Effects of 1, 6-Bis[4-(4-amino-3-hydroxyphenoxy)phenyl]diamantane (DPD), a reactive oxygen species and apoptosis inducing agent, on human leukemia cells in vitro and in vivo

Abstract

1, 6-Bis[4-(4-amino-3-hydroxyphenoxy)phenyl]diamantine (DPD), a new cytostatic and differentiation inducing agent, was found to inhibit the growth of several cancer cell lines in the National Cancer Institute (NCI) Anticancer Drug Screen system. Previously, we demonstrated that DPD inhibited the growth of human colon cancer cell lines both in vitro and in vivo. In this study, we examined the anticancer effects of DPD on two human leukemia cells lines. DPD exerted growth inhibitory activities in vitro against two human leukemia cell lines, the promyeloid line HL-60 and the lymphoblastic line Molt-3. The in vivo effect of tumor growth suppression by DPD was also observed in mouse xenografts. No acute toxicity was observed after an intra-peritoneal challenge of DPD in “severe combined immune-deficiency” (SCID) mice twice a week. The in vitro study showed HL-60 was more sensitive to DPD than Molt-3 through induction of G₀/G₁ cell-cycle arrest with the appearance of a hypodiploid DNA fraction. The increased superoxide (O₂⁻), dissipation of the mitochondrial membrane potential, activation of caspase 3, and increase in annexin V binding were evident before apoptosis in DPD-treated cells. The superoxide dismutase 1 (SOD1) mRNA expression was also decreased in DPD-treated HL-60 and Molt-3 cells. Thus, it appeared that inhibition of SOD might be the major cause for the production of cellular superoxide with concomitant decrease of H₂O₂ in DPD-treated cells. Addition of antioxidant can reduce DPD-induced mitochondrial damage, caspase activation, and annexin V binding in HL-60 cells. The results suggest that the cellular generation of O₂⁻ plays a role in initiating and coordinating DPD-mediated growth arrest and apoptosis of HL-60 cells. Importantly, addition of arsenic trioxide, a compound capable of reactive oxygen species (ROS) generation, significantly enhanced the in vitro activity of DPD. These results suggest that DPD appears to be a potential new modality in human leukemia therapy.

Introduction

Adamantane and diamantine derivatives possess several attractive pharmacological activities such as antibacterial, antifungal, antiviral, and anticancer effects (Aigami et al., 1975, Wang et al., 1997, Wang et al., 1998). Our previous study has found that N-1-adamantylmaleimide (AMI) and dimethyladamantylmaleimide (DMAMI) induces apoptosis and inhibits the growth of human gastric (SC-M1) and colon (Colo 205) cancer in SCID mice, respectively (Wang et al., 1998, Wang et al., 2002). In a recent study, we characterized the anticancer activities of diamantane derivatives using the 60 human cancer cell lines in NCI Anticancer Drug Screen, and evaluated the structure–activity relationship. 1, 6-Bis[4-(4-amino-3-hydroxyphenoxy) phenyl] diamantane (DPD) exhibited marked anticancer activities on the sub-panel of 60 human cancer cell lines. Strong anticancer effects of DPD were observed against one leukemia (HL-60), one NSCLC cancer (HOP-92), one colon cancer (Colo 205), one ovarian cancer (OVCAR-8), and one breast cancer (T-47D) cell line with a GI₅₀ of 0.50, 0.85, 1.31, 0.62, and 0.75 μM, respectively (Wang et al., in press). Recently, we demonstrated that administration of DPD induced G₀/G₁ arrest and differentiation in human colon cancer cells. DPD also shows in vivo anticancer activity on human colon cancer cells xenografts with no obvious acute toxicity (Wang et al., 2003).

Acute promyelocytic leukemia (APL) is typically treated with anthacycline-based chemotherapy and all-trans retinoic acid (ATRA). Approximately 85–95% of patients achieve complete remission; however, the relapse rate has been reported to be about 30–40%. APL relapse, in general, has been in part attributable to repetitive or prolonged exposure to ATRA and the possibility of additional chromosomal changes, making the disease more refractory to treat (Burry and Seki, 2002). Overall survival in adult acute lymphoblastic leukemia (ALL) is at most 50% and progression rates in high risk pediatric ALL remain at 20–30% (Pui and Evans, 1998). Therefore, developing new therapeutic drugs for refractory or recurrent leukemia is a worthwhile task. Disordered proliferation is one of the characteristics of most malignant tumors. The defective apoptotic pathways play a pivotal role in the risk of mammary tumorigenesis. Multiple genetic alterations in tumor cells affect the regulation of the cell cycle machinery (Hunter and Pines, 1994). Effective chemotherapeutic agents, therefore, may prove to be those that promote both growth arrest and apoptosis. Common pathways of apoptosis implicate critical events like changes in cellular morphology, generation of reactive oxygen species (ROS), activation of caspases as well as increased binding of annexin V. ROS play a important role in cell death by apoptosis or by necrosis (Zhou et al., 2003). For example, chemical agents such as 1-β-D-arabinofuranosylcytosine (ara-C), daunorubicin, and cisplatin were reported to induce ROS and results in apoptosis of HL-60 cells (Ikeda et al., 1999). Mitochondrial respiration is the major biochemical pathway by which O₂⁻ is produced in the cells during oxidative phosphorylation for ATP generation.

In the present study, we examined the anticancer effect of DPD on leukemia cells; the acute promyelocytic leukemia, HL-60 (p53-null) and lymphoblastic leukemia, Molt-3 (wild type p53) (Jonveaux and Berger, 1991). We evaluated the in vitro effects of DPD on proliferation and induction of apoptosis in leukemia cells. In addition, we examined the in vivo activity of DPD in human leukemia cancer cells HL-60 xenografts. Arsenic trioxide (As₂O₃) can reduce the cell division rates and induce apoptosis in APL, has been shown with efficacy in patients with all-trans retinoic acid (ATRA)-resistant or relapsed APL (Chen et al., 1997, Miller, 2002). It has recently been shown that As₂O₃ may induce apoptosis in non-APL cell lines (Rojewski et al., 2002). Oxidative damage has been suggested to be a key mechanism by which arsenic trioxide cause cell death (Miller, 2002). On the basis of the ability of As₂O₃ to cause exogenous-free radical generation in cells, we expect that its combination with DPD may enhance cytotoxic activity. The present study demonstrated that a combination of As₂O₃ with DPD enhances the cytotoxic activity in HL-60 cells as compared to that of As₂O₃, or DPD alone. These results suggest that DPD may have potential clinical implications.