Int. J. Cancer: 73, 725–728 (1997) r 1997 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer INTRACELLULAR Ca21 RELEASE MEDIATES URSOLIC ACID–INDUCED APOPTOSIS IN HUMAN LEUKEMIC HL-60 CELLS Jin Hyen BAEK1, Yong Soo LEE2, Chang Mo KANG1, Jung-Ae KIM3, Kyung Sool KWON4, Han Chul SON4 and Kyu-Won KIM1* 1Department of Molecular Biology, Pusan National University, Pusan 609-735, Korea 2Department of Physiology, College of Medicine, Kwandong University, Kangnung 210-701, Korea 3College of Pharmacy, Yeungnam University, Kyongsan 712-749, Korea 4Pusan Cancer Research Center, Pusan National University, Pusan 602-739, Korea The effect of ursolic acid (UA) on tumor cell apoptosis was investigated using HL-60 human promyelocytic leukemia cells as a model cellular system. Treatment with UA resulted in a concentration-dependent decreased cell viability assessed by MTT assay. UA also induced genomic DNA fragmentation, a hallmark of apoptosis, indicating that the mechanism by which UA induced cell death was through apoptosis. The intracellular Ca21 level was increased by treatment with UA. Intracellular Ca21 inhibitors, such as intracellular Ca21release blockers (dantrolene, TMB-8 and ruthenium red) and an intracellular Ca21 chelator (BAPTA/AM), significantly blocked the UA-induced increased intracellular Ca21 concentration. These inhibitors also blocked the effects of UA on cell viability and apoptosis. These results suggest that enhanced intracellular Ca21 signals may be involved in UA-induced apoptosis in HL-60 cells. Int. J. Cancer 73:725–728, 1997. r 1997 Wiley-Liss, Inc. Ursolic acid (UA), a pentacyclic triterpene acid, has been isolated from many kinds of medicinal plant, such as Eriobotrya japonica, Rosmarinus offıcinalis and Glechoma hederaceae. UA has been reported to produce anti-tumor activities, including inhibition of skin tumorigenesis (Huang et al., 1994) and inhibition of tumor promotion (Tokuda et al., 1986). It also induced tumor cell differentiation by regulation of the expression of differentiationspecific genes in mouse F9 teratocarcinoma cells (Lee et al., 1994). In addition, UA was shown to possess an anti-angiogenic effect in chick chorioallantoic membrane (Sohn et al., 1993) and an anti-invasive activity in HT1080 human fibrosarcoma cells (Cha et al., 1996). Apoptosis has been reported to be involved in the carcinogenic process (Thompson, 1995; Isaacs, 1993) and in tumor therapy and prevention (Bursch et al., 1992). Many of the presently used chemotherapeutic agents of cancer induce apoptosis (Miyashita and Reed, 1993). Increased intracellular Ca21 concentration has been demonstrated to act as an important mediator of apoptosis in a variety of cells (Dow, 1995). Retinoic acid, a differentiation-inducing agent, has been shown to induce tumor cell apoptosis in mouse F9 teratocarcinoma cells (Atencia et al., 1994). Since UA was reported to have an activity of tumor cell differentiation (Lee et al., 1994), in this study, we investigated the possibility that UA can induce tumor cell apoptosis using HL-60 cells as a model cellular system. We also examined the role of intracellular Ca21 signals in the apoptosis induced by UA. MATERIAL AND METHODS Material The HL-60 human promyelocytic leukemia cell line was purchased from the ATCC (Rockville, MD). The powder for RPMI 1640 medium, FBS and antibiotics (penicillin and streptomycin mixture) were purchased from GIBCO (Grand Island, NY). UA, dantrolene (Dant), 3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8), ruthenium red (RR), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) and diphenylamine (DPA) were obtained from Sigma (St. Louis, MO). Bis-(o-aminophenoxy)-ethane-N,N,N8,N8-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) and 1-(2,5-carboxyoxazol-2-yl-6-aminobenzfuran-5oxyl)-2-(28-amino-58-methylphenoxy)-ethane-N,N,N8,N8-tetraace- toxymethyl ester (Fura-2/AM) were from Molecular Probes (Eugene, OR). Unless otherwise indicated, propidium iodide (PI) and all other chemicals were the purest grade available and were obtained from Sigma. UA and TMB-8 were dissolved in ethanol. Dant and BAPTA/AM were dissolved in DMSO, and other drugs were dissolved in distilled water. These stock solutions were sterilized by filtration through 0.2 µm disc filters (Gelman, Ann Arbor, MI). Cell culture HL-60 cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml of streptomycin at 37°C in a humidified incubator under 5% CO2/95% air. Cells were split in a 1:5 ratio every 2 or 3 days. Cells were treated with different concentrations of UA. UA was dissolved in ethanol with a final concentration of 0.1% (v/v). Cell viability test (MTT staining) Cell viability was assessed as described by Mosmann (1983). Briefly, cells were incubated in 100 µl of media in 96-well plates with the indicated concentration of the drugs for 24 hr at an initial cell density of 3 3 105 cells/ml. An appropriate volume of drug vehicle was added to untreated cells. After each period of incubation, 10 µl of MTT solution (5 mg MTT/ml in H2O) were added and cells incubated for a further 4 hr. One hundred microliters of acid-isopropanol (0.04 N HCl in isopropanol) were added to each culture and mixed by pipetting to dissolve the reduced MTT crystals. Relative cell viability was obtained by scanning with an ELISA reader (Molecular Devices, Menlo Park, CA) with a 570 nm filter. DNA isolation and electrophoresis HL-60 cells were collected by centrifugation (200g, 5 min), washed twice in PBS (pH 7.4) and resuspended at a density of 2 3 106 cells/200 µl in lysis buffer containing 50 mM NaCl, 5 mM EDTA and 150 mM Tris-HCl (pH 8.0). To each 200 µl of cell suspension, 20 µl of proteinase K (25 µg/µl) and 20 µl of 10% SDS were added. Samples were incubated at 50°C for 48 hr. Ten microliters of RNase A (100 U/200 µl) were added and the incubation continued for a further 60 min. DNA was then extracted with phenol:chloroform (24:1), precipitated by the addition of 0.1 vol of 5 M NaCl and 1 vol of isopropyl alcohol and visualized by electrophoresis in 1.5% agarose. The pattern of DNA fragmentation was photographed under UV light, after staining the gel with ethidium bromide (0.5 µg/ml in TAE buffer). Quantitative analysis of fragmented DNA For quantitative DNA analysis, HL-60 cells were collected and washed twice with PBS. Cells were resuspended in lysis buffer Contract grant sponsors: Korea Science and Engineering Foundation and the Ministry of Education, Korea. *Correspondence to: Department of Molecular Biology, Pusan National University, Pusan 609-735, Korea. Fax: 82-51-513-9258. E-mail: [email protected] Received 29 April 1997; Revised 4 July 1997 BAEK ET AL. 726 FIGURE 1 – Decreased cell viability by UA in HL-60 human promyelocytic leukemia cells. Cell viability was assessed by MTT staining. Results are expressed as the percent change of the control condition in which cells were grown in medium containing drug-free vehicle. Data points represent the mean values of 4 replicates, with the bars indicating SEM. containing 1 mM EDTA, 10 mM Tris-HCl (pH 8.0) and 0.2% Triton X-100 and incubated on ice for 30 min (Sentman et al., 1991). Low and high molecular weight DNAs were separated by centrifugation at 15,000 g at 4°C. The supernatant was collected and the pellet resuspended in 0.5 ml of lysis buffer. DNA from both supernatant and pellet was precipitated by addition of 1 N perchloric acid. The DPA method (Bhalla et al., 1992) was used for measurement of DNA content. The percent change of DNA fragments was calculated with the following equation: % Fragment 5 [A570 of small DNA/(A570 of small DNA 1 A570 large DNA) 3 100]. Measurement of [Ca21]i Aliquots of HL-60 cells, cultured for 3–5 days, were washed in Eagle’s basal salt solution (EBSS). Then, 2 µM Fura-2/AM were added and the cells incubated for 60 min at room temperature (22°–23°C). Unloaded Fura-2/AM was removed by centrifugation at 150 g for 3 min. Cells were resuspended at a density of 2 3 106/ml in Ca21-free Krebs-Ringer buffer (KRB) containing 125 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 mM NaHCO3 , 25 mM HEPES and 6 mM glucose (pH 7.4); transferred to a quartz cuvette; and stirred continuously. Fluorescence emission (510 nm) was monitored with the excitation wavelength cycling between 340 and 380 nm at 37°C using a Hitachi F2000 fluorimeter. At the end of an experiment, maximum and minimum values of fluorescence at each excitation wavelength were obtained by first lysing the cells with 0.1% Triton X-100 (maximum) and then adding 10 mM EGTA (minimum). With the maximum and minimum values, the 340:380 nm fluorescence ratios were converted into free Ca21 concentration using the Fura-2 Ca21-binding constant (224 nM) and the formula described by Grynkiewicz et al. (1985). parisons; p values less than 0.05 are considered to be statistically significant. Data analysis All experiments were performed 4 times. All data were displayed as percentages of the control condition. All control experiments were carried out in the same media containing drug-free vehicle. Data were expressed as mean 6 SEM and analyzed using 1-way ANOVA and the Student-Newman-Keul’s test for individual com- Induction of apoptosis by UA As shown in Figure 1, UA decreased cell viability of HL-60 cells in a dose-dependent manner. Ethanol alone, at a final concentration of 0.1% (v/v) used for the dissolution of UA, did not affect cell FIGURE 2 – UA induces apoptotic cell death in HL-60 human promyelocytic leukemia cells. Cells were treated for 24 hr without or with each concentration of UA. DNA was isolated from the cells and analyzed by 1.5% agarose gel electrophoresis (a). Lane M represents DNA marker. Dose-dependent effect of UA on DNA fragmentation in HL-60 human promyelocytic leukemia cells (b). Cells were treated for 24 hr without or with each concentration of UA. The amount of fragmented DNA was measured by the DPA method. Results are expressed as the percent change of DNA fragments compared to the control condition in which cells were grown in medium containing drug-free vehicle. Data points represent mean values of 4 replicates, with the bars indicating SEM. RESULTS UA-INDUCED APOPTOSIS IN HL-60 CELLS 727 viability. To determine whether cell death induced by UA occurs through an apoptotic pathway, we tested morphological changes with PI staining. We observed that morphological changes corresponding to apoptosis, including cell shrinkage and cytoplasmic and nuclear membrane blebbing, were elicited by 10 µM of UA, which induced extensive decreased cell viability (data not shown). An assay for genomic DNA fragmentation, a hallmark of apoptotic cell death, was performed. As shown in Figure 2a, treatment with UA produced DNA fragmentation, indicating that UA induced apoptosis in the HL-60 cells. In addition, UA increased the quantity of DNA fragments in a dose-dependent manner in HL-60 cells (Fig. 2b). Effect of UA on [Ca21]i Increased [Ca21]i has been shown to induce Ca21/Mg21dependent endonuclease activation and apoptosis (Thompson, 1995). To investigate the interrelationship between the UA-induced increased [Ca21]i and apoptosis, we monitored the changes of [Ca21]i by UA in HL-60 cells. Treatment of cells with 10 µM UA increased [Ca21]i in 2 phases, initial increase and late sustained increase (Fig. 3a). We tested the effect of intracellular Ca21 release blockers on the UA-induced increased intracellular Ca21. The UA-induced increased intracellular Ca21 was inhibited significantly by treatment with Dant or TMB-8, inhibitors of intracellular Ca21 release (Rittenhouse-Simmons and Deykin, 1978; Zhang and Melvin, 1993), or RR, an inhibitor of intracellular Ca21 release, specifically from the ryanodine-sensitive Ca21 pools (Ehrlich et al., 1994) (Fig. 3b–d). BAPTA/AM, an intracellular Ca21 chelator (Zuker and Steinhardt, 1978), showed results similar to those depicted in Figure 3e. FIGURE 4 – Effect of intracellular Ca21 inhibitors on UA-induced decreased viability of HL-60 human promyelocytic leukemia cells. Intracellular Ca21 inhibitors (25 µM Dant, 5 µM TMB-8, 0.5 µM RR and 0.1 µM BAPT/AM) were added to the cells 4 hr before treatment with UA. Cell viability was assessed by MTT staining. Results are expressed as the percent change of the control condition in which cells were grown in medium containing drug-free vehicle. We also tested the drug-free vehicles and did not find any effects of those alone. Data represent mean values of 4 replicates, with the bars indicating SEM. #p , 0.05 compared to control. *p , 0.05 compared to UA alone. Effect of intracellular Ca21 inhibitors on UA-induced apoptosis To investigate whether these intracellular Ca21 inhibitors also block UA-induced apoptosis in HL-60 cells, we examined the effects of these agents on the UA-induced decreased cell viability and DNA fragmentation. As shown in Figure 4, pre-treatment with these intracellular Ca21 inhibitors significantly blocked the UA- FIGURE 5 – Effect of intracellular Ca21 inhibitors on UA-induced DNA fragmentation in HL-60 human promyelocytic leukemia cells. Intracellular Ca21 inhibiors (25 µM Dant, 5 µM TMB-8, 0.5 µM RR and 0.1 µM BAPTA/AM) were added to the cells 4 hr before treatment with UA. The amount of fragmented DNA was measured by the DPA method. Results are expressed as the percent change of DNA fragments compared to the control condition in which cells were grown in medium containing drug-free vehicle. We also tested the drug-free vehicles and did not find any effects of those alone. Data points represent mean values of 4 replicates, with the bars indicating SEM. #p , 0.05 compared to control. *p , 0.05 compared to UA alone. induced decreased cell viability. DNA fragmentation induced by treatment of 10 µM UA also was decreased significantly by pre-treatment with these intracellular Ca21 inhibitors (Fig. 5). In both experiments, we did not find any effects of the Ca21 inhibitors alone (data not shown). DISCUSSION FIGURE 3 – Effect of intracellular Ca21 inhibitors on UA-induced increased [Ca21]i in HL-60 human promyelocytic leukemia cells. Data represent intracellular Ca21 changes with time. Arrows show the time points for addition of 10 µM UA. Intracellular Ca21 inhibitors were applied 3 min before fluorescence measurements. In this study, we found that UA induced apoptosis in HL-60 cells (Figs. 1, 2). The apoptosis-inducing activity of UA also was found in some other tumor cell lines, such as F9 teratocarcinoma cells and HepG2 human hepatocellular carcinoma cells (data not shown). Previous reports demonstrated that UA has anti-tumor activities, such as inhibition of tumorigenesis (Huang et al., 1994), inhibition of tumor promotion (Tokuda et al., 1986) and induction of tumor cell differentiation (Lee et al., 1994). UA also has been shown to 728 BAEK ET AL. inhibit effectively angiogenesis (Sohn et al., 1993) and invasion (Cha et al., 1996), which are importantly involved in tumor metastasis (Claffey et al., 1996; Meitar et al., 1996). Thus, these previous and present results suggest that UA is an effective anti-cancer agent acting at the various stages of tumor development. Increased [Ca21]i appears to be involved in apoptosis in a variety of cells (Dow, 1995). Although the precise mechanism by which intracellular Ca21 mediates apoptosis is not known, the endogenous Ca21/Mg21-dependent endonuclease, which cleaves doublestrand DNAs at nucleosome linker regions, remains an attractive target for the effect of Ca21 (Wyllie, 1980; Arends et al., 1990). This endogenous enzyme appears to be activated by mobilization of cytosolic Ca21 (McConkey et al., 1989). The results of the present study show that UA increases [Ca21]i in HL-60 cells in 2 phases, initial and late sustained increases (Fig. 3). Although the source of UA-induced increased [Ca21]i is not defined clearly in this study, various intracellular Ca21 release blockers inhibited the UA-induced increased [Ca21]i (Fig. 3), suggesting that UA may mobilize intracellular Ca21 from the endoplasmic reticulum in HL-60 cells. The UA-induced decreased cell viability and apopto- sis were significantly decreased by several intracellular Ca21 release blockers (Figs. 4, 5), indicating that UA mobilizes intracellular Ca21, which then activates the apoptotic processes in tumor cells. However, the precise mechanism of the apoptosis-inducing action of UA is unknown at present. 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