Affiliations of authors: Departments of Molecular Pathology (YZ, CG-P, DAC, RX, HP, PH), Leukemia (DAC, DMH, ZE, MJK), Neuro-Surgery (SK), and Pathology (JL), The University of Texas M. D. Anderson Cancer Center, Houston, TX; College of Pharmacy, Division of Medicinal & Natural Products Chemistry, The University of Iowa, Iowa City, IA (YK, WY, CL, ZJ)
Correspondence to: Peng Huang, MD, PhD, Department of Molecular Pathology, Box 089, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030 (e-mail:phuang{at}mdanderson.org).
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ABSTRACT |
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The present study was conducted to test the anticancer activity and selectivity of OSW-1 in various cancer and nonmalignant cells in culture and to investigate its underlying mechanisms. We first used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously (15) to determine the cytotoxic activity (i.e., the IC50 values, defined as the concentrations that cause a 50% loss of cell viability) of OSW-1 in six different cancer cell lines, including human leukemia and lymphoma cell lines, ovarian cancer SKOV3 cells, malignant brain tumor cells (U87-MG), and pancreatic cancer cells (AsPC-1), and in several types of nonmalignant cells. As shown in Table 1, OSW-1 exhibited potent cytotoxicity in all six malignant tumor cell lines. The IC50 values were less than 0.1 nM in all but the pancreatic cell line. By contrast, OSW-1 had higher IC50 values in nonmalignant cells including normal lymphocytes from four healthy donors, normal human fibroblasts, and immortalized normal human astrocytes than the malignant cells (Table 1). Overall, the mean IC50 value in the MTT assay for the three types of nonmalignant cells was 3.2 nM, which was approximately 30-fold greater than the mean IC50 value for the six malignant cell lines tested (0.11 nM, P = .046). Analysis for granulocyte-macrophage colony-forming capacity using normal blood samples also showed that OSW-1 was less toxic to the normal cells (IC50 = 1.44 nM, 95% confidence interval [CI] = 1.30 to 1.56) than to leukemia cells (0.11 nM, 95% CI = 0.10 to 0.12) in the clonogenic assay in semisolid medium.
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To investigate the mechanism of this anticancer activity, we used DNA microarray analysis to examine potential changes in gene expression in cells that were incubated with OSW-1 in vitro. Expression of genes coding for several mitochondrial metabolic enzymes, including NADH dehydrogenase 1 subcomplexes 1 and 4, NADH dehydrogenase 1
subcomplex 7, COX6A, COX7B, and cytochrome c oxidase subunit IV isoform 1, were consistently increased in expression after the cancer cells were treated with OSW-1, with a t score of greater than 7.0 by using ArrayVision software (Imaging Research, Inc.; Ontario, Canada). These results suggested that OSW-1 might disturb mitochondrial metabolic function. However, direct analysis of mitochondrial respiratory rate using an oxygen consumption assay (16) showed that treatment with OSW-1 did not induce a substantial change in mitochondrial respiratory activity (data not shown). Consequently, we used transmission electron microscopy to examine the mitochondrial ultrastructure of OSW-1-treated cells. As shown in Fig. 1, substantial changes in mitochondrial morphologyincluding a blurred membrane outline, disappearance and/or disorganization of the cristae, and paling of the mitochondrial matrixwere observed in leukemia cells (HL-60) that had been incubated with OSW-1. These ultrastructural changes were visible as early as 6 hours after incubation with OSW-1, and they became more apparent as the incubation time was prolonged up to 14 hours (Fig. 1, B). Interestingly, the nuclear membranes appeared intact even at 14 hours, suggesting that the effect of OSW-1 was specific to the mitochondrial membranes. Further evidence that OSW-1 damaged mitochondrial membranes came from the finding that treatment of HL-60 leukemia cells with OSW-1 led to the loss of mitochondrial transmembrane potential, as revealed by flow cytometric analysis of cells that had been labeled with the membrane potential-sensitive fluorescent dye rhodamine-123 (Fig. 2, B).
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The results described above suggested that the increase in cytosolic calcium may be a consistent biochemical event resulting from OSW-1 treatment. To further evaluate the role of increased cytosolic calcium in OSW-1-induced apoptosis, we used the cell-permeable calcium chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) to test if chelation of cytosolic Ca2+ would alter the amount of drug-induced cell death. As illustrated in Fig. 1F, exposure of HL-60 cells to OSW-1 (1 nM) resulted in a loss of 61% of cells within 24 hours. Addition of 1 µM BAPTA-AM, which by itself did not alter cell viability at 24 hours (Fig. 1, G), substantially suppressed OSW-1-induced apoptosis, resulting in a loss of only 14% of the cells (Fig. 1, H). These data suggest that the increase of cytosolic calcium may be a critical event in mediating OSW-1-induced apoptosis.
If damage to mitochondria by OSW-1 were critical in causing apoptosis, cells that have adapted to survive with mitochondrial defects should be less sensitive to this compound than cells that rely on intact mitochondria. To test this possibility, we compared the OSW-1 sensitivity of a subclone of human leukemia cells (C6F) that are known to have mitochondrial DNA mutations and functional defects (16,18) with that of the parental HL-60 cells. Flow cytometric analysis showed that HL-60 cells were sensitive to 0.5 nM OSW-1 (Fig. 2, A). A large subpopulation of cells with sub-G1 DNA content was observed at 24 hours after OSW-1 treatment, indicating DNA fragmentation and apoptosis. By contrast, C6F cells treated with the same concentration of OSW-1 did not generate a substantial number of apoptotic cells. An additional assay was then used to further compare the apoptotic responses to OSW-1 in HL-60 and C6F cells. That is, flow cytometric analysis of cells stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide showed that 1 nM OSW-1 caused substantial apoptosis in HL-60 cells, but much less apoptosis in C6F cells (Supplementary Fig. 1; available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97/issue23).
Because mitochondrial respiratory chain activity generates reactive oxygen species, which can cause cellular damage and induce apoptosis, we tested if OSW-l might affect reactive oxygen species generation in HL-60 and C6F cells. Analysis of superoxide using a hydroethidium assay (16, 19) revealed that a substantial amount of superoxide was produced only in the parental HL-60 cells at the late stage of apoptosis 1824 hours after treatment with 0.5 nM OSW-1 (Supplementary Fig. 1, A; available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97/issue23), suggesting that generation of reactive oxygen species may be secondary to mitochondrial damage and apoptosis. Only a small amount of reactive oxygen species was generated in C6F cells treated at the same concentration of OSW-1 (Supplementary Fig. 1, B; available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97/issue23). Analysis of mitochondrial transmembrane potential () showed that OSW-1 induced a time-dependent loss of
in HL-60 cells but not in C6F cells (Fig. 2, B). Colony formation assay also showed that C6F cells were substantially more resistant to OSW-1 than the parental HL-60 cells. For instance, treatment with 0.3 nM OSW-1 induced a complete loss of colony-forming ability in HL-60 cells, whereas 35% of C6F cells treated with 0.3 nM still formed colonies (data not shown).
Our finding that OSW-1 appears to act by damaging mitochondria and induce apoptosis through a calcium-dependent mechanism raised the possibility that this compound could be effective in cancer cells that are resistant to conventional anticancer agents. Consequently, we tested the cytotoxic effect of OSW-1 in primary leukemia cells isolated from patients with chronic lymphocytic leukemia (CLL) who were refractory to the chemotherapeutic agent fludarabine. Peripheral blood samples were obtained from CLL patients after obtaining written informed consent as required by the institutional review board. CLL cells from two patients refractory for fludarabine were extremely resistant to F-ara-A (the active component of fludarabine for in vitro study), with IC50 values of greater than 30 µM in a 72-hour incubation (Supplementary Fig. 2, A; available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97/issue23). These fludarabine-resistant CLL cells were extremely sensitive to OSW-1, with IC50 values of less than 0.3 nM. Analysis of primary leukemia cells from 26 CLL patients who were either sensitive (n = 18) or resistant (n = 8) to fludarabine showed that the fludarabine-resistant CLL cells were sensitive to OSW-1, with a mean IC50 value of 0.15 nM (95% CI = 0.02 to 0.28), which was similar to the IC50 value of 0.23 nM (95% CI = 0.04 to 0.42) in fludarabine-sensitive CLL cells (P = .47) (Supplementary Fig. 2, B; available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol97/issue23). Further analysis of the in vitro cytotoxic data from a total of 34 patient samples revealed that there was no statistically significant difference in OSW-1 sensitivity with respect to patient's gender, Rai stage, or prior chemotherapeutic history (data not shown).
In summary, we found that OSW-1 possesses highly potent anticancer activity against several human malignant cell lines and primary leukemia cells from patients with CLL. This compound exhibited a unique mechanism of action, in which structural and functional damage to mitochondria triggers activation of the Ca2+-dependent apoptosis pathway. Moreover, OSW-1 appeared less toxic to normal or nonmalignant cells than to tumor cells in vitro. The exact mechanisms responsible for such selectivity remain unclear. It is possible that cancer cells have alterations in mitochondria and in calcium regulation that are not found in normal cells, making them more vulnerable to OSW-1. It should also be noted that, although nonmalignant cells appeared less sensitive to OSW-1 than cancer cells, the IC50 values for the normal cells were in the nanomolar range, suggesting that this compound is still toxic to normal cells. Thus, it is essential to perform vigorous animal toxicology studies before considering clinical evaluation of this compound. Targeting strategies such as antibody-mediated drug delivery may improve therapeutic selectivity and should be considered in any future development of this potent compound as a potential novel anticancer agent.
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NOTES |
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Present address: Dennis A. Carney, Department of Haematology, Peter MacCallum Cancer Centre and University of Melbourne, Melbourne, Victoria, Australia.
We gratefully acknowledge Dr. Paul Chiao for providing AsPC-1 cells. This work was supported in part by grants CA85563, CA105073, CA109041, and CA16672 from the National Cancer Institute, the National Institutes of Health, and a grant from the Lockton Family Foundation.
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Manuscript received March 4, 2005; revised September 21, 2005; accepted October 27, 2005.
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