Induction of CDK Inhibitors (p21 and p27) and Bak in the b-Lapachone-Induced Apoptosis of Human Prostate Cancer Cells

b-Lapachone, a novel anti-neoplastic drug, induces various cancer cells to undergo apoptosis. In a previous report, we showed that b-lapachone-induced apoptosis of HL-60 cells is mediated by oxidative stress. However, in the present study, we found that b-lapachone-induced apoptosis of human prostate cancer (HPC) cells may be independent of oxidative stress. In contrast to the 10-fold b-lapachone-induced increase in H2O2 production seen in HL-60 cells, only a 2to 4-fold increase was observed in HPC cells. N-acetyl-L-cysteine (NAC), a thiol antioxidant, inhibited the apoptosis in DU145 cells after 12 h exposure to b-lapachone. Nonetheless, NAC, along with other antioxidants, failed to exert similar effect in HPC cells subjected to b-lapachone treatment for 24 h. Under this premise, we suggest that the oxidative stress may not play a crucial role in b-lapachone-mediated HPC cell apoptosis. Here we demonstrate that damage to genomic DNA is the trigger for the apoptosis of HPC cells induced by b-lapachone. According to our results, b-lapachone stimulates DNA dependent kinase expression and poly(ADP-ribose) polymerase cleavage in advance of significant morphological changes. b-Lapachone promotes the expression of cyclin-dependent kinase (cdk) inhibitors (p21 and p27), induces bak expression, and subsequently stimulates the activation of caspase-7 but not of caspase-3 or caspase-8 during the apoptosis of HPC cells. Taken together, these results suggest that the signaling pathway involving the b-lapachone-induced apoptosis of HPC cell may be by DNA damage, induction of cdk inhibitors (p21 and p27), and then subsequent stimulation of caspase-7 activation. b-Lapachone (3,4 dihydro-2,2-dimethyl-2H-naphtho[1,2-b] pyran-5,6-dione), a natural plant product extracted from the lapacho tree (Tabebuia avellanedae), has been shown to have a variety of pharmacological effects, including antiviral, antibacterial, antifungal, and antiparasitic activities (Docampo et al., 1979; Goncalves et al., 1980; Schaffner-Sabba et al., 1984). Recently, it was also shown to have a significant antineoplastic effect on various cancer cells (Li et al., 1995; Chau et al., 1998; Lai et al., 1998). b-Lapachone is a DNA Topo I inhibitor and produces its cytotoxicity by directly interacting with the Topo I enzyme, rather than by stabilizing the DNA-cleavable complex (Li et al., 1993). Furthermore, it increases the sensitivity of tumor cells to DNAdamaging agents, such as methylmethanesulfonate or ionizing radiation (Boorstein and Pardee, 1983), and interrupts the cell cycle (Boothman and Pardee, 1989). It is also suggested to be a DNA Topo IIa inhibitor, because it suppresses the growth of yeast and blocks the DNA-unwinding enzyme Topo IIa in vitro (Frydman et al., 1997; Neder et al., 1998). However, in contrast to other Topo-inhibitors, such as camptothecin or etoposide, as shown by alkaline or neutral filter elution studies, b-lapachone does not bind covalently to the Topo I-DNA complex or other protein-DNA complexes (Li et al., 1993; Frydman et al., 1997; Wuerzberger et al., 1998). In terms of its chemical structure, b-lapachone is a lipophilic O-naphthoquinonic compound. Many quinonic compounds are widely used as chemotherapeutic agents and exert their cellular toxicity by stimulating intracellular free radical production in mitochondrial and microsomal fractions (Dubin et al., 1990; Fry and Pudney, 1992; Henry and Wallace, 1995). This work was supported by Grants NSC89-2314-B-010-029 from the National Science Council, VTY88-G5–04 from Veterans General Hospital, TsinHua, Yang-Ming Research Program and awarded by Medical Research and Advancement Foundation in Memory of Dr. Chi-Shuen Tsou. ABBREVIATIONS: b-lapachone, 3,4 dihydro-2,2-dimethyl-2H-naphtho[1,2-b] pyran-5,6-dione; Topo, topoisomerase; ROS, reactive oxygen species; cdk, cyclin-dependent kinase; HPC, human prostate cancer; DCFH-DA, 29,79-dichlorofluorescein diacetate; HE, hydroethidine bromide; NAC, N-acetyl-L-cysteine; AO, acridine orange; TUNEL, terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling; PI, propidium iodide; DNA-PK, DNA-dependent kinase; DAB, diaminobenzidine; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; PARP, poly(ADP-ribose) polymerase. 0026-895X/01/5904-784–794$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 4 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 134/893399 Mol Pharmacol 59:784–794, 2001 Printed in U.S.A. 784 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from Moreover, the b-lapachone-mediated cell death of human leukemic cells involves the up-regulation of intracellular ROS generation and the subsequent initiation of c-Jun amino terminal kinase and caspase-3 activation (Chau et al., 1998; Planchon et al., 1999; Shiah et al., 1999). Currently, it is still unclear whether b-lapachone mediates apoptosis in cancer cell lines by acting as a DNA Topo inhibitor or a free radical generator. The progression of the cell cycle is regulated by a number of cyclin-dependent kinases (cdks). Binding of cdks to cyclins results in the activation of the cdks and passage through specific cell cycle checkpoints. The proteins p21 and p27 act primarily by inhibiting the kinase activities of cdk, thus blocking cell cycle progression (Zi et al., 1998; Robson et al., 1999). Prostate cancer is an epidemic disease in the world and is also the second leading cause of cancer deaths in American men. The number of patients who died from prostate cancer in 1997 accounts for 14% of all cancer deaths in American men (Parker et al., 1997). The main curative treatment for prostate cancer at early stage includes surgery, radiation therapy, and hormone ablation. However, the results of hormone ablation and prostatectomy for patients with metastatic prostate cancer have been disappointing because most metastatic prostate cancer cells become androgen-independent and escape apoptosis induced by androgen ablation and by many cytotoxic drugs (Marcelli et al., 1999). The aims of the present study, therefore, were to study 1) whether the cytotoxic effect of b-lapachone on androgenindependent human prostate cells is mediated by ROS production and 2) whether b-lapachone interrupts the cell cycle by the induction of cdk inhibitors. In addition, changes in expression of members of the bcl-2 protein family and caspase activity were also examined, because their involvement in the apoptotic pathway is well known. In the present study, we demonstrate that the b-lapachone-induced cell death of HPC cells may not be mediated by ROS production. Results using DCFH-DA and HE probes showed that b-lapachone does not induce a large amount of ROS production in these cells. Furthermore, b-lapachoneinduced cell death is not prevented by antioxidant treatment. Western blotting and immunocytochemical studies showed that b-lapachone induces DNA-PK expression and PARP cleavage, these events happening in advance of any morphological changes. In addition, b-lapachone up-regulates cdk inhibitors (p21 and p27), promotes bak expression, and induces activation of caspase-7, but not of caspase-3 or caspase-8. Materials and Methods Chemicals. b-Lapachone, prepared according to the procedures described by Schaffner-Sabba et al. (1984), was dissolved as a 20 mM stock solution in ice-cold absolute alcohol and stored in aliquots at 220°C. 29,79-Dichlorofluorescein diacetate (DCFH-DA) and hydroethidine bromide (HE), obtained from Molecular Probes (Fremont, CA), were dissolved in DMF at a concentration of 2 mM. Other chemicals were obtained from Sigma (St. Louis, MO). Cell Cultures. The human prostate cancer cell lines (DU145 and PC-3) used in this study were obtained from the American Type Culture Collection (Manassas, VA). Cells (2 3 10) were grown for 24 h at 37°C in a humidified 5% CO2 atmosphere in RPMI medium supplemented with 10% FCS, 2 mM L-glutamine, and 100 U/ml each of penicillin and streptomycin. They were then washed twice with prewarmed PBS and cultured in serum-free medium for an addi-