Molecular Toxicology, Faculty of Medicine, Sir Alexander Fleming Building, Imperial College of Science, Technology and Medicine, Exhibition Road, London, SW7 2AZ, UK
Received August 1, 2002; accepted September 12, 2002
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ABSTRACT |
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Key Words: Cryptolepis sanguinolenta; cryptolepine; cytotoxicity; hprt mutation; alamar blue; Trypan blue; clonogenicity.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Chemicals.
Synthetic CLP (purity >99%) was a kind donation from Dr. J. Addae-Kyeremeh, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. CSE and CLP were dissolved in cell culture media and filtered through sterile 0.2 µM filter for cell culture studies. Unless otherwise indicated in the text, all other chemicals were purchased from Sigma Chemical Co. (Poole, England.)
Cell line and conditions.
The V79-MZ cell, a Chinese hamster lung fibroblast cell line was propagated in Dulbeccos modified Eagles medium (DMEM); HCT116, a human colon adenocarcinoma cell line in RPMI; SKOV3, a human ovary adenocarcinoma cell line in Macoys 5a; MCF7, a human breast adenocarcinoma in minimal essential medium (MEM), and MDA MB 361, a human breast adenocarcinoma cell line in L-15 media, all from Invitrogen Corporation (Paisley, Scotland, UK). The media were supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, and 100 units of penicillin/streptomycin, also from Invitrogen.
Routinely, cells were maintained at 37°C in a humidified atmosphere of 5% CO2/95% air, harvested by trypsinization with trypsin-EDTA (Invitrogen) and centrifugation, and subcultured as appropriate for the individual cell lines. Cell numbers were determined by counting with a haemocytometer and viability was assessed using Trypan blue exclusion.
Growth inhibition (resazurin reduction assay).
Growth inhibition was assessed by serial measurements of the fluorescence intensity following the reduction of resazurin (alamar blue) by viable cells (Fields and Lancaster, 1993). Briefly, V79 cells were seeded into 24-well multiwell dishes at a density of 5 x 104 cells per well in culture medium and then incubated overnight before treatment with vehicle, CSE, or CLP for 4, 24, or 72 h. After the incubation period, 1% v/v alamar blue was added to each well and incubated for a further 1 h. The fluorescence intensity was then measured using a plate reader, Fluostar® with excitation at 530 nm and emission at 590 nm. Under these conditions the cells remain viable and continue to grow, allowing serial fluorescent measurements to be recorded.
Cell viability (Trypan blue staining).
In order to estimate the percentage of dead V79 cells after treatment with CSE or CLP, floating cells in the medium of each flask were transferred to centrifuge tubes. After detachment of the adherent cells with trypsin, the cells were mixed with the corresponding floating cells before centrifugation. The cells were then stained with 0.4% Trypan blue, and the number of Trypan blue positive and negative cells were counted on a haemocytometer by light microscopy.
Colony survival (clonogenic assay).
For survival studies, treated cells were trypsinized, centrifuged, and reseeded at 100 cells per well in 6-well multidishes in drug-free medium. After incubation for at least 7 days, the wells were stained with methylene blue in 50% methanol, and colonies that contained 50 or more cells were scored as survivors. Cell survival was expressed as a percentage of appropriate vehicle-treated controls.
Flow cytometry analysis.
Vehicle-, CSE-, or CLP-treated V79 cultures were harvested by centrifugation, washed twice with PBS, and fixed in 70% ethanol at 20°C for 24 hours. Cells were then resuspended in 1 ml of PBS solution containing 5 µg/ml propidium iodide (PI) and 100 µg/ml RNase and incubated in a water bath at 37°C for 30 minutes. Samples were then analyzed on a Becton Dickinson FACScan flow cytometer. Using the Cellquest software, the percentage of cells at different phases of the cell cycle was determined. PI was excited at 488 nm, and fluorescence analyzed at 620 nm.
Hprt mutation assay.
The assay was performed according to the method of Yadollahi-Farsani et al. (1996)) with modifications. Prior to drug treatment, V79-MZ cells were maintained in culture medium supplemented with HAT (5 x 10-5 M hypoxanthine, 4 x 10-7 M aminopterin, and 5 x 10-6 M thymidine) for 72 h to reduce the frequency of preexisting hprt mutants in the cell population. Cells in exponential phase (1.5 x 106) were seeded into 75-cm2 flasks in culture medium 24 h before treatment with CSE, CLP, or the positive control, ethylmethane sulphonate (EMS) for a further 24 h.
To determine cell survival, treated cell cultures were trypsinized and reseeded at 100 cells/well in 2 ml of culture medium in 6-well plates. At the end of 7 days, wells were stained with methylene blue in 50% methanol and colonies that contained more than 50 cells were scored as survivors. Cell survival was expressed as a percentage of vehicle-treated controls.
Resistance to the lethal effects of the purine analogue 6-thioguanine (6-TG) was used as the genetic marker for the measurement of mutant frequency (MF). The treated cells were maintained in exponential growth for 7 days to allow phenotypic expression of induced mutants. Following the expression period, 1 x 106 cells were plated at 2 x 103 cells/cm2 in culture media containing 5 µg/ml of 6-TG for a period of 710 days. The cloning efficiency at the time of mutant selection was also determined in normal culture medium (41 ± 4%) to correct for the observed mutant frequencies.
Statistical analysis.
Samples were compared using the Students t-test.
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RESULTS |
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DISCUSSION |
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It was to investigate the circumstances of this delayed cell death that we sought to determine the cycle distribution of treated cells by flow cytometry. It is intriguing that in the flow cytometry analysis, the large sub-G1 population (up to 55%) observed in samples treated for 24 h indicated that not all the dying cells were detected in the Trypan blue test. The appearance of cells in a sub-G1 population has been considered as a marker of apoptotic cell death (Darzynkiewicz et al., 1992); thus our flow cytometry study would suggest that CSE and CLP induce apoptosis in V79 cells as reported previously for CLP in HL-60 leukemia cells (Dassonneville et al., 2000
). Many DNA-damaging agents, including mutagens and carcinogens, cause cell-cycle arrest at G1, S-phase, and G2 checkpoints, which may be followed by apoptotic cell death (Barry et al., 1990
; Hartwell and Kastan, 1994
), depending on the intensity of the damage and the repair machinery at the disposal of the cell. The cell cycle checkpoints allow sufficient time for DNA repair, yet apoptosis may ensue to eliminate overwhelmed DNA-damaged cells (Lowe and Lin, 2000
; Offer et al., 2002
). Interestingly, in our experiments the sub-G1 population increased in the absence of any obvious block in G1, S, or G2/M phases. The p53 status of a cell is an important determinant of cell cycle arrest (Diller et al., 1990
; Kuerbitz et al., 1992
; Livingstone et al., 1992
). It induces cell cycle arrest at the G1/S DNA damage checkpoint through the transcriptional activation of the CDK inhibitor p21Cip1/WAF1 (Brugarolas et al., 1995
; El-Diery et al., 1993
). Recent evidence also suggests that p53 and p21 are required for maintaining a G2 arrest following DNA damage (Flatt et al., 2000
). The V79 cells used in the current study are reported to have a mutated, nonfunctional p53 (Chaung et al., 1997
). This might have contributed to the inability of the cells to evoke a distinct arrest at any phase of the cycle.
Our data not only indicate toxicity of the agents to V79, a typical mammalian cell line, but several organ-specific human cancer cell lines. The reason for the differential sensitivity of the cell lines is not immediately clear from the studies, but the cell type and the balance of expression of apoptotic and antiapoptotic proteins in the individual cell lines could contribute at least in part to the differential sensitivity. It is, however, noteworthy that both agents inhibited colony formation, a major factor for anticancer agents. If this property were to occur in vivo, our results would suggest that CSE and CLP could be used against human cancers.
One of the major problems associated with anticancer agents is the possibility of genotoxicity resulting from DNA interaction. The results of the hprt mutation assay would appear to indicate that CLP is not mutagenic at concentrations that invoke substantial cytotoxicity. The significant mutation frequency observed at the highest concentration of CSE could suggest the presence of a minor component, which is probably more mutagenic than CLP, the activity of which becomes apparent at high concentrations of CSE administration. Several minor alkaloids have been isolated from the plant (Paulo et al., 1994) whose biological activities have yet to be determined. The reason for the poor mutagenic action of CLP in the face of the reported DNA intercalation (Bonjean et al., 1998
; Lisgarten et al., 2002
) could be due to its topoisomerase-II inhibitory properties. Topoisomerase-II inhibition is manifest as fragmented DNA. This is a clastogenic event resulting in large deletions, which are poorly detectable in the hprt assay system (Kulling and Metzler, 1997
) since their lethality frequently occurs through effects on neighboring essential genes (Moore et al., 1989
). The hprt gene locus is known to be rich in GC sites, and CLP has been demonstrated to have high affinity for GC-rich regions of DNA (Bonjean et al., 1998
). CLP binding and consequent damage to the hprt gene itself could be the reason for the inability of CLP to induce hprt mutants. The profound cytotoxicity of CLP probably precedes the phenotypic expression of induced mutant clones. Thus, alternative genotoxicity assessment is warranted, perhaps using an autosomal locus target as well as clastogenicity assays.
Overall, our studies indicate that CSE and CLP are potently cytotoxic to V79 cells and a number of organ-specific human cancer cell lines. The demonstration of a broad-spectrum activity on a variety of cancer cell lines, apoptotic cell death, and apparently low genotoxicity suggest that these agents may have potential as candidates for cancer chemotherapy.
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ACKNOWLEDGMENTS |
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NOTES |
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