proliferation of MCF-7 breast cancer cells were studied to determine if the complexes have cell death inducing capabilities. Cells treated with 10 M of complex (1) or (3) showed more than a 90% decrease in viability and proliferation 24 hours after treatment when compared to the untreated control (
(74) The values obtained show strong cytotoxic activity, with complex (1), complex (3) and complex (4) being the strongest. Silver(I) bidentate pyridyl phosphine complexes have also shown in vitro cytotoxicity that strongly depends upon their lipophilicity.
(75) In the light of the decreased viability, the morphology of the cells was studied 24 hours after treatment with 10 M of complexes (1), (2), (3), (4) and (5) to determine if apoptotic or necrotic features were present. The morphology of the cells was compared to the apoptotic control treated with 100 M Cisplatin (
(76) The morphology of the cells treated with the respective silver(I) complexes (1)-(5) reveal features comparable to the cells treated with the apoptotic inducer. Shrinkage of the cells, chromatin condensation as well as blebbing and formation of apoptotic bodies indicative of apoptosis were observed (
(77) 4.2 SNO-Oesophageal Cancer Cell Assays
(78) The AlamarBlue Viability and Proliferation Assay of SNO-Oesophageal Cancer Cells
(79) An AlamarBlue Viability (Serotec, UK) assay was performed after a 24 hour treatment with complexes (1)-(5) and their ligands (Ligand-1 to Ligand-5, respectively) to evaluate the cellular effect the instant complexes (and their ligands) may have on the cancer cells. AlamarBlue dye (10%) was incubated with trypsinized cells over a 2 hour period before the fluorescence was measured with a Synergy HT Multi-Detection Microplate reader (BioTek, Winooski, Vt.) at wavelengths of 530 nm and (excitation) and 590 nm (emission). This fluorescent based assay in particular involves the metabolic oxidation-reduction reaction of resazurin to a reduced resorufin product that is directly proportional to the amount of viable cells.
(80) The percentage cellular viability of differentially treated SNO-oesophageal cells was determined using an AlamarBlue assay, as depicted in
(81) Morphological Features of the SNO-Oesophageal Cancer Cells Following Differential Treatments as Evaluated by Inverse Light Microscopy
(82) Morphological studies were done by means of an Axiovert 25 inverted microscope (Carl Zeiss, Gttingen, Germany) with Axio Vision 3.1 software (Carl Zeiss, Gttingen, Germany) using an magnification of 100.
(83) After 24 hours of treatment with complexes (1)-(5) including their ligands the cells were examined under said Axiovert 25 inverted light microscope. This was done to determine how the complexes of the present invention influence morphology which was compared to 100 M Cisplatin (apoptotic control) and 15% H.sub.2O.sub.2 (necrotic control). These results would indicate whether apoptosis or necrosis was induced in the cells by complexes (1)-(5) of the present invention.
(84) Light microscope images indicating the morphology of the differentially treated SNO-oesophageal cancer cells is shown in
(85) Flow Cytometric Analysis of Apoptosis and/or Necrosis for SNO-Oesophageal Cancer Cells
(86) After the 24 hour treatment period the cells were analysed by means of flow cytometry to confirm if the cells had undergone apoptosis or necrosis.
(87) To determine if either apoptosis or necrosis occurred, the cells were double labelled with Annexin-V FITC and Propidium Iodide (PI) by means of an Annexin-V FITC assay kit (Serotec, UK). This was done according to manufacturer's instructions with a few adjustments. Briefly, the cells (310.sup.5 cells/ml) were washed twice with cold phosphate buffered saline (PBS), followed by the addition of 100 l 1 binding buffer. Two and a half microliters of Annexin-V along with 5 l PI were added in the dark and was incubated for 15 minutes at room temperature. After incubation, 400 l 1 binding buffer was added and cells were analysed using the FACSAria flow cytometer (BD Biosciences, San Jose, Calif.) with FACSDiva software (BD Biosciences, San Jose, Calif.) 492 nm (excitation) and 520 nm for Annexin and 488 nm and 575 nm(emission) for PI.
(88) The cell membrane contains a negatively charged phosphatidylserine (PS) on the inner leaflet and is exposed when apoptosis or necrosis takes place. Annexin-V interacts with PS in both apoptotic and necrotic cells, making these cellular deaths undistinguishable. This is overcome by using PI as an alternative label that interacts with the exposed DNA of necrotic cells only, leading to the fluorescent detection of both these labels. Pie charts (
(89) Pie charts representing the four different flow cytometric quadrants of Annexin-V and FITC to determine the mode of cellular death induced in differentially treated SNO-oesophageal cells are represented in
(90) Statistical Analysis
(91) Data represented were expressed as the Standard Error of Mean (SEM) where at least 3 biological and 3 technical repeats were used. The Student's T-test was used to calculate the significant difference (*P<0.05 and **P<0.001) of the treatments with respect to the untreated control except in the flow cytometric data.
(92) Discussion
(93) The effects of the silver(I) complexes (1)-(5) on the viability and proliferation of SNO-oesophageal cancer cells were studied to determine if the compounds have cell death inducing capabilities.
(94) The various 10 M silver(I) thiocyanate phosphine complexes (1)-(5) significantly (P<0.0001) decreased the percentage cellular viability when compared to the control. Complexes (3) and (5) appear to be highly toxic to the SNO-oesophageal cells followed by complexes (4), (2) and (1). The ligands of complexes (1)-(5) where included to evaluate their effects on the SNO-oesophageal cells in the absence of AgSCN and to determine if the same phenomena occurred as when the AgSCN main backbone is present.
(95) Surprisingly the cells' viability ranged from 95-79% indicating the ligands low toxicity. Although the ligands of complexes (2) and (5) significantly decreased (P<0.001) the viability to 86.4% and 80.7% respectively it seems that the whole silver(I) thiocyanate phosphine complex (attached to its ligand) is required to induce cancer cell death. This is contradictory to Baguley et al. (2008) findings where the 2-pyridyl and 4-pyridyl ligands had similar toxicities in a range of ovarian cancer cells, than the entire silver or gold bidentate phosphine compounds. Complexes (1)-(5) and their ligands where dissolved in 0.1% DMSO that decreased the percentage viability with approximately 2.2% signifying its minimal influence in cancer cell growth.
(96) As can be observed from the light microscope images indicating the morphology of the differentially treated SNO-oesophageal cancer cells (
(97) As shown by the flow cytomtric analysis depicted in
5. The Toxicity Effects of Complexes (1)-(5) on a Non-Cancerous Cell Line
(98) To evaluate the relative toxicity on non-tumorigenic tissues, the ideal is to compare the effect of the compounds to a similar cell line that is non-cancerous. The Applicant is currently in the process of purchasing these cell lines and will evaluate the toxicity of the compounds in the near future. However, the Applicant did evaluate the effect of the complexes of the instant invention on peripheral blood mononucleocytes (PBMCs) isolated from fresh whole blood samples from a healthy donor. This already gives a good indication of the potential toxicity of complexes (1)-(5) in an in vivo environment.
(99) The percentage viability of differentially treated PBMCs (peripheral blood mononucleocytes) for complexes (1)-(5) was determined by the Trypan blue viability assay, as shown in
6. Conclusion
(100) The Applicant has found that both the MCF-7 breast cancer cells and SNO-oesophageal cancer cells that were treated with the silver(I) thiocyanate phosphine complexes (1)-(5) of the present invention revealed a significant degree of induction of apoptosis, and to a certain extent, necrosis. When comparing the relative dosages used in comparison to the known chemotherapeutic drug, Cisplatin, the effective dosages inducing cell death are 10 times more effective. The PBMCs, used as a control non-tumorigenic reference, showed a lesser degree of induced cell death, and in the case of complex (3), a significantly lower degree of toxicity.
B: Silver(I) Nitrate Triphenylphosphine Complexes of the Invention and the Toxicity Evaluation Thereof on SNO-Oesophageal Cancer Cells
1. General
(101) Silver nitrate, triphenylphosphine, acetonitrile and ethanol, were obtained from Sigma-Aldrich. Infrared spectra were recorded on a Bruker Tensor 27 FT-IR spectrophotometer, using a Pike Golden Gate ATR accessory. .sup.1H NMR (400 MHz) and .sup.13C NMR (75 MHz) spectra were recorded on a BrukerAvance III 400 MHz spectrometer, using tetramethylsilane as an internal standard. Melting points were recorded on a Stuart Scientific Melting Point apparatus SMP10, and are uncorrected.
(102) For the biological studies, SNO-oesophageal cancer cells were gifted by the University of Pretoria, RSA. Dulbecco's modified Eagles media (DMEM), Foetal Bovine Serum (FBS), antibiotic supplements were all purchased from Highveld Biological (Kelvin, RSA). Cell culture graded dimetyl sulpoxide (DMSO) were obtained from AppliChem (Darmstad, Germany). Cisplatin (CDDP) was purchased from Molekula (Dorset, UK) while hydrogen peroxide (H.sub.2O.sub.2) was purchased from Minema (Gauteng, RSA). AlamarBlue dye and the Annexin-V FITC assay kit were obtained from Serotec (Oxford, UK). The CaspaseGlo 3/7 assay was purchased Promega. All reagents were used as supplied.
2. Synthesis of Complexes
(103) Preparation of Silver(I) Nitrate Triphenylphosphine Complexes of the Invention
(104) Silver(I) nitrate triphenylphosphine complexes, referred to herein as complexes (6), (7), (8), (9), were prepared according to literature procedures. Microanalysis was performed by Mr. K. S. Mothwa in the Department of Chemical Technology, University of Johannesburg, RSA on a Thermo Flash 2000 series CHNS/O, Organic Elemental Analyser.
Example 1: AgNO3(PPh3) Complex (6)
(105) AgNO.sub.3 (0.60 g, 3.5 mmol) was added to a solution of PPh.sub.3 (0.93 g, 3.5 mmol) in acetonitrile (50 cm.sup.3). The mixture was heated under reflux until all the reagents dissolved. The solution was filtered while hot, and the solvent was reduced to 20 cm.sup.3. The solution was allowed to cool to room temperature, after which colorless crystals were obtained. Yield: 61%; mp 174 C.; IR: .sub.max/cm.sup.1: 1375, 1302, 1042, 812. .sup.1H NMR: (400 MHz, CDCl.sub.3): (ppm) 7.53 (t, .sub.1=7.542, .sub.2=7.525, .sub.3=7.511 .sup.1J=6.2 Hz), 7.43 (t, .sub.1=7.457, .sub.2=7.429, .sub.3=7.411 .sup.1J=9.2 Hz). .sup.13C{H} NMR: (100 MHz, CDCl.sub.3): (ppm) 133.4 (d, .sub.1=133.53, .sub.2=133.36, .sup.1J(CP)=12.7 Hz), 131.38, 131.0 (d, .sub.1=131.06, .sub.2=131.01, .sup.1J=3.8 Hz), 129.3 (d, .sub.1=129.37, .sub.2=129.27, .sup.1J=7.5 Hz). .sup.31P{H} NMR: (161 MHz, CDCl.sub.3): (ppm) 11.1 (d, .sub.1=13.24, .sub.2=9.03, .sup.1J(PAg)=677.81 Hz). Elemental Analysis: (Anal. Calcd. For C.sub.18H.sub.15PAgNO.sub.3: C, 50.03; H, 3.50; N, 3.24. Found: C, 50.12; H, 3.47; N, 2.48).
Example 2: AgNO3(PPh3)2 Complex (7)
(106) AgNO.sub.3 (0.30 g, 1.7 mmol) was added to a solution of PPh.sub.3 (0.93 g, 3.5 mmol) in acetonitrile (50 cm.sup.3). The mixture was heated under reflux until all the reagents dissolved. The solution was filtered while hot, and the solvent was reduced to 20 cm.sup.3. The solution was allowed to cool to room temperature, after which colourless crystals were obtained. Yield: 54%; mp 220 C.; IR: .sub.max/cm.sup.1: 1397, 1291, 1029, 817. .sup.1H NMR: (400 MHz, CDCl.sub.3): (ppm) 7.52 (t, .sub.1=7.539, .sub.2=7.521, .sub.3=7.503 .sup.1J=7.2 Hz), 7.43 (t, .sub.1=7.448, .sub.2=7.430, .sub.3=7.411 .sup.1J=7.4 Hz), 7.35 (t, .sub.1=7.369, .sub.2=7.345, .sub.3=7.325 .sup.1J=8.8 Hz). .sup.13C{H} NMR: (100 MHz, CDCl.sub.3): (ppm) 133.5 (d, .sub.1=133.59, .sub.2=133.43, .sup.1J(CP)=12.0 Hz), 131.55, 131.27, 130.96, 129.3 (d, .sub.1=129.34, .sub.2=129.26, .sup.1J=6.0 Hz). .sup.31P{H} NMR: (161 MHz, CDCl.sub.3): (ppm) 8.9. Elemental Analysis: (Anal. Calcd. For C.sub.36H.sub.30P.sub.2AgNO.sub.3: C, 62.26; H, 4.35; N, 2.02. Found: C, 62.22; H, 4.35; N, 1.20).
Example 3: AgNO3(PPh3)3 Complex (8)
(107) AgNO.sub.3 (0.20 g, 1.17 mmol) was added to a solution of PPh.sub.3 (0.93 g, 3.5 mmol) in acetonitrile (50 cm.sup.3). The mixture was heated under reflux until all the reagents dissolved. The solution was filtered while hot, and the solvent was concentrated to 20 cm.sup.3. The solution was allowed to cool to room temperature, after which colourless crystals were obtained. Yield: 72%; mp 233 C.; IR: .sub.max/cm.sup.1: 1382, 1308, 1027, 824. .sup.1H NMR: (400 MHz, CDCl.sub.3): (ppm) 7.45 (t, .sub.1=7.472, .sub.2=7.454, .sub.3=7.436 .sup.1J=7.2 Hz), 7.30 (t, .sub.1=7.318, .sub.2=7.300, .sub.3=7.281 .sup.1J=7.4 Hz), 7.17 (t, .sub.1=7.192, .sub.2=7.168, .sub.3=7.147 .sup.1J=9 Hz). .sup.13C{H} NMR: (100 MHz, CDCl.sub.3): (ppm) 133.3 (d, .sub.1=133.36, .sub.2=133.20, .sup.1J(CP)=12.0 Hz), 132.18, 131.98, 130.47, 129.0 (d, .sub.1=129.07, .sub.2=128.98, .sup.1J=6.8 Hz). .sup.31P{H} NMR: (161 MHz, CDCl.sub.3): (ppm) 6.41. Elemental Analysis: (Anal. Calcd. For C.sub.54H.sub.45P.sub.3AgNO.sub.3: C, 67.79; H, 4.74; N, 1.46. Found: C, 67.83; H, 4.73; N, 0.68).
Example 4: AgNO3(PPh3)4 Complex (9)
(108) A solution of AgNO.sub.3 (0.25 g, 1.5 mmol) in acetonitrile (5 cm.sup.3) was added to a solution of PPh.sub.3 (1.93 g, 7.5 mmol) in ethanol (50 cm.sup.3). The mixture was heated under reflux for 24 hours. The solution was filtered while hot, and the solvent was concentrated to 20 cm.sup.3. The solution was allowed to cool to room temperature, after which colourless crystals were obtained. Yield: 73%; mp 225 C. IR: .sub.max/cm.sup.1: 1338, 829. .sup.1H NMR: (400 MHz, CDCl.sub.3): (ppm) 7.43 (t, .sub.1=7.445, .sub.2=7.426, .sub.3=7.408 .sup.1J=7.4 Hz), 7.27 (t, .sub.1=7.292, .sub.2=7.274, .sub.3=7.255 .sup.1J=7.4 Hz), 7.13 (t, .sub.1=7.149, .sub.2=7.127, .sub.3=7.106 .sup.1J=8.6 Hz). .sup.13C{H} NMR: (100 MHz, CDCl.sub.3): (ppm) 133.2 (d, .sub.1=133.31, .sub.2=132.82, .sup.1J(CP)=12.8 Hz), 132.96, 132.82, 130.16, 128.9 (d, .sub.1=128.98, .sub.2=128.89, .sup.1J=6.8 Hz). .sup.31P{H} NMR: (161 MHz, CDCl.sub.3): (ppm): 3.9 (s) Elemental Analysis: (Anal. Calcd. For C.sub.72H.sub.60P.sub.4AgNO.sub.3: C, 70.94; H, 4.96; N, 1.15. Found: C, 70.41; H, 4.81; N, 0.00) For ease of reference, the chemical structures of complexes (6)-(9) are provided herein below.
(109) ##STR00003##
(110) TABLE-US-00001 TABLE 1 Infrared bands (cm.sup.1) for nitrate vibrational modes for complexes (6)-(9) where s = strong, m = medium and w = weak Assignment (6) (7) (8) (9) Asym NO str. (.sub.1) 1375 s 1397 s 1382 s 1338 s 1307 s 1291 s 1308 s Sym NO str. (.sub.3) 1042 w 1029 m 1027 m Out-of-plane def. (.sub.2) 812 m 817 m 824 m 829 w
3. Cell Culturing and Treatment
(111) The SNO-oesophageal cancer cells were cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% Foetal bovine serum (FBS), 0.8% Penicillin/Streptomycin/Fungizone and 0.2% Gentamycin. The SNO-oesophageal cancer cells were subcultured every 48 hours and incubated at 37 C. under a 5% CO.sub.2 humidified atmosphere. After 48 hours the cells were trypsinized and plated (610.sup.5 cells) in 3.5 cm culture dishes and left to cultivate for 24 hours.
(112) The cells were thereafter treated with complexes (6)-(9) at a concentration of 10 M prepared in 0.1% DMSO. Treatment with either 25% H.sub.2O.sub.2 or 100 M CDDP served as controls for necrosis and apoptosis respectively. The CDDP was prepared in 0.9% NaCl while the H.sub.2O.sub.2 was diluted with supplemented DMEM prior to treatment.
(113) Several assays were used to evaluate the effect of complexes (6)-(9) on cell death with respect to the SNO-oesophageal cancer cells. These included cell viability assays such as the AlamarBlue viability and proliferation assay, microscopy to evaluate cell morphology and flow cytometric analysis of apoptosis and/or necrosis.
4. Cell Viability Determination and Morphological Studies
(114) 4.1 SNO-Oesophageal Cancer Cells Assays
(115) The AlamarBlue Viability and Proliferation Assay of SNO-Oesophageal Cancer Cells
(116) To determine whether complexes (6) to (9) exhibit toxic properties in SNO-oesophageal cancer cells, an AlamarBlue fluorescent based assay was prepared (
(117) Morphological Features of the SNO-Oesophageal Cancer Cells Following Differential Treatments as Evaluated by Inverse Light Microscopy
(118) An Axiovert 25 inverted microscope was used to examine if these complexes affect the morphological features of the malignant cells in any other way. This was done at a magnification of 100 with the use of Axio Version 3.1 software (Carl Zeiss, Gottingen, Germany).
(119)
(120) Flow Cytometric Analysis of Apoptosis and/or Necrosis for SNO-Oesophageal Cancer Cells
(121) Approximately 310.sup.5 cells were washed with ice cold phosphate buffered saline solution (PBS). The cells were double labeled using an Annexin-V FITC assay kit adjusting the protocol. A 1 binding buffer (100 l) was used to fix the cells. Annexin-V (2.5 l) and Propidium Iodide (PI5 l) labels were added respectively followed by a 15 minute incubation, at room temperature, in the dark. After the incubation period, 400 l of the 1 binding buffer was added, where counting of the cellular events was made using an FACSAria flow cytometer (BD Biosciences, San Jose, Calif.) with FACSDiva software (BD Biosciences, San Jose, Calif.). This technique in particular identifies whether predicted apoptotic cell death (or necrosis) was induced by these complexes.
(122) As observed in
(123) Apoptotic Pathway Investigation by Means of Caspase 3/7 Assay for SNO-Oesophageal Cancer Cells
(124) The Caspase-Glo3/7 assay was used to confirm that the structural changes observed are due to apoptotic cell death. In this luminescent based assay, a 1:1 ratio of the Caspase-Glo reagent was added to treated cells into a microtiter plate. The plate was gently agitated for 30 seconds and incubated for 30 minutes at room temperature. The whole luminescent intensity was measured using a Synergy HT Multi-Detection Microplate reader, where the intensity of the luminescence is proportional to the Caspase-3/7 concentration.
(125)
(126) Statistical Analysis
(127) The data obtained was analyzed using Microsoft Excel, using the Student's T-test. All data is presented as the standard error of mean (SEM), represented by the error bars and the p-value being significant at <0.05* and <0.01**, where n represents the number of biological repeats.
(128) Chemical Analysis Discussion
(129) The adducts of silver(I) nitrate and triphenylphosphine in a 1:1-1:4 ratio (complexes (6)-(9)) were prepared according to literature procedures. All complexes were spectroscopically characterized by IR, .sup.1H, .sup.13C and .sup.31P-NMR, and this data is in accordance with literature. In addition, microanalytical data provided proof of purity of the bulk sample. Since silver(I) nitrate phosphine complexes show a rich structural diversity, the identity of the prepared complexes was established by determining the unit cell for all complexes and comparing these with the literature values. All samples appeared to be identical to those reported previously.
(130) Complex (6) is reported to be a polymeric compound, and complex (7) and (8) are coordinated complexes where the nitrate is found within the coordination sphere of the silver atom. Despite this solid-state behaviour, it was assumed that all complexes form separate cation-anion pairs in solution. A low-temperature re-determination of complex (7) confirms the nature of this complex as well as its coordination mode. The molecular diagram is presented in
(131) Biological Studies Discussion
(132) Toxicity Profiles of Compounds
(133) The silver(I) nitrate phosphine complexes (6)-(9) appeared to be highly toxic when compared to the vehicle control. The viability decreased especially when the cells were exposed to complexes (6)-(8), representing a ligand ratio of 1:1, 1:2 and 1:3 respectively. The degree of toxicity seems to increase as the ligand to metal ratio increases, but complex (9) on the other hand, with a ligand ratio of 1:4 only induced 31.25% cancer cell death. There is currently no supporting literature and data on these observations based on the ratio, but does however confirm structure-activity relations in coordinated chemistry and toxicology (Reedijk, 2003).
(134) Morphological Analysis
(135) The morphological analysis by light microscopy gives insight to the structural changes including indication of the targeted cell death pathways like apoptosis or necrosis. As discussed herein before, apoptosis occurs when cellular damage has exceeded the capacity of repair (McCabe and Dlamini, 2005) leading to rapid condensation and budding of cells also known as apoptotic bodies. Necrosis results in cellular swelling, organelle dysfunction, mitochondrial collapse and disintegration (Kerr et al., 1994). Both an apoptotic (100 M CDDP) and a necrotic (25% H.sub.2O.sub.2) control were included with complexes (6)-(9) (
(136) From
(137) Determination of the Mode of Cell Death
(138) In apoptotic cells, the phospholipid phosphatidylserene (PS) is translocated from the inner to the outer layers of the plasma membrane exposing PS to the external environment (Elmore, 2007). Annexin-V is a calcium dependent phospholipid-binding protein, having a high affinity for PS and can be conjugated with FITC flourochromes. It interacts with the PS of both apoptotic and necrotic cells making them undistinguishable. Propidium Iodide (PI) is an alternative flourochrome which only binds to the PS of necrotic cells and acts as the discriminating label. In this case, an Annexin-V/PI assay was done to confirm the observations in
(139) Flow cytometry showed that complexes (6)-(8) induced 20%, 30.6% and 24% apoptotic cell death respectively. These cellular events were observed in the early (Q4) and late Quadrants (Q2) with minimal or no necrotic cell death in (Q1). A large majority of the cancer cells where still viable and intact. Complex (9) had minimal cell death although 15.3% of the cells underwent apoptosis. The 1% DMSO (dot blot not included) had no affect on the cancer cells and 97.95% were still viable which was located in (Q3).
(140) Confirmation of Apoptotic Cell Death by Monitoring Caspase-3 Cleavage
(141) CaspaseGlo 3/7 is an apoptotic assay and was utilized based on the principle that activated Caspases in apoptotic cells cleave the synthetic substances to release free chromophores measured spectrophotometrically. Other metals in cancer research, such as Iron and Copper have also been shown to activate Caspase-3, -6, and -7 as initiator Caspases (Jimenez and Velez-Pardo, 2004). Caspase-3/7 assay was utilised as confirmation of apoptosis.
(142) Caspase induction was observed for the positive apoptotic control Cisplatin (2 fold increase), which has been shown to induce Caspase-3, -8 and -9 dependant apoptosis (Sadler and Guo, 1998). The levels of Caspase-3/7 increased with most treatments. Complex (9) had a low cytotoxicity (
(143) Complex (9) in particular could target other factors in the malignant cells which results in Caspase cleavage via an alternative mechanism. This therefore shows that apoptosis is induced and is Caspase dependent, however further studies are to be made in differentiating between the extrinsic and intrinsic pathways of apoptosis. Silver nanoparticles induce apoptosis via a mitochondria- and Caspase-dependent pathway. They decrease Bcl-2 expression and increase Bax expression in a time dependent manner. The change in the Bax/Bcl-2 ratio leads to the release cytochrome C from the mitochondria into the cytosol. These nanoparticles activate Caspase-9 and -3 in a time-dependent manner from 6 hours to 48 hours respectively (Piao et al., 2011). Silver induced mitochondrial membrane damage has shown to result in the induction of programmed cell death, resulting in decreased ATP production (Teodoro et al., 2011). It has also been shown that silver compounds enter fibroblasts and hepatocytes and cause DNA damage leading to the induction of apoptosis (Piao et al., 2010).
5. Conclusion
(144) A number of silver(I) nitrate triphenylphosphine complexes were synthesised and their in vitro cytotoxicity has been assessed against SNO-oesophageal cancer cells. It was found that these complexes induce apoptosis as the mode of cell death based on microscopy as well as dye exclusion experiments. The cytotoxicity profile of the silver(I) complexes were found to be superior as compared to Cisplatin. The degree of apoptotic cell death has been observed to be dependent on the ratio of ligands bound to the metal. Flow cytometry experiments and Caspase analysis confirmed that the mode of cell death was apoptosis. A major barrier to the continued development of these compounds as anti-cancer agents is the lack of a defined mechanism.
C: Toxicity Evaluation of Silver(I) Chloride, Bromide and Cyanide Triphenylphosphine Complexes of the Invention and the Toxicity Evaluation Thereof on SNO-Oesophageal Cancer Cells
1. General
(145) Silver chloride, silver bromide and silver cyanide, triphenylphosphine, acetonitrile and ethanol were obtained from Sigma-Aldrich. Infrared spectra were recorded on a Bruker Tensor 27 FT-IR spectrophotometer, using an ATR accessory with a ZnSe crystal. .sup.1H NMR (400 MHz) and .sup.13C NMR (75 MHz) spectra were recorded on a Bruker Avance III 400 MHz spectrometer, using tetramethylsilane as an internal standard. Melting points were recorded on a Stuart Scientific Melting Point apparatus SMP10, and are uncorrected. Gentomycin, Hanks balanced salt solution and trypsin were obtained from Highveld Biological, Kelvin, RSA. AlamarBlue and Annexin-V FITC assay kits were obtained from Serotec, Oxford, UK. All reagents were used as supplied.
(146) All compounds were prepared according to literature procedures. Microanalysis was performed by Dr Edith Antunes in the Department of Chemistry, Rhodes University, RSA on a Thermo Flash 2000 series CHNS/O, Organic Elemental Analyser.
2. Synthesis of Complexes
Preparation of Silver(I) Chloride, Bromide and Cyanide Triphenylphosphine Complexes of the Invention
Example 1: AgCl(PPh3)3 Complex (10)
(147) AgCl (0.50 g, 3.5 mmol) was added to a solution of PPh.sub.3 (2.76 g, 10.5 mmol) in acetonitrile (50 ml). The mixture was refluxed until all the reagents dissolved. The solution was filtered while hot, and the solvent was concentrated to 20 ml. The solution was allowed to cool to room temperature, after which colourless crystals suitable for XRD were obtained. Yield: 65%; mp 190 C.; IR: .sub.max/cm.sup.1: 3054 (w), 2117 (w), 2081 (w), 1978 (w), 1881 (w), 1831 (w), 1773 (w), 1664 (w), 1570 (w), 1478 (s), 1433 (s), 1326 (m), 1306 (m), 1281 (w), 1184 (w), 1156 (w), 1091 (s), 1069 (m), 1026 (w), 996 (w), 913 (w), 854 (w), 741 (s), 691 (s). .sup.1H NMR: (400 MHz, CDCl.sub.3): (ppm) 7.40 (t, J=7.2 Hz), 7.31 (t, J=7.2 Hz), 7.23 (t, J=8.2 Hz). .sup.13C{H} NMR: (100 MHz, CDCl.sub.3): (ppm) 133.3 (d, J(CP)=13.5 Hz), 131.51, 129.47, 128.7 (d, J=3.0 Hz). .sup.31P{H} NMR: (161 MHz, CDCl.sub.3): (ppm) 25.62. Elemental Analysis: (Anal. Calcd. For C.sub.54H.sub.45AgClP.sub.3: C, 69.73; H, 4.88; N, 0. Found: C, 69.74; H, 4.91; N, 0).
Example 2: AgBr(PPh3)3 Complex (11)
(148) AgBr (0.30 g, 1.6 mmol) was added to a solution of PPh.sub.3 (1.26 g, 4.8 mmol) in acetonitrile (50 ml). The mixture was refluxed until all the reagents dissolved. The solution was filtered while hot, and the solvent was concentrated to 20 ml. The solution was allowed to cool to room temperature, after which colourless needles suitable for XRD were obtained. Yield: 60%; mp 192 C.; IR: .sub.max/cm.sup.1: 3047 (w), 2113 (w), 1981 (w), 1955 (w), 1885 (w), 1805 (w), 1670 (w), 1584 (w), 1570 (w), 1477 (s), 1432 (s), 1382 (s), 1307 (s), 1181 (w), 1156 (w), 1095 (s), 1069 (m), 1024 (m), 996 (m), 920 (w), 841 (w), 739 (s), 689 (s). .sup.1H NMR: (400 MHz, CDCl.sub.3): (ppm) 7.59 (t, J=6.0 Hz), 7.65 (d, J=2.8 Hz), 7.35 (t, J=5.0 Hz). .sup.13C{H} NMR: (100 MHz, CDCl.sub.3): (ppm) 134.0 (d, J(CP)=12.4 Hz), 133.0 (d, J(CP)=16.2 Hz), 130.72, 129.3 (d, J=6.8 Hz). .sup.31P{H} NMR: (161 MHz, CDCl.sub.3): (ppm) 25.48. Elemental Analysis: (Anal. Calcd. For C.sub.54H.sub.45AgBrP.sub.3: C, 66.55; H, 4.65; N, 0. Found: C, 65.20; H, 4.58; N, 0).
Example 3: AgCN(PPh3)3 Complex (12)
(149) AgCN (0.15 g, 1.12 mmol) was added to a solution of PPh.sub.3 (0.6 g, 2.24 mmol) in acetonitrile (50 cm.sup.3). The mixture was heated under reflux for 48 hours. The solution was filtered while hot, and the solvent was reduced to 20 cm.sup.3. The solution was allowed to cool to room temperature, after which colorless needles suitable for XRD were obtained. Yield: 75%; mp 193 C.; IR: .sub.max/cm.sup.1: 3056 (w), 2323 (w), 2119 (m), 1891 (w), 1823 (w), 1670 (w), 1585 (w), 1478 (s), 1433 (s), 1309 (m), 1182 (w), 1182 (w), 1156 (w), 1092 (s), 1069 (m), 1026 (w), 997 (w), 917 (m), 849 (w), 740 (s), 691 (s). .sup.1H NMR: (400 MHz, CDCl.sub.3): (ppm) 7.19 (t, J=7.0 Hz), 7.18 (m, J=7.0 Hz), 3.33. .sup.13C{H} NMR: (100 MHz, CDCl.sub.3): (ppm) 133.8 (d, J(CP)=12.5 Hz), 133.4 (d, J=12.0 Hz), 130.58, 129.3 (d, J=14.9 Hz). .sup.31P{H} NMR: (161 MHz, CDCl.sub.3): (ppm) 25.53. Elemental Analysis: (Anal. Calcd. For C.sub.37H.sub.30AgNP.sub.2: C, 67.49; H, 4.59; N, 2.13. Found: C, 67.07; H, 4.99; N, 2.11).
(150) For ease of reference, the chemical structures of complexes (10)-(12) are provided herein below:
(151) ##STR00004##
3. Cell Culturing and Treatment
(152) SNO-oesophageal cancer cells were a gift from the University of Pretoria, RSA. They were cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% Foetal bovine serum (FBS), 1.8% Penicillin/Streptomycin/Fungizone and 0.4% Gentamicin sulphate. The SNO-oesophageal cancer cells were subcultured every 48 hours and incubated at 37 C. under a 5% CO.sub.2 humidified atmosphere. After 48 hours the cells were trypsinized and plated (610.sup.5 cells) in 3.5 cm culture dishes and left to cultivate for 24 hours.
(153) SNO-oesophageal cancer cells were treated with 14 M silver(I) complexes (10)-(12) for 24 hours. The treatments were prepared in Dimethyl sulfoxide (DMSO) and didn't exceed 1%. For comparative purposes, 100 M Cisplatin (CDDP) and 25% H.sub.2O.sub.2 was included which respectively serves as the apoptotic and necrotic controls. Cisplatin was prepared in 0.9% NaCl, whereas H.sub.2O.sub.2 was prepared in the supplemented DMEM media right before treatment. An untreated negative control (UC) was exposed to similar conditions as the treated cells and monitored during the experimental study.
(154) Several assays were used to evaluate the effect of complexes (10)-(12) on cell death with respect to the SNO-oesophageal cancer cells. These included cell viability assays such as the AlamarBlue viability and proliferation assay, microscopy to evaluate cell morphology and flow cytometric analysis of apoptosis and/or necrosis.
4. Cell Viability and Morphological Studies
(155) 4.1 SNO-Oesophageal Cancer Cells Assays
(156) The AlamarBlue Viability and Proliferation Assay of SNO-Oesophageal Cancer Cells
(157) An AlamarBlue (Serotec, UK) assay was done to investigate the proliferation capabilities of the differentially treated cells. AlamarBlue dye (10%) was incubated with trypsinized cells over a 2-hour period before the fluorescence was measured with a Synergy HT Multi-Detection Microplate reader (BioTek, Winooski, Vt.) at wavelengths of 530 nm and (excitation) and 590 nm (emission). This assay, in particular involves the metabolic oxidation-reduction reaction of resazurin to a reduced resorufin product that is directly proportional to the amount of viable cells.
(158)
(159) Morphological Features of the SNO-Oesophageal Cancer Cells Following Differential Treatment as Evaluated by Inverse Light Microscopy
(160) Morphological studies where done after 24 hours of treatment to determine how these compounds influence/change specific features of the malignant SNO-oesophageal cancer cells, compared to the untreated (UC) and 1% DMSO vehicle control. The treated cancer cells including their controls were examined under an Axiovert 25 inverted light microscope (Carl Zeiss, Gttingen, Germany) with Axio Vision 3.1 software (Carl Zeiss, Gttingen, Germany) using a 100 magnification. In
(161) Flow Cytometric Analysis of Apoptosis and/or Necrosis for SNO-Oesophageal Cancer Cell Assays
(162) To determine if either apoptosis or necrosis occurred, the cells were double labelled with Annexin-V FITC and Propidium Iodide (PI) by means of an Annexin-V FITC assay kit (Serotec, UK). This was done according to manufactures instructions with a few adjustments. As mentioned herein above, Annexin-V FITC binds to the inner leaflet of PS when apoptosis and necrosis takes place, making the cellular deaths undistinguishable. This is overcome by using PI as an alternative label that interacts with exposed DNA of necrotic cells only, leading to the flow cytometric detection of both these labels.
(163) The cells (310.sup.5 cells/ml) were washed twice with cold phosphate buffered saline (PBS), followed by the addition of 100 l 1 binding buffer. Two and a half microliters of Annexin-V along with 5 l PI were added in the dark and was incubated for 15 minutes at room temperature.
(164) After incubation, 400 l 1 binding buffer was added and cells were analysed using the FACSAria flow cytometer (BD Biosciences, San Jose, Calif.) with FACSDiva software (BD Biosciences, San Jose, Calif.) 492 nm (excitation) and 520 nm for Annexin and 488 nm and 575 nm(emission) for Propidium Iodide.
(165) SNO-oesophageal cancer cells were treated with complexes (10)-(12) including controls for 24 hours. Dot blots in
(166) The untreated control had minimal cell death consisting of an average viability of 93.97%. This was similar to the 1% DMSO vehicle control. Cellular death was minimal in the controls, but on the contrary apoptosis and necrosis was induced in the malignant cells treated with the different silver(I) complexes (10), (11) and (12). Late apoptotic cell death was more apparent. Complex (10) and (12) had the highest apoptotic SNO-oesophageal cancer cells with a total of over 80% (early and late stages). Complex (11) on the other hand induced apoptotic death in 47.23% (early and late stages) of the malignant cells while 38.07% of the cells seemed viable and intact. Necrotic cell death was minimal in these silver(I) treatments although complex (11) showed to have 14.73% necrotic cells positive for PI. Ligand-10 once again did not influence the cellular viability and PS externalisation like the respective treatments.
(167) Statistical Analysis
(168) Data represented were expressed as the Standard Error of Mean (SEM) where at least 3 biological and 3 technical repeats (except for the dot blots) were used. The Student's T-test was used to calculate the significant difference (*P<0.05, **P<0.001 and ***P<0.00001) of the treatments with respect to the untreated control except in the flow cytometric data where the standard deviation was calculated.
(169) Biological Studies Discussion
(170) Toxicity Profiles of Compounds
(171) When compared to the vehicle control, all treatments with silver(I) complexes (10), (11) and (12) significantly induced cancer cell death, especially complex (11) and complex (12) (P<0.00001). These complexes were even more toxic than the platinum based compound, Cisplatin which killed 74% cells at a high concentration of 100 M. The Ligand-10 treatment on the other hand was less toxic with a viability of 99.03%. This signifies their minimal influence in cancer cell targeting on their own.
(172) Morphological Analysis
(173) The untreated control and 1% DMSO treatments (A and B) represent no signs of cellular death. When compared to complexes (10), (11) and (12) (F-H) multiple signs of apoptosis that involve blebbing and apoptotic body formation (Kerr et al., 1972) can be seen. As seen from the 25% H.sub.2O.sub.2 treatment (D), necrotic cancer cells can be characterized by extensive cell swelling and bursting, which results in the release of cellular content (Kroemer et al., 2009). Minimal necrotic cell death was observed in the cancer cells treated with complexes (10), (11) and (12). Ligand-10 (E) on the other hand showed minimal cell death similar to apoptosis although the majority seems viable.
5. Conclusion
(174) These silver(I) phosphine compounds, Ag(PPh.sub.3).sub.3Cl (10), Ag(PPh.sub.3).sub.3Br (11) and Ag(PPh.sub.3).sub.3CN (12) were used to treat SNO-oesophageal cancer cells. All complexes were found to be highly toxic to the cancer cells. AlamarBlue fluorescence assays combined with morphological studies confirmed that the silver(I) complexes (10), (11) and (12) resulted in apoptosis of the SNO-oesophageal cancer cells. These results were confirmed by flow cytometric analysis.
Example D: Silver(I) Bromide Triphenylphosphine and Silver(I) Nitrate Triphenylphosphine Complexes of the Invention and the Toxicity Evaluation Thereof on A375 Malignant Melanoma Cancer Cells
1. General
(175) Silver bromide, silver nitrate, triphenylphosphine, acetonitrile and ethanol were obtained from Sigma-Aldrich. Infrared spectra were recorded on a Bruker Tensor 27 FT-IR spectrophotometer, using an ATR accessory with a ZnSe crystal. .sup.1H NMR (400 MHz) and .sup.13C NMR (75 MHz) spectra were recorded on a Bruker Avance III 400 MHz spectrometer, using tetramethylsilane as an internal standard. Melting points were recorded on a Stuart Scientific Melting Point apparatus SMP10, and are uncorrected. Gentomycin, Hanks balanced salt solution and trypsin were obtained from Highveld Biological, Kelvin, RSA. AlamarBlue and Annexin-V FITC assay kits were obtained from Serotec, Oxford, UK. All reagents were used as supplied.
(176) All compounds were prepared according to literature procedures. Microanalysis was performed by Dr Edith Antunes in the Department of Chemistry, Rhodes University, RSA on a Thermo Flash 2000 series CHNS/O, Organic Elemental Analyser.
2. Synthesis of Complexes
Example 1: AgBr(PPh3)3 Complex (13)
(177) AgBr (0.30 g, 1.6 mmol) was added to a solution of PPh.sub.3 (1.26 g, 4.8 mmol) in acetonitrile (50 ml). The mixture was refluxed until all the reagents dissolved. The solution was filtered while hot, and the solvent was concentrated to 20 ml. The solution was allowed to cool to room temperature, after which colourless needles suitable for XRD were obtained. Yield: 60%; mp 192 C.; IR: .sub.max/cm.sup.1: 3047 (w), 2113 (w), 1981 (w), 1955 (w), 1885 (w), 1805 (w), 1670 (w), 1584 (w), 1570 (w), 1477 (s), 1432 (s), 1382 (s), 1307 (s), 1181 (w), 1156 (w), 1095 (s), 1069 (m), 1024 (m), 996 (m), 920 (w), 841 (w), 739 (s), 689 (s). .sup.1H NMR: (400 MHz, CDCl.sub.3): (ppm) 7.59 (t, J=6.0 Hz), 7.65 (d, J=2.8 Hz), 7.35 (t, J=5.0 Hz). .sup.13C{H} NMR: (100 MHz, CDCl.sub.3): (ppm) 134.0 (d, J(CP)=12.4 Hz), 133.0 (d, J(CP)=16.2 Hz), 130.72, 129.3 (d, J=6.8 Hz). .sup.31P{H} NMR: (161 MHz, CDCl.sub.3): (ppm) 25.48. Elemental Analysis: (Anal. Calcd. For C.sub.54H.sub.45AgBrP.sub.3: C, 66.55; H, 4.65; N, 0. Found: C, 65.20; H, 4.58; N, 0).
Example 2: AgNO3(PPh3)3 Complex (14)
(178) AgNO.sub.3 (0.20 g, 1.17 mmol) was added to a solution of PPh.sub.3 (0.93 g, 3.5 mmol) in acetonitrile (50 cm.sup.3). The mixture was heated under reflux until all the reagents dissolved. The solution was filtered while hot, and the solvent was concentrated to 20 cm.sup.3. The solution was allowed to cool to room temperature, after which colourless crystals were obtained. Yield: 72%; mp 233 C.; IR: .sub.max/cm.sup.1: 1382, 1308, 1027, 824. .sup.1H NMR: (400 MHz, CDCl.sub.3): (ppm) 7.45 (t, .sub.1=7.472, .sub.2=7.454, .sub.3=7.436 .sup.1J=7.2 Hz), 7.30 (t, .sub.1=7.318, .sub.2=7.300, .sub.3=7.281 .sup.1J=7.4 Hz), 7.17 (t, .sub.1=7.192, .sub.2=7.168, .sub.3=7.147 .sup.1J=9 Hz). .sup.13C{H} NMR: (100 MHz, CDCl.sub.3): (ppm) 133.3 (d, .sub.1=133.36, .sub.2=133.20, .sup.1J(CP)=12.0 Hz), 132.18, 131.98, 130.47, 129.0 (d, .sub.1=129.07, .sub.2=128.98, J=6.8 Hz). .sup.31P{H} NMR: (161 MHz, CDCl.sub.3): (ppm) 6.41. Elemental Analysis: (Anal. Calcd. For C.sub.54H.sub.45P.sub.3AgNO.sub.3: C, 67.79; H, 4.74; N, 1.46. Found: C, 67.83; H, 4.73; N, 0.68).
(179) For ease of reference, the chemical structures of complexes (13) and (14) are provided herein below:
(180) ##STR00005##
3. Cell Culturing and Treatment
(181) A375 melanoma cancer cells originate from an immortal cell line from human malignant melanoma from skin (ATCC no CRL-1619).
(182) A375 malignant melanoma cancer cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), 13.53 g/L with sodium bicarbonate (NaHCO3), 3.7 g/L supplemented with 10% fetal bovine serum (FBS), and 1% Penicillin/Streptomycin/Fungizone in 75 cm.sup.3 sterile culture flasks. Cells were incubated at 37 C. with 5% CO2 in a humidified atmosphere and were subcultured twice a week. Cells were subcultured every 48 hours and used between Passages 3 and 7. For experiments, cells were trypsinized and plated at a concentration of 210.sup.5 cells/mL using 3.0 mL supplemented DMEM in 3.5 cm.sup.3 culture dishes and left to cultivate for 24 hours.
(183) Complexes (13) and (14) under investigation were dissolved in 1.0% Dimethyl sulfoxide (DMSO). DMSO alone (vehicle control/VC) had minimal influence on cancer cell proliferation with a small decrease in the viability of approximately 6.0%. For comparative purposes, 100 M Cisplatin (CDDP) and 25% H.sub.2O.sub.2 was included to serve as positive apoptotic and necrotic controls respectively. Cisplatin was prepared in 0.9% NaCl, whereas H.sub.2O.sub.2 was prepared in the supplemented DMEM media right before treatment. An untreated negative control (UT) was exposed to similar conditions as all treated cells were monitored throughout the experimental study. The silver(I) bromide triphenylphosphine complex (13) and silver(I) nitrate triphenylphosphine complex (14) were tested with concentration ranges from 0 M (1.0% DMSO Vehicle Control/VC) to 10 M.
(184) Several assays were used to evaluate the effect of complexes (13) and (14) on cell death with respect to the A375 malignant melanoma cancer cells. These included cell viability assays such as the AlamarBlue viability and proliferation assay and the Trypan blue exclusion assay, microscopy to evaluate cell morphology and flow cytometric analysis of apoptosis and/or necrosis.
4. Cell Viability and Morphological Studies
(185) 4.1 A375 Malignant Melanoma Cancer Cells Assays
(186) The AlamarBlue Viability and Proliferation Assay of A375 Malignant Melanoma Cancer Cells
(187) An AlamarBlue (Serotec, UK) assay was done to investigate the proliferation capabilities of the differentially treated cells. AlamarBlue dye (10%) was incubated with trypsinized cells over a 2-hour period before the fluorescence was measured with a Synergy HT Multi-Detection Microplate reader (BioTek, Winooski, Vt.) at wavelengths of 530 nm and (excitation) and 590 nm (emission).
(188) Biological repeats were then combined into 1 standard curve to determine IC50 value for the respective complex.
(189)
(190)
(191)
for both.
(192) The Trypan Blue Exclusion Assay of A375 Malignant Melanoma Cancer
(193) The Trypan blue dye exclusion assay [Sigma, USA] works on the principle that viable cells exclude the diazo dye as their cellular membranes are intact, whereas, dead cells absorb and are stained blue by the dye, therefore, it is a cellular viability indicator. Approximately 10 L of a cell to Trypan blue reagent (1:1, 10%) mixture was pipetted onto Bio-Rad slide and counted with Bio-Rad Cell Counter [Bio-Rad, USA]. Whole cell number, live/viable cell number and % viability recorded.
(194)
(195) Morphological Features of the Following Differential Treatments as Evaluated by Inverse Light Microscopy
(196) Morphological studies were conducted using a Zeiss Axiovert 25 Inverted Light Microscope [Carl Zeiss, Germany] with photographs taken with an Axio Cam Camera and Axio Vision 3.1 software [Carl Zeiss, Germany] at 100 magnification.
(197) Morphological features of A375 malignant melanoma cancer cells evaluated by inverse light microscopy following differential treatments are indicated in
(198) Celltitre-Glo Luminescent Cell Viability Assay for A375 Malignant Melanoma Cancer Cells
(199) For purposes of the Celltitre-Glo Luminescent Cell Viability [Promega, USA] assay, a 1:1 mixture of cell sample to Celltitre-Glo substrate/buffer mix reagent, in duplicate, was added to 96-well black plate, shaken for 30 seconds at room temperature and then further incubated for 30 minutes. Whole luminescence was then measured with a Synergy HT Multi-Detection Microplate Reader. The CytoTox-ONE Homogenous Membrane Integrity [Promega, USA] assay is a rapid fluorescent assay that measures the level of lactate dehydrogenase leaked from damaged membranes, therefore, the amount of fluorescence measured is directly proportional to the amount of lysed/damaged cells. In triplicate, a 1:1 cell sample to CytoTox-ONE substrate/buffer mix reagent was added to a 96-well clear plate, shaken for 30 seconds followed by incubation for 10 minutes. A CytoTox-ONE stop solution was added to each sample followed by a further 30 second shake on an orbital shaker. A positive control consisting of untreated cells lysed with a lysis solution for 1 hour was used, i.e. provided maximum LDH levels. The fluorescence was then measured with a Synergy HT Multi-Detection Microplate Reader at an excitation wavelength of 530 nm and emission wavelength of 590 nm.
(200)
(201) Apoptopic Pathway Investigation by Means of Caspase 3/7 Assay for A375 Malignant Melanoma Cancer Cells
(202) Caspase-GLO 3/7, 8 and 9 Assays are luminescent assays that measure Caspase activities, which have key effector functions in intrinsic and extrinsic apoptosis, therefore the amount of luminescence measured is proportional to the amount of Caspase activity present. The assays are based on the principle that after Caspase cleavage, a substrate containing the tetrapeptide DEVD sequence binds with Caspase and is released for a luminescent reaction with a thermostable luciferase enzyme. In duplicate, a 1:1 cell sample to Caspase-Glo substrate/buffer mix reagent was added into 96-well black plates, then shaken for 30 seconds at room temperature followed by a 1 hour incubation. Whole luminescence was then measured with a Synergy HT Multi-Detection Microplate Reader.
(203)
(204) Flow Cytometric Analysis of Apoptosis and/or Necrosis for A375 Malignant Melanoma Cancer Cells
(205) To determine if either apoptosis or necrosis occurred, the cells were double labelled with Annexin-V FITC and Propidium Iodide (PI) by means of an Annexin-V FITC assay kit (Serotec, UK). This was done according to manufacture's instructions with a few adjustments. The cells (310.sup.5 cells/ml) were washed twice with cold phosphate buffered saline (PBS), followed by the addition of 100 l 1 binding buffer. Two and a half microliters of Annexin-V along with 5 l PI were added in the dark and was incubated for 15 minutes at room temperature. After incubation, 400 l 1 binding buffer was added and cells were analysed using the FACSAria flow cytometer (BD Biosciences, San Jose, Calif.) with FACSDiva software (BD Biosciences, San Jose, Calif.) 492 nm (excitation) and 520 nm for Annexin and 488 nm and 575 nm(emission) for Propidium Iodide.
(206) The CBA077 Innocyte Flow Cytometric Cytochrome C Release Kit [Merck, RSA] with anti-cytochrome C primary antibody and anti-IgG FITC secondary antibody was used, as per manufacturer's instructions, to determine the relocalisation of cytochrome C from the mitochondria to the cytoplasm in order to analyze the regulation of apoptotic signalling in cells in all test samples and controls; this assay is based on the selective permeabilisation of the cellular membrane for the release of cytosolic components whilst leaving the mitochondrial membrane intact and a FACSAria flow cytometer and FACSDiva software was used for the analyses. This assay also indicates whether the intrinsic apoptotic pathway is being activated as cytochrome C is associated with this pathway. A mouse IgG antibody was used as an isotype control.
(207) Statistical Analysis
(208) Data represented were expressed as either the Standard Error of Mean (SEM) for bar charts or as the Standard Deviation (STDEV) for line graphs. At least 3 biological with 3 technical repeats (n=9) were used for all studies. The Student's T-test [Microsoft Excel 2010, USA] was used to calculate the significant difference (*p<0.05; **p<0.001; ***p<0.0001) of the treatments with respect to the vehicle control except in the flow cytometric data.
(209) Biological Studies Discussion
(210) Cellular Viability
(211) Cellular viability, i.e. complex (13) and complex (14)-induced toxicity in both controls and differentially treated A375 malignant melanoma cancer cells, was determined by measuring metabolic mitochondrial activity using an AlamarBlue Cell Viability Assay. All values were normalised and represented as percentages of the 1.0% DMSO Vehicle Control (VC) that was set to 100%. Representative bar graphs in
(212) Morphological Analysis
(213) A375 cancer cell morphology, after various treatments, were compared to the untreated negative control as well as to the positive apoptotic and necrotic controls in order to determine if the various treatments were causing cell death and whether apoptosis was occurring. The morphology of the A375 malignant melanoma cancer cells (
(214) Cellular Viability and Cytotoxicity Investigation
(215) Cellular viability, i.e. silver complex-induced toxicity, in both controls and differentially treated A375 malignant melanoma cancer cells, was determined by measuring metabolic mitochondrial activity using the AamarBlue Cell Viability Assay, membrane integrity Trypan Blue Viability Assay as well as ATP levels being measured using the Celltitre-Glo Luminescent Cell Viability Assay. Included is the CytoTox-ONE Membrane Integrity LDH Assay as this cytotoxicity assay goes hand in hand with analysing ATP levels.
(216) As can be seen in
(217) In
(218) It is difficult to compare these results to those in literature concerning cellular viability and mode of programmed cell death (PCD), in the malignant melanoma cell line, due to the fact that the silver(I)complexes (13) and (14) are under investigation with no reported studies on these compounds being available. With that said, comments can be made on anticancer metal complexes, in general, on biochemical changes observed and recorded in cancer cells. Conventional cytotoxic chemotherapies used, in conjunction with adjuvants such as interferon and interleukin inhibitors, to treat malignant melanomas are alkylating agents dacarbazine (DITC), carmustine, temozolomide as well as Pt-based analogues.
(219) Apoptotic Pathway Investigation
(220) Caspase-GLO 3/7, 8 and 9 assays were performed, not only to confirm apoptosis was occurring, but to also deduce whether intrinsic or extrinsic apoptosis was occurring, i.e. these assays were performed to more closely examine the mode of PCD. Caspase 8 plays a key initiator role in the extrinsic apoptotic pathway, whereas Caspase 9 is a key initiator in the intrinsic apoptotic pathway, therefore, measuring their relevant levels in differentially treated A375 cancer cells might shed light as to which apoptotic pathway is occurring. Caspase 3/7 assay is performed to confirm whether apoptosis is occurring as these enzymes play key effector roles in the apoptotic pathway.
(221) It can be clearly seen in
(222) Caspases form the central component of the apoptotic proteolytic system and can be divided into 3 sub groups namely: 1) initiator Caspases such as 8 and 9 that are initiators of the extrinsic and intrinsic apoptotic pathways respectively, 2) apoptotic executioners such as Caspases 3 and 7 and 3) and Caspases that participate in cytokine maturation such as Caspases 1 and 4 (Lin et al., 2000; Rao et al., 2002). Therefore, it could be deduced that 24 hour treatments might be too long a time for Caspase 8 and 9 detection, i.e. apoptosis is already in its late stages as indicated by the increased levels of Caspase 3/7. Caspase 3/7 enzymes are involved later in the apoptotic pathway, therefore it makes sense that these enzyme levels are increased in A375 cancer cells. Intracellular substrates are cleaved by effector Caspases like 3 and 7, resulting in cell death and the typical morphological and biochemical features of apoptosis as seen in
Example E: Various Ratios of Silver(I) Nitrate Triphenylphosphine Complex Prepared According to Example B and the Toxicity Evaluation Thereof on Fibroblast Cells and A-549 Lung Cancer Cells
1. General
(223) The silver(I) nitrate triphenylphosphine complexes under investigation in this study were synthesized according to the procedure set forth in Example B (complexes (6)-(9)). For purposes of this study, the toxicity evaluation of various molar ratios of silver(I) nitrate salt: phosphine ligand on fibroblast cells and A-549 lung cancer cells was assessed.
(224) For the biological studies, fibroblasts (Sigma-Aldrich) were maintained with FM (ScienCell Research Laboratories). Lung cancer (A-549) cells were obtained from the University of Pretoria, RSA.
2. Cell Culturing and Treatment
(225) A-549 lung cancer cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS), 1 mM pyruvate, 2 mM L-glutamine (Highveld Biological, Kelvin, RSA), 1% antibiotics (penicillin, streptomycin, amphotericin (Highveld Biological, Kelvin, RSA) and gentamycin (Invitrogen Life Technologies, Eugene, Oreg.)).
(226) Cells (110.sup.4 cells/well) were plated in 96-well flat-bottomed plates and incubated for 24 h at 37 C. with 5% CO.sub.2. All compounds were preheated for 30 min prior treatment. After 24 h compounds (2.5 M) were mixed with supplemented media and incubated for 24 h. For the untreated control supplemented media was used. Since all compounds were dissolved in DMSO, a 1% DMSO was used as a vehicle control.
(227) Several assays were used to evaluate the effect of complexes (6)-(9) (whereby molar ratios of 1:1, 1:2, 1:3 and 1:4 were evaluated) on cell death with respect to the fibroblast cells and A-549 lung cancer cells. These included cell viability assays such as the AlamarBlue viability and proliferation assay and microscopy to evaluate cell morphology.
3. Cell Viability and Morphological Studies
(228) 3.1 A-549 Lung Cancer Cell Assays
(229) The AlamarBlue Viability and Proliferation Assay of A-549 Lung Cancer Cells
(230) The A-549 lung cancer cells were incubated at 37 C. for 3 h in the presence of 10% AlamarBlue viability and proliferation assay (Invitrogen Life Technologies, Eugene, Oreg.). This was performed on the untreated and treated cells to determine viability. Fluorescence was measured at 530-560 nm (excitation) and 590 nm (emission).
(231)
(232) Morphological Features of the A-549 Lung Cancer Cells Following Differential Treatments as Evaluated by Inverse Light Microscopy
(233) Morphological studies were conducted using a Zeiss Axiovert 25 Inverted Light Microscope [Carl Zeiss, Germany] with photographs taken with an Axio Cam Camera and Axio Vision 3.1 software [Carl Zeiss, Germany] at 100 magnification.
(234) Morphological features of A-549 lung cancer cells evaluated by inverse light microscopy following differential treatments are indicated in
(235)
(236) Biological Studies Discussion
(237) Cellular Viability
(238) Silver(I) nitrate triphenylphosphine adducts were prepared with silver(I) nitrate to phosphine ligand molar ratios of 1:1 to 1:4 (complexes (6)-(9)) and used to treat A-549 lung cancer cells. All 1:1, 1:2, 1:3 and 1:4 molar ratios were found to be toxic to the A-549 lung cancer cells however the 1:4 molar ratio (complex (9)) was observed to have a higher toxicity level in relation to the other ratios. Morphological studies, combined with AlamarBlue cell viability assay confirmed that ratios of 1:1, 1:2, 1:3 and 1:4 (complexes (6)-(9)) resulted in apoptosis of the A-549 lung cancer cells.
FINAL CONCLUSION
(239) Metal-based drugs have important roles in cancer chemotherapy. The platinum based drugs Cisplatin and carboplatin have a wide range of applications in cancer chemotherapy and other metals such as gold(I) and silver(I) are known to possess anti-tumour activity. The interplay of parameters such as the geometrical flexibility of silver(I), bite angle, electronic properties of the phosphine ligand and the coordination mode of the supporting ligands consequently make the study of silver(I) chemistry very attractive. The Applicant has prepared silver(I) monophosphine complexes having anticancer properties for selectively inhibiting the activity of cancer cells. These results indicate that the silver(I) complexes of the present invention may represent the first, promising step in the designing of target-specific chemotherapeutic drugs. Further studies to investigate the mode of apoptotic cell death of these complexes, validated by the data presented herein.
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