The Effects of Crinum asiaticum on the Apoptosis Induction and the Reversal of Multidrug Resistance in HL-60/MX2

The present study investigated the anti-proliferative and chemosensitizing effects of Crinum asiaticum var. japonicum against multi-drug resistant (MDR) cancer cells. The 80% methanol extract, chloroform (CHCI3) fraction and butanol (BuOH) fraction of C asiaticum inhibited the growth of mitoxantrone (MX) resistant HL-60 (HL-60/MX2) cells. When HL-60/MX2 cells were treated with the CHCI3 and BuOH fractions, DNA ladder and sub-G1 hypodiploid cells were observed. Furthermore, the fractions reduced Bcl-2 mRNA levels, whereas Bax mRNA levels were increased. These results suggest that the inhibitory effect of C. asiaticum on the growth of the HL-60/MX2 cells might arise from the induction of apoptosis. Treatment of HL-60/MX2 cells with the fractions markedly decreased the mRNA levels of the multi-drug resistance protein-1 and breast cancer resistance protein. The CHCI3 fraction and hexane fraction increased MX accumulation in HL-60/MX2 cells. These results imply that the CHCI3 fraction of C asiaticum plays a pivotal role as a chemosensitizer. We suggest that components of C asiaticum might have a therapeutic potential for the treatment of MDR leukemia.


INTRODUCTION
The development of multi-drug resistance (MDR) by tumor cells is a major obstacle to successful cancer chemotherapy (Riordan et al., 1979). MDR is the phenomenon by which exposure of tumor cells to a single cytotoxic agent results in cross-resistance to other, structurally unrelated, classes of cytotoxic compounds. MDR is multifactorial, and the strategies proposed to reverse MDR include targets from the apoptosis pathway, efflux transporters and so on. Increased expression of efflux proteins, which belong to the ATP-binding cassette (ABC) family of proteins, is a common feature of MDR (Allikmets et al., 1996;Goottesman et al., 1993), and overexpression of efflux proteins is associated with resistance to numerous anticancer agents (Larsen et al., 2000). The best known and weil studied are P-glycoprotein (P-gp; MDR1) encoded by the mdr1 gene (Endicott et al., 1989), multidrug resistance-associated protein-1 (MRP-1) encoded by the mrp1 gene (Cole et al., 1992), and breast cancer resistance protein (BCRP) encoded by the abcg2 gene (Doyle et al., 2003).
Another mechanism of MDR is related to resistance to apoptosis of multi-drug resistant cells induced by cytotoxic agents. The cellular moleeules involved in this mechanism include anti-apoptotic proteins such as BcI-2, which are overexpressed, and pro-apoptotic proteins such as Bax, which are down-regulated (Seilers et al., 1999).
Many studies have shown a link between tumor hypoxia and MDR. Under hypoxie conditions, the cytotoxicity of chemotherapeutic agents such as cisplatin, etoposide, bleomycin and mitomycin C, is reduced (Koch et al., 2003). Hypoxia-elicited chemotherapeutic resistance has been reported in a number of cell types, including fibroblasts, breast cancer cells, glioma cells and testicular germ cells (Koch et al., 2003;Kalra et al., 1993;Liang, 1996). Hypoxia-associated chemotherapeutic resistance is a broad phenomenon. Comerford et al. have reported that MDR1 gene expression and subsequent functional P-gp expression are dramatically upregulated in a hypoxia inducible factor-1 (HIF-1)-dependent manner in response to hypoxia (Comerford et al., 2002).
In this paper, we demonstrate that the 80% methanol (MeOH) extract and several solvent fractions of C. asiaticum decreased the survival rate of HL-60/MX2 cells by the induction of apoptosis, as weil as having a chemosensitizing effect that could increase the intracellular accumulation of drug and decrease the expression of MRP-1 and BCRP.

MATERIALS AND METHODS
Preparation of extract from C. asiaticum. C. asiaticum var. japonicum was collected in March 2003 at Jeju Island, South Korea. C. asiaticum was washed in distilled water, dried at room temperature and ground into a fine powder. The dried plant powder (100 g) was extracted with 3 I 80% methanol (MeOH) at room temperature for 3 days and then the supernatant was concentrated under a vacuum. The resulting crude extract (20 g) was suspended in water (1 I) and successively partitioned with hexane (1 I x 3), chloroform (CHCI 3 ; 1 I x 3), ethyl acetate (EtOAc; 1 I x 3) and n-butanol (BuOH; 1 I x 3), to give hexane (497 mg), CHCI 3 (162 mg), EtOAc (200 mg), BuOH (1160 mg) and HzO (4800 mg) fractions.
Cell cu/ture. The HL-60/MX2 cell line is a mitoxantrone resistant derivative of the human acute promye-loid leukemia cell line HL-60. The clone designated HL-60/MX2 was approximately 35 fold less sensitive to MX than HL-60 parental cells. The cell line was obtained from the American Type Culture Collection (ATCC) and was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/ml and 100 f,lg/ml, respectively) at 37°C in a humidified 5% COz atmosphere.
Cytotoxicity test. The effect of the 80% MeOH extract or the solvent fractions on the growth of HL-60/ MX2 cell was determined by measuring the metabolic activity using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (Carmichael et al., 1987). The MTT assays were performed as folIows: HL-60/MX2 (2.5 x 10 5 cells/ml) were treated for 4 days with 20, 50, 100 f,lg/ml of the 80% MeOH extract or the solvent fractions. After incubation, 0.1 mg (50 f,ll of a 2 mg/ ml solution) MTT (Sigma, Saint Louis, MO, USA) was added to each weil and the cells were then incubated at 37°C for 4 h. The plates were centrifuged at 1000 rpm for 5 min at room temperature and the media was then carefully aspirated. 150 f,ll dimethylsulfoxide was then added to each weil to dissolve the formazan crystals. The plates were read immediately at 540 nm on a micropiate reader (Amersham Pharmacia Biotech., USA). All the experiments were performed three times and the mean absorbance values were calculated. The results are expressed as the percentage of inhibition that produced a reduction in the absorbance by the treatment of crude extract or solvent fractions compared to the untreated controls.
Apoptosis assay. HL-60/MX2 cells (2.5 x 10 5 cells/ ml) were treated with 100 f,lg/ml of the 80% MeOH extract or the solvent fractions for 24 h. For the DNA fragmentation assay, the cells were collected by centrifugation and DNA was extracted using the Wizard genomic DNA purification kit (Promega, Madison, WI, USA). The DNA fragmentation pattern was analyzed by electrophoresis on a 1.5% aga rose gel containing 0.1 f,lg/ml ethidium bromide for 40 min at 100 V (Oberhammer et al., 1993). For the flow cytometric analysis to determine cell cycle phase distribution, the treated cells were washed twice with phosphate-buffered saline (PBS) and fixed in 70% ethanol for 30 min at 4°C. The cells were then rinsed with PBS and incubated in 50 f,lg/ml propidium iodide solution (PI; Sigma, Saint Louis, MO, USA) and 50 f,lg/ml RNase A in the dark for 30 min at 37°C. Flow cytometry analysis was performed using an EPICS-XL FACScan flow cytometer (Coulter, Miami, FL, USA). The DNA histograms obtained were analyzed to measure the proportion of sub-G1 hypodiploid cells (Sherwood et al., 1994).
Chemosensitizing effect of C. asiaticum. To determine MX uptake, HL-60/MX2 cells (1.0 x 10 6 cells/ml) were treated with 20, 50 and 100 Ilg/ml of the 80% MeOH extract or the solvent fractions of C. asiaticum in culture media for 30 min at 37°C. Then 5 Ilmol/I MX was added for a further 40 min incubation at 37°C, after which the cells were washed twice with ice-cold PBS and kept on ice until analysis. The measurement of cellular MX fluorescence were made using flow cytometry in which a focused argon laser beam (485 nm) excited the cells in a laminar sheath flow and their fluorescence emissions (547 nm) were collected to generate a histogram. Cellular fluorescence (x-axis) from intracellular accumulation of MX is plotted versus cell number (yaxis). In addition, HL-60/MX2 cells were treated with the solvent fractions of C. asiaticum in the presence of cyclospolin A or verapamil which are known to be chemosensitizers (List, 1996;Loor et al., 2002;Garrigues et al., 2002;Choi et al., 1998).
Statistical analyses. The student's t-test and oneway ANOVA were used to determine the statistical significance of differences between values for a variety of experimental and control groups. Data are expressed as a mean ± standard deviation (SD) from three independent experiments performed in triplicate. P-values of 0.05 or less were considered statistically significant.

RESULTS
The effect of C. asiaticum on the growth of HL-60/ MX2 cells. When HL-60/MX2 (2.5 x 10 5 cells/ml) were treated with 20, 50, 100 Ilg/ml of the 80% MeOH extract or the solvent fractions for 4 days, we found that 100 Ilg/ml of the MeOH extract, the CHCI3 fraction and the BuOH fraction inhibited cell growth by 40%, 53% and 73% respectively ( Table 1).
The effect of C. asiaticum on induction of apoptosis in HL-60/MX2 cells. DNA fragmentation, a hallmark of apoptosis, was detected by the DNA aga rose gel electrophoresis method (Fig. 1). DNA ladder bands were clearly detectable in HL-60/MX2 cells treated with the CHCI 3 fraction and the BuOH fraction. Furthermore, we quantified the cells in the sub-G1 population after treatment with the CHCI 3 fraction and the BuOH fraction by flow cytometry analysis. When treated with 100 8.9%  f.lg/ml of the fractions for 24 h, the percentage of cells in the sub-G1 fraction increased (Fig. 2). To investigate the possible mechanism underlying the induction of apoptosis by C. asiaticum, we examined the expression of BcI-2 and Bax in HL-60/MX2 cells after treatment with the MeOH extract or the solvent fractions of C. asiaticum. The expression of Bax increased markedly, while the expression of BcI-2 decreased in the cells treated with 100 mg/mi of the CHCI3 fraction or the BuOH fraction (Fig. 3).
Chemosensitizing effect of C. asiaticum in HL-60/ MX2 cells. To explore for the chemosensitizing effect of C. asiaticum in HL-60/MX2 cells, we examined whether C. asiaticum affected the intracellular accumulation of MX and mRNA expression levels of MDR-related genes. Flow cytometry revealed that the 80% MeOH extract, the hexane fraction, the CHCI3 fraction and the BuOH 1.2% L Fig. 2. The degree of apoptosis represented as the DNA content measured by flow cytometric analysis in HL-60/MX2 cells. HL-60/MX2 cells (2.5 x 10 5 cells/ml) were treated with 100 f,1g/ml of the 80% MeOH extract, the CHCI 3 fraction and the BuOH fraction from C. asiaticum for 24 h. For the measurement of the sub-G1 hypodiploid cells, the flow cytometric analysis was performed as described in materials and methods. HL-60/MX2 cells (1.0 x 10 6 cells/ml) were treated with 1 00 ~g/ml of the 80% MeOH extract or several solvent fractions from C. asiaticum and f10w cytometry analysis of MX accumulation was performed as described in the materials and methods. The results are expressed as the percentage of cell population that produced cellular f1uorescence by intracellular accumulation of MX. All experiments were performed in triplicate. Data are presented as a mean ± SD from three separate experiments. *p< 0.05, **p < 0.01 compared with the untreated control. fraction of C. asiaticum increased accumulation of MX in HL-60/MX2 cells (Table 2). Moreover, 100 fl9/ml of the CHCI 3 fraction or the hexane fraction caused a right shift in the fluorescence peak associated with MX (Fig.  4). When HL-60/MX2 cells were treated with the CHCI 3 fraction or the hexane fraction of C. asiaticum in the presence of cyclospoline A or verapamil, known to be chemosensitizing compounds, no additive or synergistic effects on MX accumulation were observed (Fig. 5).
We also investigated the expression of MDR1, MRP-1 and BCRP in HL-60/MX2 cells. The 295 bp mRNA of MDR1 was not detected in HL-60/MX2 cells. When HL-60/MX2 cells (2.5 x 10 5 cells/ml) were treated with 100 fl9/ml of the 80% MeOH extract or the solvent fractions  Fig. 6. RT-PCR analysis of MDR-1, MRP and BCRP expression in HL-60/MX2 cells treated with the 80% MeOH extract or several solvent fractions from C. asiaticum. HL-60/MX2 cells (2.5 x 10 5 cells/ml) were treated with 100 f,1g/ml of the 80% MeOH extract or the solvent fractions from C. asiaticum for 48 h. RT-PCR analysis of MDR-1, MRP and BCRP were performed after synthesizing the cDNA as described in the materials and methods.
of C. asiatieum for 48 h, the CHCI 3 fraction and the BuOH fraction decreased the mRNA expression levels of MRP-1 and BCRP (Fig. 6).

DISCUSSION
In present study, we examined whether C. asiatieum could overcome anti-cancer drug resistance in HL-60/ MX2 cells. To the best of our knowledge, this study is first to show that an extract of C. asiatieum inhibited the proliferation of HL-60/MX2 cells by induction of apoptosis, increased MX accumulation and down-regulated MRP-1 and BCRP expression. We observed that the 80% MeOH extract, the CHCI 3 fraction and the BuOH fraction from C. asiatieum decreased the survival rate of HL-601MX2 cells. The extract also inhibited the proliferation of HL-60, but not HEL-299, which is anormal cell line (data not shown). The inhibitory effect of its extract on the growth of HL-60 cells might result from induction of apoptosis (in submission).
Strategies such as apoptosis induction, demethylation of genes affecting drug sensitivity, modulation of glutathione levels and increase of intracellular drug accumulation have been used to overcome MDR (Fojo et al., 2003). Among these strategies, targeting BeI-2, Bel-XL and other proteins involved in apoptosis have appeared to be promising approaches to enhance chemotherapy efficacy (Fojo et al., 2003;Yang et al., 2003). Today, we recognize that apoptosis or programmed cell death is the outcome of a complex interplay of pro-and anti-apoptotic molecules. Pro-survival BeI-2 and its help-ers compete with Bax and other pro-apoptosis proteins to regulate the release of proteins and cytochrome c from mitochondria, which in turn activate 'initiator' caspases (Hanahan et al., 2000). High levels of BeI-2 expression have been found in several human tumors, and levels of BeI-2 expression have been correlated with the aggressiveness of the malignancies (Pettersson et al., 1992). BeI-2 has been shown to block cytotoxic agents, whereas the inhibition of Bc1-2 function, by BeI-2 anti sense oligonueleotides (ASO) for example, precipitates apoptosis (Heere-Ress et al., 2002). Since BeI-2 functions by forming a heterodimer with its proapoptotic partner, Bax, the BeI-2:Bax ratio is proportional to the relative sensitivity or resistance of the cells to various apoptotic stimuli (Oltvai et al., 1993). Here, we showed that the CHCI 3 fraction and the BuOH fraction from C. asiatieum fraction reduced BeI-2 mRNA levels, whereas Bax mRNA level increased. These results suggest that the inhibitory effects of C. asiatieum on the growth of HL-60/MX2 cells may arise from the induction of apoptosis via down-regulation of BeI-2 levels. Crinamine was identified from BuOH fraction of C. asiatieum (Park, 2001) and reported to induce apoptosis in hepatoma cancer cells (McNulty et al., 2007). In further study, it remains to be identified whether crinamine could induce apoptosis of HL-60/MX2.
Another approach that has been the subject of many studies is the use of chemosensitizers to modulate MDR chemo-resistant cells so that they become sensitive to chemotherapeutic agents. These chemosensitizers have a broad spectrum of chemical structures, which cause great difficulty in identifying the chemosensitizing properties of their structures. Several reviews have iIIustrated the required chemical structures of MRP-1 modulators (Boumendjel et al., 2005). In this study, we examined the chemosensitizing properties of C. asiatieum. MX accumulation was increased in HL-60/ MX2 cells when treated with the CHCI3 fraction and the hexane fraction of C. asiatieum. The addition of cyelosporine A or verapamil, which are competitive inhibitors of MDR1 (List, 1996;Loor et al., 2002;Garrigues et al., 2002;Choi et al., 1998), failed to show additive or synergistic effects on the accumulation of MX due to the CHCI 3 fraction and the hexane fraction of C. asiatieum. We found the expression of MRP-1 and BCRP, but not MDR1 in HL-60/MX2 cells as previously reported (Harker et al., 1989). Active components from the CHCI 3 fraction and the hexane fraction of C. asiatieum might cause accumulation of MX via the inhibition of MRP-1 or BCRP. Furthermore, treatment of the HL-60/MX2 cells with the CHCI3 fraction and the BuOH fraction resulted in marked decreases of MRP-1 and BCRP mRNA levels. From these results, an active compound (or compounds) from the CHCI3 fraction of C. asiaticum seems to play a pivotal role as a chemosensitizing mediator. The C. asiaticum has four alkaloids from BuOH fraction and two flavonoids from CH 2 CI 2 fraction (Park, 2001). The alkaloids were identified as (+)-crinamine, (5S, 16S)-N-demethylgalanthamine, (5S,16R)-N-demethylgalanthamine and Iycorine. The two flavonoids were identified as 4',7'-dihydroxy flavan and 4',7'-dihydroxy-4-methoxy chalcone. In particular, crinamine from C. asiaticum has been reported to induce apoptosis in hepatoma cancer cells (McNulty et al. , 2007) and inhibit HIF-1 activity (Kim et al., 2006). Comerford et al. have shown that inhibition of HIF-1 expression resulted in significant inhibition of hypoxia-inducible MDR1 expression (Comerford et al., 2002). The purified chemosensitizing principle from the CHCI3 fraction of C. asiaticum remains to be identified and further studied for its mechanism of action in relation to HIF-1.
In conclusion, the CHCI 3 fraction of C. asiaticum might have cytotoxic effects on HL-60/MX2 cells by induction of apoptosis and chemosensitizing effects via the increase of MX accumulation and the down-regulation of MRP-1 and BCRP expression. The components of C. asiaticum should be studied in more detail in order to explain the molecular mechanism involved in apoptosis and modulation of efflux transporters connected with HIF-1.