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OSW-1: a Natural Compound With Potent Anticancer Activity and a Novel Mechanism of Action

Yan Zhou, Celia Garcia-Prieto, Dennis A. Carney, Rui-hua Xu, Helene Pelicano, Ying Kang, Wensheng Yu, Changgang Lou, Seiji Kondo, Jinsong Liu, David M. Harris, Zeev Estrov, Michael J. Keating, Zhendong Jin, Peng Huang

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).


    ABSTRACT
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The naturally occurring compound 3{beta},16{beta},17{alpha}-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-{beta}-D-xylopyranosyl)-(1->3)-(2-O-acetyl-{alpha}-L-arabinopyranoside) (OSW-1) is found in the bulbs of Ornithogalum saudersiae and is highly cytotoxic against tumor cell lines. Using various human cancer and nonmalignant cell lines, we investigated the anticancer activity and selectivity of OSW-1 and its underlying mechanisms of action. OSW-1 exhibited extremely potent cytotoxic activity against cancer cells in vitro. Nonmalignant cells were statistically significantly less sensitive to OSW-1 than cancer cells, with concentrations that cause a 50% loss of cell viability 40–150-fold greater than those observed in malignant cells. Electron microscopy and biochemical analyses revealed that OSW-1 damaged the mitochondrial membrane and cristae in both human leukemia and pancreatic cancer cells, leading to the loss of transmembrane potential, increase of cytosolic calcium, and activation of calcium-dependent apoptosis. Clones of leukemia cells with mitochondrial DNA defects and respiration deficiency that had adapted the ability to survive in culture without mitochondrial respiration also were resistant to OSW-1. In vitro analysis revealed that OSW-1 effectively killed primary leukemia cells from chronic lymphocytic leukemia patients with disease refractory to fludarabine. The promising anticancer activity of OSW-1 and its unique mechanism of action make this compound worthy of further investigation for its potential to overcome drug resistance.


Previous studies have shown that 3{beta},16{beta},17{alpha}-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-{beta}-D-xylopyranosyl)-(1->3)-(2-O-acetyl-{alpha}-l- arabinopyranoside) (OSW-1), a compound found in the bulbs of Ornithogalum saudersiae, is highly cytotoxic against several malignant tumor cell lines in vitro (113). In addition, OSW-1 appears to prolong the life span of mice bearing P388 leukemia cells (2). The mechanism by which OSW-1 exerts its anticancer activity is poorly understood. One study suggested that OSW-1 might decrease the levels of ovarian steroids by inhibiting steroidal enzyme expression (14), but the importance of this inhibition remains unclear. The limited availability of OSW-1 resulting from the complex chemical synthesis of this compound (3,4) might in part have constrained the research progress in this area. Jin et al. have recently developed an efficient strategy for synthesizing OSW-1 with excellent yield (9,10), which should allow for more in-depth studies of this compound.

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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Table 1.  IC50 values of OSW-1 in malignant and nonmalignant human cells in vitro*

 
When cells from similar tissue types were compared, the cancer cells were approximately 40–150 times more sensitive to OSW-1 than the respective nonmalignant cells. For example, the average IC50 values (by MTT assay) for leukemia cells and normal lymphocytes were 0.04 nM (95% CI = 0.02 to 0.06) and 1.73 nM (95% CI = 0.12 to 3.35), respectively, with a selectivity index (i.e., the IC50 ratio of normal to malignant cells) of approximately 40. Similarly, the IC50 values for malignant brain tumor U87-MG cells and normal astrocytes were 0.05 nM and 7.13 nM, respectively, for a selectivity index of approximately 150. Thus, OSW-1 seems to have a preferential cytotoxicity against cancer cells in vitro. The antiproliferative effect of OSW-1 was further confirmed by another method using a Coulter particle analyzer (Beckman Coulter Inc.; Hialeah, FL) to directly count the cell numbers. By direct cell counting, the IC50 value for HL-60 cells after incubation with OSW-1 for 72 hours was 0.06 nM. This value was similar to the IC50 value of 0.04 nM obtained by the MTT assay.

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{beta} subcomplexes 1 and 4, NADH dehydrogenase 1{alpha} 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 morphology—including a blurred membrane outline, disappearance and/or disorganization of the cristae, and paling of the mitochondrial matrix—were 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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Fig. 1. Induction of mitochondrial damage and activation of Ca2+-dependent apoptosis in HL-60 leukemia cells by 3{beta},16{beta},17{alpha}-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-{beta}-D-xylopyranosyl)-(1->3)-(2-O-acetyl-{alpha}-L-arabinopyranoside) (OSW-1). A) Mitochondria of control cells, i.e., without drug treatment. B) Mitochondria of cells treated with 1 nM OSW-1 for 14 hours. The cells were first fixed in 3% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3 for 60 minutes, then stained with uranyl acetate and lead citrate in an LKB Utrastainer, and examined with a JEM 1010 transmission electron microscope (JEOL, USA, Inc.; Peabody, MA). C) Flow cytometric analysis of cytosolic calcium in cells treated with 1 nM OSW-1 for 6 hours. D) Flow cytometric analysis of cytosolic calcium in cells treated with thapsigargin (Thap) as a positive control for calcium release. E–H) Apoptosis analysis of cells treated for 14 hours with 1 nM OSW-1 in either the presence or absence of the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-acetoxymethyl ester (BAPTA-AM) (1 µM, added 1 hour before OSW-1) as indicated. Apoptosis was quantified by flow cytometric analysis in cells that were stained with annexin-V–FITC and propidium iodide as previously described (16). The percentages in the lower left quadrant indicate the percentage of intact cells. The x-axis indicates annexin-V–FITC fluorescence intensity; the y-axis indicates propidium iodide fluorescence intensity.

 


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Fig. 2. Effect of 3{beta},16{beta},17{alpha}-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-{beta}-D-xylopyranosyl)-(1->3)-(2-O-acetyl-{alpha}-L-arabinopyranoside) (OSW-1) on cellular DNA contents and mitochondrial transmembrane potential in parental HL-60 cells and respiration-deficient C6F cells. A) Cells were incubated with 0.5 nM OSW-1 for 0, 8, 16, and 24 hours as indicated. The cells were fixed and stained with propidium iodide, and cellular DNA content was analyzed by flow cytometry. Sub G1, G2, S, and G2/M refer to the DNA content in each phase of the cell cycle. B) Cells were treated with 0.5 nM of OSW-1 for 0, 4, 16, and 24 hours as indicated, labeled with 1 µM rhodamine-123 for 60 minutes, and the mitochondrial transmembrane potential was determined by flow cytometry as previously described (19). The x-axis indicates arbitrary units of mitochondrial transmembrane potential (TMP); the y-axis indicates cell counts.

 
Because mitochondria play an important role in maintaining a proper level of cytosolic calcium (17), we examined whether the mitochondrial damage resulting from exposure to OSW-1 might change cytosolic calcium levels. HL-60 cells were treated with various concentrations of OSW-1 for up to 6 hours, and cytosolic calcium was measured by flow cytometric analysis using a calcium-specific fluorescent dye (Calcium Green-1). As illustrated in Fig. 1, C, treatment with 1 nM OSW-1 induced a statistically significant increase of cytosolic calcium, from 9.3 (arbitrary units) to 17.6 (P<.001). For a positive control, cells were treated with thapsigargin, which is a high-affinity inhibitor of Ca2+-ATPase that causes an increase in cytosolic calcium. The degree of OSW-1-induced increase of cytosolic calcium was similar to that observed in cells treated with thapsigargin (Fig. 1, D). A similar increase of cytosolic calcium was also observed in human pancreatic AsPC-1 cells treated with OSW-1 (data not shown).

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 18–24 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 ({Delta}{Psi}) showed that OSW-1 induced a time-dependent loss of {Delta}{Psi} 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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Yan Zhou and Celia Garcia-Prieto contributed equally to this work.

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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