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Cytotoxicity,
Selectivity and the Activation of |
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INTRODUCTION
MATERIALS AND METHODS
DISCUSSION
REFERENCES
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Cytotoxicity, Selectivity and the Activation of
Apoptosis by Platinum and Palladium
Compounds in Leukemia
by Rita Thomas
INTRODUCTION
For the past five years, Dr. Robert Mack Granger, Associate Professor of Chemistry at Sweet Briar College, has been successfully synthesizing a variety of Platinum and Palladium compounds with potential as anticancer agents [1]. During this period of time, Dr. Robin Lee Davies, Professor of Biology at Sweet Briar College, has been exposing a variety of both normal and malignant cells to these compounds and has been monitoring their bioactivity and cytotoxicity [1]. One of the most promising aspects of these compounds is that some of them appear to be selectively toxic-that is, they kill cancer cells more efficiently than they kill normal cells. Dr. Granger has synthesized Platinum and Palladium compounds in both the +2 and +4 oxidation states [1]. The varying oxidation states allow Dr. Granger to attach a variety of ligands to the compounds, causing a change in their behavior [2]. For example, to increase the compounds' selectivity for malignant cells, a ligand containing asparagine (an essential amino acid for cancer cells) may be attached [3]. These compounds have been extensively tested on ovarian, breast, and uterine cancers, and only a small amount of experimentation has ever been done with leukemia. With this in mind, coupled with the promising results for the compounds' selectivity and cytotoxicity obtained in previous studies, I wanted to test these compounds against leukemia cells.
The three main questions I hoped to address for the Platinum and Palladium compounds were: 1.) Does the type of metallic center (Pt or Pd) affect the cytotoxicity and selectivity of the compounds for leukemia cells? 2.) How do the number and types of ligands present in the compound affect cytotoxicity and selectivity? 3.) What is the mechanism of cancer cell death? In other words, are Dr. Granger's compounds causing malignant cells to undergo apoptosis or necrosis? The answer to this last question is important, because it will give us a general idea of where and how the compounds attach to the cells, allowing us to further theorize how the compound will react in the human body. If it is discovered that the cells become apoptotic, or program their own death, then there is strong evidence that the compound is being absorbed by the cell and is in some way attaching to the cellular DNA [1]. Once the compound is attached, it may cause irreparable damage to the nucleic acids, thereby causing the cell to commit suicide [2]. However, if the cells undergo necrosis, or death of a cell other than by suicide, the compounds are probably not attaching to the DNA.
MATERIALS AND METHODS
Cells
Three cell lines were used in this experiment, of which two were malignant and one was normal:
A human promyelocytic leukemia, designated HL-60, obtained from a 36-year-old Caucasian female with acute promyelocytic leukemia [8]. The HL-60 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and was maintained in 75-mL tissue culture flasks with RPMI Medium supplemented with 20% heat-inactivated fetal bovine serum.
A chronic myelogenous leukemia, designated K-562, isolated from a 53-year-old Caucasian female with chronic myelogenous leukemia in terminal blast crisis [9]. The cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and was maintained in 75-mL tissue culture flasks with Iscove's Modified Dulbecco's Medium supplemented with 10% heat-inactivated fetal bovine serum.
Apparently normal Epstein-Barr virus transformed lymphoblasts from a 32-year-old Caucasian female, repository number GM00893B, were acquired from the NIGMS Human Genetic Cell Repository (CIMR, Camden, NJ) and cultured in 75-mL flasks with RPMI Medium 1640 supplemented with 15% heat-inactivated fetal bovine serum [7]. · A human promyelocytic leukemia, designated HL-60, obtained from a 36-year-old Caucasian female with acute promyelocytic leukemia [8]. The HL-60 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and was maintained in 75-mL tissue culture flasks with RPMI Medium supplemented with 20% heat-inactivated fetal bovine serum.
All three cell lines were incubated at 37º C with 5% CO2 until they were growing vigorously and present in sufficient numbers to be used in the experiment.
Exposure of Cells to Compounds
Once all of the cell lines were growing vigorously, 2-mL aliquots of each cell line were placed in 50-mL polypropylene tubes. Each tube was labeled with the cell repository number and designated as "Normal" (Control #1), "DMSO" (Dimethyl Sulfoxide Control #2), or with the proper chemical notation of the compound to which they would be exposed. The compounds tested on all cell lines are listed below:
Pd(dione)Cl4
· Pt(dione)Cl4
· [Pd(dione)3(PF6)4]
· [Pd(bipy)(dione)2(PF6)4]
· Cisplatin*
· [Pd(dione)(phen)Cl2]Cl2
· [Pd(phen)(dione)2](PF6)4
· [Pt(dione)(PF6)4] II
Figure 1. The general structure of the Pd(dione)Cl4 and Pt(dione)Cl4 "M" designates the metallic center of the compound. Image courtesy of Dr. Robert Granger.
The compounds were individually dissolved in DMSO to yield a concentration of 1.00 X 10-3 M. Four 5-mL aliquots of untreated or "normal" medium (5 mL of medium), DMSO (4.5 mL of medium + 0.5 mL of DMSO), and the compound to be used (4.5 mL of medium + 0.5 mL of dissolved compound) were made and placed in 15-mL polypropylene tubes. To begin the exposure, 0.20 mL of the prepared aliquots were added to the properly labeled 50-mL tubes containing the cells. This gave a final concentration of 1% DMSO in the Control #2, and a final compound concentration of 1.00 x 10-5 M in the experimental tubes.
MTT Assay
After an exposure time of 24 hours, the cells were subjected to a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay [4]. The MTT assay distinguished between the viable and non-viable cells in each sample, because only live cells absorb the yellow-colored MTT and convert it into a blue formazan [4]. Therefore, the amount of blue formazan present corresponded directly to the number of live cells in each sample. To quantify the exact amounts of formazan produced, each sample was read using a Beckman DU-600 spectrometer set at 540.0 nm.
Detection of Apoptosis
To test for the presence of apoptosis in the three cell lines treated with a metallic compound, two assays widely used for this purpose were employed:
· DNA isolation assay: During apoptosis, the cell's DNA becomes fragmented by an endonuclease, giving it a distinctive laddering appearance in gel electrophoresis [5]. This assay was performed on both experimental and control setups, using a DNA extraction kit from the Intergen Company (Purchase, NY) [6]. The assay allows for the isolation of high-molecular-weight DNA from the samples by lysing the cells, denaturing the proteins, and precipitating out the desired DNA [6]. The amount of DNA present was quantified with a spectrometer at an absorbance of 260 nm and an A260/A280 ratio was calculated [6]. After extracting the DNA, a horizontal electrophoresis was conducted using 25 mL of an agarose gel with 2.5 μL of 5-mg/mL ethidium bromide.
· Caspase Inhibitor Assay: When a cell undergoes apoptosis, its mitochondria are stimulated and release cytochrome C or Apaf-2, an apoptotic protease-activating factor [2]. Cytochrome C combined with dATP activate caspase 9, a protease, which, in turn, activates caspase 3, leading to a cascade in protease activation [2]. To determine if this characteristic of apoptosis was taking place in the cells exposed to the metallic compounds, the CaspaTagTM Fluorescein Caspase Activity Kit was employed (Intergen Company, Purchase, NY) and results were obtained through the use of fluorescence microscopy. The compound Pd(dione)Cl4, against which HL-60 showed the lowest percent survival and GM00893B showed the highest percent survival, was used in this assay, along with the similarly structured Pt(dione)Cl4. This assay contains the inhibitor FAM-VAD-FMK. This inhibitor, labeled with a caroxyfluorocein (FAM), irreversibly binds to caspases 1, 8, 9, 5, 7, 3, 6, 4, and 2 and allows cells with activated caspases to be observed through fluorescence microscopy [2].
RESULTS
Cytotoxicity and Selectivity of Compounds
Based on the data acquired from the MTT assay, the compound most selective and cytotoxic for HL-60 is Pd(dione)Cl4. This particular compound resulted in a 26.81% survival rate for human promyelocytic cells and an amazing survival rate of 77.31% for the apparently normal lymphoblasts, GM00893B (Figure 6). However, Pd(dione)Cl4 did not perform as well against K-562, a chronic myelogenous leukemia, as it did against HL-60. The K-562 cell line exhibited an average cell survival of 153.44% when exposed to Pd(dione)Cl4 (Figure 4). In addition to this monodione, [Pd(phen)(dione)2](PF6)4 also exhibited a degree of selectivity for HL-60, resulting in a survival rate of 20.96% (Figure 6).
As Figure 3 shows, the compounds least selective and cytotoxic for HL-60 are Cisplatin and [Pd(dione)(phen)Cl2]Cl2, which caused survival rates of 105.42% and 102.61%, respectively. None of the compounds tested on K-562 resulted in a survival rate lower than 102.00% (Figure 4).
A sharp contrast to the results obtained for Pd(dione)Cl4 is the extremely high survival
rate of HL-60 cells exposed to Pt(dione)Cl4 (60.82%-Figure 6).
Figure 2. 2-mL aliquots of cell line GM00893B were exposed to 1.00 x 10-5 M concentrations of Platinum and Palladium compounds for 24 hours in 50-mL polypropylene tubes. An MTT assay was used to distinguish between viable and non-viable cells. A Beckman DU-600 spectrometer set at 540.0 nm quantified the results. *The data shown here are an average of 3 trial runs per compound.
Evidence of Apoptosis· DNA Extraction
The results obtained with the DNA extraction kit were inconclusive. Although three trial extractions were conducted with HL-60 Control #1 and HL-60 exposed to Pd(dione)Cl4, and the protocol designated by the kit was followed, no DNA was isolated from the experimental setup. DNA was successfully extracted from HL-60 Control #1 and gelled with electrophoresis. Analysis of these gels suggested that no DNA laddering was evident in Control #1, as was expected.
Figure 3. 2-mL aliquots of cell line HL-60 were exposed to 1.00 x 10-5 M concentrations of Platinum and Palladium compounds for 24 hours in 50-mL polypropylene tubes. An MTT assay was used to distinguish between viable and non-viable cells. A Beckman DU-600 spectrometer set at 540.0 nm quantified the results. *The data shown here are an average of 3 trial runs per compound.
Figure 4. 2-mL aliquots of cell line K-562 were exposed to 1.00 x 10-5 M concentrations of Platinum and Palladium compounds for 24 hours in 50-mL polypropylene tubes. An MTT assay was used to distinguish between viable and non-viable cells. A Beckman DU-600 spectrometer set at 540.0 nm quantified the results. *The data shown here are an average of 3 trial runs per compound. Please note that no trial experiments were conducted for K-562 and [Pd(phen)(dione)2](PF6)4 due to time constraints.· Caspase Inhibitor and Fluorescence Microscopy
As shown in Figure 5, HL-60 cells that were exposed to Pd(dione)Cl4 and Pt(dione)Cl4 underwent apoptosis. This is known because these cells appeared bright red when viewed with fluorescence microscopy. This indicates that the caspase inhibitor had irreversibly bound to an activated caspase in the cell, meaning that the cell was, in fact, in an apoptotic state.
(a) (b)
(a)
(b)
Figure 5. Photos taken with a fluorescent microscope of treated leukemia cells tested with a caspase assay. (a) A HL-60 cell exposed to Pd(dione)Cl4 for 24 hours. (b) A HL-60 cell exposed to Pt(dione)Cl4 for 24 hours. Both cells appear bright red when viewed with fluorescence microscopy, which indicates that these cells had activated caspases-they were apoptotic.
Figure 6. The data shown here are averages of 3 experimental trials for the cytotoxicity and selectivity of the Pd(dione)Cl4 and Pt(dione)Cl4 compounds for each cell line. The results for [Pd(phen)(dione)2](PF6)4 are based on a single trial experiment with GM00893B and HL-60. The compound was not tested on K-562 because of time contraints.DISCUSSION
This study has tested the effectiveness of Dr. Robert Granger's Platinum and Palladium compounds against two leukemia cell lines (HL-60 and K-562) along with the non-malignant cell line GM00893B. Based on the obtained results, it is evident that Pd(dione)Cl4 is the compound most cytotoxic and selective for HL-60, while, at the same time, it is responsible for a cell death rate of less than 25.00% in GM00893B (Figure 6). None of the tested compounds were effective in destroying K-562 (Figure 4). The compound [Pd(phen)(dione)2](PF6)4 resulted in an impressively low survival rate for HL-60 and an average survival rate for GM00893B (Figure 6). However, only one trial experiment was conducted with this compound, and further studies are expected to be carried out to see if these results are replicable.
The severe difference in the selectivity of Pd(dione)Cl4 and Pt(dione)Cl4 for HL-60 was not expected. Because Platinum and Palladium possess similar chemical properties, and because they are bound to the same type of ligand, one would expect the two metals to react similarly with HL-60 and yield analogous results. Therefore, based on these chemical similarities, the type of metallic center used in the monodione compound should not have as great an effect on the results as that shown in Figure 6. In an effort to understand why this happened, I plan to review the protocol I used in both of these exposures and conduct further experimental trials of these two compounds with HL-60 and GM00893B.
A positive identification of apoptosis in HL-60 exposed to Pd(dione)Cl4 and Pt(dione)Cl4 (Figure 5) strongly suggests that these compounds are destroying this particular cell line by binding to the DNA, causing irreversible damage to the nucleic acids, and stimulating programmed cell death. I hope to conduct further studies on the concept of the metallic compounds binding to DNA by exposing the cells to the compounds, isolating the affected DNA, and analyzing the specific amounts of Palladium and Platinum bound to the DNA using an atomic absorption spectrometer (AAS). However, instead of testing only HL-60, I will also test GM00893B. Knowing the amount of the metallic compound attached to HL-60's and GM00893B's DNA is important because it will explain why Pd(dione)Cl4 and Pt(dione)Cl4 are more selective for HL-60 than the other compounds. I will also test for apoptosis in cells exposed to the selective compound [Pd(phen)(dione)2](PF6)4. This will provide my fellow researchers and myself with a deeper understanding of exactly how these compounds interact with cells, which, in turn, will provide us with a probable explanation of how these compounds can be expected to react within the human body.
ACKNOWLEDGMENTS
I would like to thank my advisor, Dr. Robin Lee Davies, for the guidance and encouragement she gave me during this study and Dr. Robert Mack Granger for supplying me with the compounds I needed. I also thank the Virginia Foundation for Independent Colleges for providing me with the Minority Fellowship, which allowed me to conduct my research, and, finally, the Sweet Briar Summer Honors Program.
REFERENCES
1. Davies, Robin L. Personal Communication. 16 Feb. 2001.
2. Eastman, Alan. (1999). "The mechanism of action of cisplatin: from adducts to apoptosis, in cisplatin." Chemistry and Biochemistry of a Leading Anticancer Drug. Ed. B. Lippert, Weinheim: Wiley-VCH. 111-134.
3. Jakoby, William B. Pastan, Ira H. (1979). Methods in Ezymology Vol. 58. San Diego: Academic Press, 1979.
4. Mossman, T. (1983). "Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays." J. Immunol. Meth. 65: 55-63.5. O'Neill, Ciaran F. Hunakova, Luba. Kelland, Lloyd R. (1999). "Cellular pharmacology of cis and trans pairs of platinum complexes in cisplatin-sensitive and -resistant human ovarian carcinoma cells." Chemico-Biological Interactions 123: 11-29.
6. Viale, Maurizio, Cafaggi, Sergio, Pardoi, Brunella, Esposito, Mauro. (1995). "Cytotoxicity and cellular accumulation of a new cis-diammineplatinum (II) complex containing procaine in murine L1210 cells sensitive and resistant to
cis-diamminedichloroplatinum (II)." Cancer Chemother Pharmacol 35:371-376.7. "GM00893B." Catalog Detail. National Institute of General Medical Sciences. Online. Internet. Accessed 9 June 2001.
Available: http://locus.umdnj.edu/nigms/nigms_cgi/display.cgi?GM008938. "HL-60." Catalog Detail. American Type Culture Collection. Online. Internet. Accessed 6 June 2001.
Available : http://phage.atcc.org/cgi-bin/searchengine/longview.cgi?view=ce%2C429096%2CCCL-240&view=ce%2C438893%2CCCL-243&text=leukemia9. "K-562." Catalog Detail. American Type Culture Collection. Online. Internet. Accessed 6 June 2001.
Available: http://phage.atcc.org/cgi-bin/searchengine/longview.cgi?view=ce%2C429096%2CCCL-240&view=ce%2C438893%2CCCL-243&text=leukemia
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This page updated July 3, 2002
