Effects of Naturally Occurring Polymethyoxyflavonoids on ... - CiteSeerX

Cancer Investigation, 28:220–229, 2010 ISSN: 0735-7907 print / 1532-4192 online c Informa Healthcare USA, Inc. Copyright DOI: 10.3109/07357900902744486

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ORIGINAL ARTICLE Cellular and Molecular Biology

Effects of Naturally Occurring Polymethyoxyflavonoids on Cell Growth, P-Glycoprotein Function, Cell Cycle, and Apoptosis of Daunorubicin-Resistant T Lymphoblastoid Leukemia Cells Kimiko Ishii,1 Sachiko Tanaka,1 Keisuke Kagami,1 Kayo Henmi,1 Hiroo Toyoda,2 Toshikazu Kaise,3 and Toshihiko Hirano1

Departments of Clinical Pharmacology1 and Clinical Molecular Genetics,2 School of Pharmacy, Department of Environmental Chemodynamics, School of Life Sciences,3 Tokyo University of Pharmacy and Life Sciences, Horinouchi, Hachioji, Tokyo, Japan

ABSTRACT Effects of polymethoxyflavonoids tangeretin and nobiletin and the related polyphenolic compounds baicalein, wogonin, quercetin, and epigallocatechin gallate on the cell growth, P-glycoprotein function, apoptosis, and cell cycle of human T lymphoblastoid leukemia MOLT-4 and its daunorubicin-resistant cells were investigated. The IC50 values of these compounds on the cell growth were 7.1–32.2 µmol/L, and the inhibitory effects were observed to be almost equal to the parent MOLT-4 and the daunorubicin-resistant cells. Tangeretin and nobiletin showed the strongest effects with the IC50 values of 7.1–14.0 µmol/L. These polymethoxyflavonoids inhibited the P-glycoprotein function and significantly influenced the cell cycle (p < .05), whereas they did not induce apoptosis.

INTRODUCTION The success of chemotherapy in cancer treatment is frequently limited by intrinsic or acquired multidrug resistance (MDR) due to the increased expression of a plasma membrane P-glycoprotein (P-gp) (1, 2). This protein is a transporter dependent on adenosine triphosphate (ATP) that effluxes a number of structurally unrelated anticancer agents out of cells, thereby

Keywords: Naturally occurring polymethoxyflavonoids, MOLT-4 cell line, Daunorubicin resistance, P-glycoprotein function, Cell cycle, Apoptosis. Correspondence to: Toshihiko Hirano, PhD Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences 1432-1 Horinouchi, Hachioji Tokyo 192-0392, Japan email: [email protected]

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reducing intracellular drug concentration and permitting the tumor cells to survive against high concentrations of drugs that otherwise would be toxic. Inhibiting P-gp function with diverse pharmacological agents circumvents the MDR phenotype. Compounds such as verapamil, cyclosporine, and certain kinds of flavonoids were reported to sensitize MDR cells to cytotoxic agents (3–5). Rhodamine 123 (Rh123) has been observed to accumulate in the mitochondria of cells and is used as a standard functional indicator of MDR (6–9). The function of P-gp can thus be evaluated by inhibition of Rh123 efflux with MDR modulators (7, 8). MOLT-4, a human T lymphoblastoid leukemia cell line, has been used extensively for studies of leukemia cell biology and anti-leukemia therapy (10, 11). We have developed an MDR cell line MOLT-4/daunorubicin (DNR) from a T lymphoblastoid leukemia MOLT-4 cell line by exposing the parent cells to stepwise increasing concentrations of DNR over 3 months (12). This resistant subline MOLT-4/DNR has been revealed to overexpress functional P-gp and MDR1 mRNA (12). Moreover,

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centration of 10 mmol/L with ethanol and diluted to working concentrations before use. Test-compound solutions were made at a concentration of 5 mmol/L with ethanol and diluted to working concentrations before use. Annexin V–FITC Apoptosis Detection Kit I from BD PharMingen (San Diego, CA) was used.

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Nobiletin Figure 1. Chemical structures of tangeretin and nobiletin.

MDR in MOLT-4/DNR has been shown to be closely related to the expression of P-gp and MDR1 mRNA (12), and therefore, this subline will be a suitable model to investigate the agents which modify P-gp function and drug resistance. Anticancer efficacies and P-gp-inhibiting activity of naturally occurring flavonoids and other related polyphenolic compounds have been reported by many researchers including us (4, 5, 13–15). Polymethoxyflavonoids tangeretin and nobiletin (Figure 1) originated from citrus and are also known to have several pharmacological activities including anticancer efficacies (14, 16–19). However, the effects of tangeretin and nobiletin on the growth of human T lymphoblastoid leukemia cells expressing functional P-gp and the molecular mechanisms of their action on these cells have little been investigated so far to the best of our knowledge. Thus, the present study was undertaken to evaluate antiproliferative effects of naturally occurring polymethoxyflavonoids and other related polyphenolic compounds on a P-gp-expressing MDR cell line MOLT-4/DNR and provide an insight into their action mechanisms by examining the cell growth, P-gp function, cell cycle, and apoptosis in these cells treated with the agents.

MATERIALS AND METHODS Reagents Fetal bovine serum and RPMI-1640 medium were purchased from Gibco BRL Co. (Grand Island, NY). Rh123, DNR, tangeretin, nobiletin, baicalein, wogonin, quercetin, and epigallocatechin gallate were obtained from Sigma Chemical Co. (St. Louis, MO). DNR stock solutions were made at a con-

MOLT-4 and MOLT-4/DNR cells were maintained in RPMI1640 medium containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin as we described previously (20, 21). The leukemia cells were washed with and resuspended in the above medium to 5 × 105 cells/mL, and then 196 µL of the cell suspension were placed in each well of a 96-well flat-bottom plate. Four microliters of ethanol solution containing each test compound and 4 µL of ethanol solution containing DNR were added to yield final concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 µmol/L. Four microliters of ethanol were added to the control wells. The cells were incubated for 72 hr in 5% CO2 /air at 37 ◦ C in a humidified chamber. Then, the cells were pulsed with 18.5 KBq/well of [3 H]thymidine for an additional 20 hr of incubation and collected on a glass-fiber filter paper using a multiharvester device and dried. The radioactivity retained on the filter was further processed for liquid scintillation counting. The mean of the counts for a duplicate of each sample was determined. Suppressive effects of test compounds were estimated from the percentage of cell growth evaluated by the amount of [3 H]thymidine incorporated into cells in the presence of the agents, as compared to cell growth in the absence of the agents. IC50 (ng/mL) values were determined from linear regression of at least four points in different concentrations of the agents.

Dye efflux function of leukemic cells A total of 5 × 105 cells were collected and centrifuged at 1300 rpm for 5 min at 4◦ C, resuspended in buffer containing 2 µmol/L Rh123, and made to stand for 10 min in 5% CO2 /air at 37◦ C. After washing, the cells were incubated in presence or absence of test compounds for 90 min in 5% CO2 /air at 37◦ C. After incubation, the cells were washed twice in a washing buffer and resuspended in 400 µL staining buffer. The remaining intracellular Rh123 fluorescence intensity was determined by flow cytometry (Becton Dickinson) (12).

Analysis of dye efflux data The data were analyzed with a FACSCalibur flow cytometer, using CellQuest software (Becton Dickinson) (12). Efflux of Rh123 by leukemia cells was assessed by analyzing changes in cellular fluorescence after efflux in the presence or absence of tangeretin or nobiletin. Rh123 is excited at a wavelength of 488 nm. Fluorescence intensity of Rh123 in the cells was detected on fluorescence channel one (FL1) of the FACSCalibur, using a 1,024 log channel scale. The mean cellular fluorescence of the cells after efflux in the presence or absence of

Methoxyflavone Effects on Drug-Resistant Leukemia

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polymethoxyflavonoids was recorded. Rh123 intensity was assessed by calculating its difference in fluorescence intensity in the cells after efflux in the presence or absence of the polymethoxyflavonoids, measured both by using the Kolmogorov– Smirnov (KS) statistic D. The KS statistic measures the difference between two distribution functions and generates a D value ranging between 0 and 1.0. A higher D value indicates greater difference between the distribution functions.


Cell cycle analysis MOLT-4 and MOLT-4/DNR cells were washed with and resuspended in RPMI-1640 medium to 5 × 105 cells/mL, and then 980 µL of the cell suspension were placed in each well of a 24-well flat-bottom plate. Then, 20 µL of ethanol solution containing tangeretin or nobiletin were added to give final agent concentrations of 1, 10, and 50 µmol/L. After culturing of the cells for 72 hr in 5% CO2 /air at 37◦ C, cells were placed in 1.5-mL tube, washed twice in phosphate buffered saline containing 1% fetal bovine serum, and then fixed in 70% ethanol at −20◦ C for 24 hr. After the fixation, the cells were washed twice with the above buffer and treated with 1 mg/mL of RNase at 37◦ C for 30 min. Subsequently, 10 µL of propidium iodide (PI) solution (Becton Dickinson) was added to this cell suspension (5 × 105 cells per 60 µL), and the tube containing the cell suspension was incubated for 20 min at room temperature in the dark. Subsequently, the cells were resuspended with 800 µL of phosphate buffered saline containing 0.4% paraformaldehyde and 1% fetal bovine serum and were analyzed by flow cytometer (Becton Dickinson) within 1 hr after staining. A total of 30,000 non-gated cells were analyzed.

Apoptosis assays After MOLT-4 and MOLT-4/DNR cells (5 × 105 /mL) were incubated in the presence of indicated concentrations of tangeretin or nobiletin for 72 hr in 5% CO2 /air at 37◦ C, 1 mL of this cell suspension was placed in a 1.5-mL tube, and the cells were washed twice in cold phosphate buffered saline (pH 7.2). Cells in each tube were resuspended with 500 µL of phosphate buffered saline containing 1% fetal bovine serum, and 5 µL of Annexin V–FITC solution and 5 µL of PI solution were added to the tube. Then, the cells were incubated for 15 min at room temperature in the dark. Subsequently, 400 µL of binding buffer was added to each tube, and the cells were analyzed by flow cytometry (Becton Dickinson) within 1 hr after staining (20, 21). A total of 30,000 non-gated cells were analyzed.

Statistics Comparison of the data between two groups was carried out by Student’s t test. Comparison of the data in multiple groups was carried out by Bonferroni/Dun multiple comparison. In each case, p values less than .05 were considered to be significant. 222

RESULTS Effects of polymethoxyflavonoids and other phenolic compounds on parental MOLT-4 and resistant MOLT-4/DNR cell growth DNR effects on growth of MOLT-4 and MOLT-4/DNR cells were examined after 72 hr in culture, as assessed by [3 H]thymidine incorporation into the cells [Figure 2(a)]. The IC50 value of DNR on MOLT-4 cell growth was 0.25 µmol/L, while the IC50 value of DNR on MOLT-4/DNR cell growth was 3.44 µmol/L, which was 13.9 times higher than the IC50 value of DNR on MOLT-4 cell growth (p < .05). Thus, DNR was effective against parental MOLT-4 cells but less effective against MOLT-4/DNR cells, as expected. Then, the parental MOLT-4 and subline MOLT-4/DNR cells were continuously treated with 0.01–100 µmol/L of polymethoxyflavonoids or other phenolic compounds for 72 hr, and the cell growth was assessed. In contrast to the effects of DNR, growth of MOLT-4 and MOLT-4/DNR cells was suppressed almost equally by these compounds in a dose-dependent manner. The IC50 values of these compounds against the growth of parent and subline cells were deviated from 7.1 to 32.2 µmol/L (Table 1). Among those, polymethoxyflavonoids tangeretin and nobiletin exhibited the strongest effects with the IC50 values ranging from 7.1 to 14.0 µmol/L [Table 1; Figures 2(b) and (c)]. Chemical structures of these flavonoids were presented in Figure 1. The suppressive effects of the polymethoxyflavonoids against MOLT-4/DNR cells were rather stronger than those against the parent cells, and the difference of the effects of tangeretin at 10 µmol/L was statistically significant (p = .035, n = 5). As described above, MOLT-4/DNR cell line was less sensitive to DNR at the concentrations of 0.1–1.0 µmol/L, but when MOLT-4/DNR cells were cultured in the presence of DNR combined with 5 µmol/L tangeretin or nobiletin, the growth of MOLT-4/DNR cells was extensively inhibited (Figure 3). The dose–response curves of DNR combined with tangeretin or nobiletin against the growth of MOLT-4/DNR cells shifted to almost the same level as those against the growth of parent MOLT-4 cells. In this experiment shown in Figure 3, the IC50 Table 1. Comparison for the IC50 Values of DNR, Polymethoxyflavonoids, and Other Phenolic Compounds Against the Proliferation of Parent MOLT-4 and DNR-Resistant MOLT-4/DNR Cells IC50 ( SD) Values (µM)a Test Compound DNR Tangeretin Nobiletin Baicalein Wogonin Quercetin −(−) Epigallocatechin Gallate aIC 50

MOLT-4 Cells

MOLT-4/DNR Cells

0.25 14.0 (1.1) 12.6 (0.3) 23.4 (1.3) 30.7 (2.9) 29.9 (2.2) 30.0

3.44 7.1 (1.1) 7.6 (1.8) 32.2 (5.1) 29.6 (3.6) 28.2 (4.2) 31.0

values with SD are the mean (SD) values of three independent experiments.

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Figure 2. Effects of (a) DNR, (b) tangeretin, and (c) nobiletin on in vitro growth of MOLT-4 (open circles) and MOLT-4/DNR (closed circles) cells. Cells were treated with different concentrations of DNR or the polymethoxyflavonoids for 4 days, and the cell growth was estimated by [3 H]thymidine incorporation. Values are the mean of three independent experiments. Statistically significant differences were observed between MOLT-4 and MOLT-4/DNR cells, as indicated with *p < .05 and **p < .01, by the Mann–Whitney’s U test.

value of DNR alone against the proliferation of MOLT-4/DNR cells was 3.07 µmol/L, which was 11.3 and 12.8 times lower (the IC50 values came to 0.27 and 0.24 µmol/L), when the cells were cultured in the presence of 5 µmol/L tangeretin and nobiletin, respectively. In contrast, the IC50 value of DNR against the proliferation of the parent MOLT-4 cells was 0.29, which was not extensively changed (the IC50 values came to 0.24 and 0.20 µmol/L, respectively) in combination with 5 µmol/L tangeretin or nobiletin (data not shown).

Effects of tangeretin and nobiletin on the P-gp function of MOLT-4/DNR cells Our previous examination revealed that only 2.5% of MOLT4 cells express P-gp, whereas 93.7% of MOLT-4/DNR cells express P-gp (20). To evaluate the P-gp efflux function, 2 µmol/L Rh123 as a P-gp substrate was used in P-gp efflux experiments. After the cells were incubated for 90 min, the intercellular Rh123 levels of MOLT-4/DNR cells rapidly decreased, whereas the parental MOLT-4 cells still retained the dye (Figure 4). This result showed that MOLT-4/DNR cells possess high level of efflux activity. When MOLT-4/DNR cells were cultured in the presence of tangeretin or nobiletin at concentrations of 5, 10, and 50 µmol/L for 90 min, the intracellular Rh123 accumulation was partially enhanced as shown in Figure 5.

Mean fluorescence intensity of Rh123 retained in cells before and after the incubation of the cells with or without 5, 10, and 50 µmol/L tangeretin or nobiletin was estimated, and the KS statistic measuring the difference of the Rh123 fluorescence intensity before and after the incubation was carried out to generate the D values. The D values in the presence of 5, 10, and 50 µmol/L tangeretin were 0.16, 0.30, and 0.66, while the values in the presence of 5, 10, and 50 µmol/L nobiletin were 0.20, 0.43, and 0.66, respectively. (The basal D values of fluorescence intensity for the parental MOLT-4 cells and the DNR-resistant MOLT-4/DNR cells calculated in the presence or absence of a positive-control P-gp inhibitor cyclosporine (5 µmol/L) were 0 and 1.0, respectively.) Thus, these results confirm the observations that the polymethoxyflavonoids inhibited efflux of Rh123 from MOLT-4/DNR cells—though the effects were not potent as that of cyclosporine.

Tangeretin and nobiletin did not efficiently induce apoptosis in cells of MOLT-4 and MOLT-4/DNR cell line Parental MOLT-4 and the drug-resistant MOLT-4/DNR cell lines were cultured in the presence of tangeretin or nobiletin at concentrations of 1, 10, and 100 µmol/L for 3 days and stained with Annexin V and PI, and the percentages of


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Figure 4. Comparison of R123 efflux ability between MOLT-4 (open area) and MOLT-4/DNR (closed area) cells. Cells of each cell line were suspended in buffer containing 2 µmol/L Rh123 and stood for 10 min. After washing, the cells were incubated for 90 min, and the remaining intracellular Rh123 fluorescence intensity was determined by flow cytometry. MOLT-4/DNR cells have high ability to efflux the dye; thereby the fluorescent intensity extensively decreased after the incubation (closed area).

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late apoptosis (with necrosis) in both the parent and the drugresistant MOLT-4/DNR cells.] Thus, tangeretin and nobiletin did not efficiently induce apoptosis in cells of both MOLT-4 and MOLT-4/DNR cell line, even at the relatively high concentration (100 µmol/L), which can almost completely suppress the growth of these cells (see Figure 2).

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Figure 3. Dose–response curves of DNR against the proliferation of MOLT-4 (open circles) and MOLT-4/DNR (triangles) cells in presence (closed triangles) or absence (open triangles) of (a) tangeretin and (b) nobiletin.

apoptotic cells in an Annexin V-positive and PI-negative area were estimated (Figures 6 and 7). [Mean (SD) percentages of apoptotic cells in MOLT-4 and MOLT-4/DNR cells treated with 1–100 µmol/L tangeretin or nobiletin are shown in Table 2.] The percentage of apoptotic cells in MOLT-4 cell line treated with polymethoxyflavonoids was not largely different from that of untreated cells (Figure 6). Similarly, the percentage of apoptotic cells in MOLT-4/DNR cell line treated with polymethoxyflavonoids was not largely different from that of untreated cells (Figure 7). [These polymethoxyflavonoids especially tangeretin rather reduced—instead of increasing—the Table 2. Percentages of Apoptotic Cells in MOLT-4 and MOLT-4/DNR Cells Treated With 1, 10, and 100 µmol/L of Tangeretin or Nobiletin

Effects of tangeretin and nobiletin on cell cycle of MOLT-4 and MOLT-4/DNR cells MOLT-4 and MOLT-4/DNR cells were cultured in presence of 1, 10, and 50 µmol/L tangeretin or nobiletin for 3 days and stained with PI, and the percentages of cells in apoptosis, G1 phase, S phase, and G2 /M phase were estimated with flow cytometry (Figures 8 and 9). Tangeretin increased percentage of the S phase cells of the parent MOLT-4 cell line with a significant effect at 50 µmol/L (p < .05) (Figure 8). Tangeretin tended to increase the G1 phase cells of MOLT-4/DNR cell line, but the effects were not statistically significant (Figure 9). Nobiletin increased the percentage of G1 phase cells with a significant effect at 50 µmol/L (p < .05), whereas the flavonoid decreased the percentage of G2 /M phase cells with a significant effect at 50 µmol/L (p < .05), in both parental MOLT-4 and the drugresistant MOLT-4/DNR cell line (Figures 8 and 9).

Percentages of Apoptotic Cells (SD)a Test Compound Control Tangeretin

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1 µmol/L 10 µmol/L 100 µmol/L 1 µmol/L 10 µmol/L 100 µmol/L

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DISCUSSION

1.6 (0.4) 3.3 (1.7) 2.5 (1.3) 8.0 (2.5) 4.0 (2.1) 3.0 (1.8) 5.6 (1.9)

2.2 (2.0) 3.3 (3.4) 3.1 (3.2) 4.5 (1.7) 3.0 (1.0) 4.3 (1.6) 2.9 (0.7)

The data described above show that naturally occurring polymethoxyflavonoids and polyphenolic compounds inhibit growth of both T lymphoblastoid leukemia MOLT-4 cells and P-gpexpressing DNR-resistant MOLT-4 (MOLT-4/DNR) cells almost equally, although the precise action mechanism of these compounds remains to be elucidated. The data also showed that the suppressive effects of the polymethoxyflavonoids tangeretin and nobiletin were strongest among the compounds tested with

values are the mean (SD) of four independent experiments.

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Figure 5. Effect of tangeretin (upper three figures) or nobiletin (lower three figures) on the dye efflux function of MOLT-4/DNR cells. MOLT4/DNR cells were suspended in buffer containing 2 µmol/L Rh123 and stood for 10 min. After washing, the cells were incubated for 90 min in the presence (open area) or absence (closed area) of 5, 10, and 50 µmol/L tangeretin or nobiletin, and the remaining intracellular Rh123 fluorescence intensity was determined by flow cytometry.

Figure 6. Dot plots showing apoptotic MOLT-4 cells cultured in the (b, c) presence or (a) absence of 1, 10, and 100 µmol/L (b) tangeretin or (c) nobiletin. The percentage given in each dot plot is the percentage of apoptotic cells and necrotic cells. The dots in the lower right quadrant in each figure are the AnnexinV+/PI− cells, indicating apoptotic cells, whereas the dots in the upper right quadrant are the AnnexinV+/PI+ cells, indicating necrotic cells. Typical data of four independent experiments are shown (see also Table 2).


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Figure 7. Dot plots showing apoptotic MOLT-4/DNR cells cultured in the presence or absence of 1, 10, and 100 µmol/L tangeretin (upper three figures) or nobiletin (lower three figures). The percentage given in each dot plot is the percentage of apoptotic cells and necrotic cells. The dots in the lower right quadrant in each figure are the AnnexinV+/PI− cells, indicating apoptotic cells, whereas the dots in the upper right quadrant are the AnnexinV+/PI+ cells, indicating necrotic cells. Typical data of four independent experiments are shown (see also Table 2).

the IC50 values of 7.9–16.5 µmol/L. It further showed that these flavonoids influence the cell cycle—but not apoptosis—of these cells. MOLT-4/DNR cell line was derived from parental MOLT-4 cell line by exposing the cells to a stepwise concentration of DNR in our laboratory (12), which was shown to be persistently resistant to antiproliferative effect of DNR. The IC50 value of DNR on cells of MOLT-4/DNR cell line was 13.9 times higher than that of DNR on cells of parental MOLT-4 cell line. The resistance of MOLT-4/DNR cells to DNR has been reported to closely correlate with the expression of functional P-gp (12). MDR is recognized as one of the most common causes for failure of chemotherapy in treating cancer patients (22). P-gp is an ATP-binding cassette (ABC) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells (23). The fluorescent dye Rh123 has been found to be transported by P-gp (7, 22), and the flow cytometric measurement of cellular Rh123 uptake/efflux is an efficient tool to assess the functional activity of P-gp in tumor samples (12). In the present study, we revealed that both parental MOLT-4 and resistant MOLT-4/DNR cells were sensitive to the suppressive effects of tangeretin and nobiletin, and the effects were additive in combination with DNR. Since both of these flavonoids partially suppress the efflux function in MOLT-4/DNR cells in a concentration range of 10–50 µmol/L, the additive antiproliferative effects of these flavonoids in combination with DNR are suggested to be mediated via suppression of P-gp function. Indeed, the additive effects were not observed against the proliferation of the parental MOLT-4 cells expressing less efflux 226

activity, suggesting that the additive effects of these flavonoids in combination with DNR are mainly due to the inhibition of P-gp function in MOLT-4/DNR cells. Polymethoxyflavonoids were reported to influence cell cycle and induce G1 arrest in human breast and colon cancer cells (18). DNR is known to block DNA replication and RNA synthesis, and therefore, the cell-cycle-modulating effects of these flavonoids are possibly additive (or synergistic) to the pharmacological action of DNR against MOLT-4 and MOLT-4/DNR cell growth. However, such additive effects of polymethoxyflavonoids and DNR were not sufficiently observed in the parent MOLT-4 cells as described above. In MOLT-4/DNR cells, the inhibition of R123 efflux by the concentrations of the polymethoxyflavonoids exhibiting synergic effects in combination with DNR on the cell growth (< 10 µmol/L) did not appear to be efficient enough. Therefore, the data suggest that the synergic effect of the polymethoxyflavonoids with DNR observed in MOLT-4/DNR cells was not obtained solely by the inhibition of P-gp function but may have been obtained by additional cytostatic effects of the polymethoxyflavonoids. The lack of any significant cell death at concentrations that have profound inhibitory effects on proliferation, and induce cytostatic effects (G1 /S accumulation), suggests that cytostasis and not cytotoxicity is the most relevant biological effect of tangeretin and nobiletin. The ability of these polymethoxyflavonoids to inhibit growth of MOLT-4 and MOLT-4/DNR cells in the absence of toxicity suggests that they interact selectively with mediators of cell cycle events of these cells, and that interactions promoting toxicity are excluded. Thus, the

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Figure 8. Cell cycle distribution in the flavonoid-treated MOLT-4 cells. Cells were treated with 1, 10, and 50 µmol/L tangeretin or nobiletin, stained with PI, and analyzed by flow cytometry. Each bar represents the mean ± SD of three independent experiments; ∗ p < .05 by Bonferroni test.

Annexin V apoptosis assays in the present study showed that these compounds did not induce early apoptosis, rather it appears that they reduced the late apoptosis (with necrosis) especially in those treated with tangeretin (Figures 6 and 7). Previous studies have shown that the polymethoxyflavonoids do not induce apoptosis in some human cell lines (18). In addition, Takano et al. (24) showed that tangeretin protects cells against endoplasmic reticulum stress and neurotoxin. While molecular background of the polymethoxyflavonoid effects to reduce apoptosis in the present study remains to be elucidated, their selective interaction with mediators of cell cycle events and their consequent cytostatic effects may contribute to reduce the apoptosis events of these cells. Treatment of MOLT-4 and MOLT-4/DNR cells with the polymethoxyflavonoids, especially with nobiletin, tends to arrest cell cycle of these cells to increase the G1 phase cells (Figures 8 and 9). In a previous report, tangeretin was found to induce cell

cycle G1 arrest through inhibition of cyclin-dependent kinases 2 and 4 activities, as well as Cdk inhibitors p21 and p27 in human colorectal carcinoma cells (25). In addition, tangeretin and nobiletin were also reported to induce G1 cell cycle arrest—but not apoptosis—in human breast and colon cancer cells as described above (18). Thus, the data of our present study were consistent with those of the previous reports, and our data suggest that such cytostatic effects of the polymethoxyflavonoids can be obtained in MDR cells despite the fact that these cells highly expressed the functional P-gp molecules. In the present study, polyphenolic compounds, baicalein, wogonin, quercetin, and epigallocatechin gallate were also examined for their antiproliferative effects on MOLT-4 and MOLT4/DNR cells. Most of these naturally occurring compounds have been suggested to possess anticancer efficacy (13, 14, 26–28), and the data of the present study support such observations. The IC50 values of these compounds were 26.2–87.0 µmol/L,

Figure 9. Cell cycle distribution in the flavonoid-treated MOLT-4/DNR cells. Cells were treated with 1, 10, and 50 µmol/L tangeretin or nobiletin, stained with PI, and analyzed by flow cytometry. Each bar represents the mean ± SD of three independent experiments; ∗ p < .05 by Bonferroni test.


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which were less effective as compared to the IC50 values of tangeretin and nobiletin. However, most of these compounds were equally effective against the proliferation of parent MOLT-4 and the drug-resistant MOLT-4/DNR cells, suggesting that the compounds were not excluded by functional P-gp expressed on the MOLT-4/DNR cells. Although further examinations concerning the antiproliferative mechanism(s) of these compounds have not been performed in the present study, many reports have suggested apoptosis-inducing effects and/or P-gp-modifying activities of the related compounds in cancer cells (14, 27, 28). The present study, in conclusion, shows that naturally occurring polymethoxyflavonoids and polyphenolic compounds exhibit suppressive efficacy on in vitro growth of both human T lymphoblastoid leukemia MOLT-4 cells and the Pgp-expressing DNR-resistant MOLT-4 (MOLT-4/DNR) cells. Among those, tangeretin and nobiletin showed the strongest effects with IC50 values less than 20 µmol/L. These polymethoxyflavonoids influence cell cycle and inhibit growth of the parent and functional P-gp-expressing cells almost equally, although the precise action mechanism of these compounds remains to be elucidated.

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DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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