Reversal of multidrug resistance by 5,5'-dimethoxylariciresinol-4-O-β-D-glucoside in doxorubicin-resistant human leukemia K562/DOX.

OBJECTIVE: The objective of this study was to investigate the reversal effects of 5,5'-dimethoxylariciresinol-4'-O-β-D-glucoside (DMAG) extracted from traditional Chinese medicines Mahonia on multidrug resistance (MDR) of human leukemia cells to chemotherapeutic agents.
MATERIALS AND METHODS: MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to determine the effect of DMAG on doxorubicin sensitivity to K562/DOX cells. Propidium iodide /Hoechst 33342 double staining assay was used to investigate the effect of DMAG on doxorubicin-induced cellular apoptosis. Intracellular accumulation of doxorubicin and rhodamine 123 assay were performed to evaluate the effect of DMAG on drugs efflux activity of P-glycoprotein.
RESULTS: DMAG significantly enhanced the doxorubicin cytotoxicity to K562/DOX cells. In the presence of 1.0 μM of DMAG, the IC50 of doxorubicin decreased from 34.93 ± 1.37 μM to 12.51 ± 1.28 μM. DMAG of 1.0 μM significantly enhanced doxorubicin-induced cell apoptosis in K562/DOX cells and the enhancement was time-dependent. A significant increase in accumulation of doxorubicin in the presence of DMAG was observed. After treatment of the K562/DOX cells for 1 h with 15.0 μM doxorubicin alone, the fluorescence intensity was 33093.12. With the addition of 1.0 μM of DMAG, the fluorescence intensity of doxorubicin was 2.3-fold higher. A significant increase of accumulation of rhodamine 123 in the presence of DMAG was also observed. With the addition of 1.0 μM of DMAG, the fluorescence intensity was increased by 49.11% compared with rhodamine 123 alone.
CONCLUSION: DMAG was shown to effectively enhance chemosensitivity of resistant cells, which makes it might be a suitable candidate for potential MDR-reversing agents.

Introduction

Multidrug resistance (MDR) of cancer cells is one of the primary obstacles to the success of chemotherapy. Cancer cells may be intrinsically resistant to chemotherapeutic drugs, or they may acquire resistance, which can be induced by chemotherapeutic drugs during treatment.[1] The classical mechanism of MDR development involves the overexpression of P-glycoprotein (P-gp), a 170 kDa membrane protein encoded by MDR1 gene, which facilitates the efflux of chemotherapeutic agents from cancer cells.[234] P-gp is known to facilitate the efflux of a broad range of cytotoxic drugs including anthracyclines (doxorubicin, daunorubicin and epirubicin), Vinca alkaloids (vincristine, vinblastine), epipodophyllotoxins (etoposide, teniposide) and taxenes (paclitaxel, docetaxel).[5]

MDR mediated by P-gp overexpression leads to the chemoresistance and the decreased survival in leukemia patients.[67] For the sake of increasing success of chemotherapy and achieving better clinical outcome in leukemia, a number of strategies to overcome P-gp transporter mediated MDR have been proposed. Significant effort has been aimed at the development of MDR reversers. Although a large number of chemical MDR modulators and inhibitors aim to overcome MDR by interfering expression and function of P-gp, their insufficient efficacy, unacceptable toxicities and unpredictable pharmacokinetic interactions limit their usage in clinics. Consequently, development or discovery of safe and effective MDR reversal agents is exigent.

Currently, much effort is being extended to identifying natural medicine from plant origins. Traditional Chinese medicines (TCM) have been applied for the treatment of cancers in China and beyond for many years.[89] Moreover, TCM has become a supplementary force to conventional chemotherapy and can improve the quality-of-life and prolong survival time on cancer patients when used in combination with conventional chemotherapeutic drugs.[1011] The compounds from TCM could circumvent the limitations of chemical MDR modulators and inhibitors due to their minimal toxicities or side-effects in clinical practice.[8]

Mahonia is a kind of TCM. Both root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal and anti-inflammatory effects. Mahonia bealei (Fort.) Carr. (Berberidaceae) leaves have been widely used as a tea leaf beverage south of the Qinling Mountains of China. Water extract of M. bealei leaves (WML) exhibits extremely high antioxidant properties and strong reducing abilities and provides protection against oxidative protein damage induced by hydroxyl radicals.[12] WML could also significantly inhibit the growth of human colon cancer (HT-29) cells in a concentration-dependent manner and it gradually increases the proportion of apoptotic cells and reduces the expression of the survivin gene. Mahonia oiwakensis can inhibit the growth of human lung cancer cells in vitro and in vivo.[13]

In the present study, we hypothesized that DMAG, an extract from TCM Mahonia, might act as a reverser of MDR. We investigated the reversal effect of DMAG on MDR cancer cells using the doxorubicin-resistant human leukemia K562/DOX cell line and doxorubicin-sensitive K562 cells. We found that DMAG was able to reverse MDR of cancer cells to doxorubicin.

Materials and Methods

Chemicals and Drugs

RPMI-1640, fetal bovine serum (FBS) and penicillin–streptomycin were purchased from Gibco. MTT, rhodamine123 (Rh123), propidium iodide (PI), Hoechst and RNase A were purchased from Sigma. Doxorubicin was purchased from Zhejiang Haizheng Pharmacy Company. Verapamil was purchased from Shanghai Hefeng Pharmaceutical Co. Ltd. DMAG was extracted from M. bealei, collected from Guizhou (China) in September 2007, identified by Professor Yue Cong (Henan University) and the Voucher No. 20070923 and deposited at the institute of pharmacy, Henan University. The sliced stems were extracted with 70% ethanol and then separated, purified and characterized.[14] Mahonia dry stems (slice) of 7.0 kg was extracted with 34 L of 70% ethanol for 5 times, each time 2.5 h, merging and decompressing recovery the extracted liquid, consequently obtained 900 g of ethanol extract. The extract was isolated with dry silica gel column chromatography followed by the expansion using chloroform-methanol-water (8:2:0.2) and divided into 8 parts, each with chloroform/methanol (3:2→methanol) gradient elution and recovery. The eighth part was purified with silica gel column and eluted with petroleum ether and ethyl acetate-methanol (4:6:0.8), consequently obtained the compound DMAG (24 mg).

Cell Lines and Cell Culture

The parental drug-sensitive K562 and doxorubicin-resistant K562/DOX cell lines were purchased from Institute of Hematology, Chinese Academy of Medical Sciences. K562 cells were maintained in RPMI-1640 medium supplemented with 10% FBS and antibiotics (100 unit/ml of penicillin and 100 μg/ml of streptomycin) at 37°C in a humidified 5% CO2 atmosphere. Same medium with the addition of 1.724 μM doxorubicin was used for K562/DOX cell culture. K562/DOX cells were incubated in doxorubicin-free medium for 2 weeks before the planned experiment.

Cell Viability Assay

The viability of K562 and K562/DOX cells treated with doxorubicin in the presence or absence of DMAG was analyzed with MTT assay. Briefly, cells were seeded into 96-well microplates (1 × 104 cells/well) and incubated for 24 h at 37°C and 5% CO2. The cells were then exposed to graded concentrations of doxorubicin alone or in combination with DMAG for 48 h. 20 μl MTT solution (5 mg/ml in phosphate buffered saline [PBS]) were added to each well and the cells were incubated for an additional 4 h at 37°C and 5% CO2. The medium with MTT solution was then removed, followed by the addition of 100 μl dimethyl sulfoxide and shaken for an extra 10 min. The absorbance was measured at 570 nm with a microplate reader. The half inhibitory concentrations (IC50) of doxorubicin in the presence or absence of DMAG were calculated.

Cell Apoptosis Analysis

The effects of doxorubicin on cell apoptosis in the presence or absence of DMAG were detected with double staining of Heochst 33342 and PI with Array Scan VTIHCS600 High-Contents. Briefly, K562/DOX or K562 cells were seeded into 96-well microplates at a density of 1 × 104 cells/well. After 24 h, cells were exposed to doxorubicin (K562/DOX cells 15.0 μM, K562 cells 0.15 μM) in the presence or absence of DMAG (verapamil as a positive control) for 24 h and 48 h, respectively and then incubated with 5 mg/L Hoechst 33342 for 10 min and 5 mg/L PI for another 1 h in the dark. The cells were washed twice with ice-cold PBS and subjected to Array Scan VTIHCS600 High-Contents to record the red and blue fluorescence produced by PI and Hoechst 33342, respectively.

Intracellular Doxorubicin Accumulation Assay

K562/DOX and K562 cells were seeded into 96-well plates at a density of 1 × 104 cells/well and allowed to grow overnight. The cells were then incubated with doxorubicin alone or in combination with DMAG for 1 h in the dark. The cells were rinsed twice with ice-cold PBS and subjected to Array Scan VTIHCS600 High-Contents to record the green fluorescence produced by doxorubicin at 480 nm.

Intracellular rhodamine 123 accumulation assay

The effect of DMAG on the efflux function of P-gp was evaluated by intracellular accumulation of rhodamine 123 (Rh123) assay. K562/DOX or K562 cells were seeded into 96-well plates at a density of 1 × 104 cells/well and allowed to grow overnight. The cells were then incubated with DMAG (verapamil as a positive control) for 1 h, followed by the addition of 5 mg/L Hoechst 33342 for 10 min and 5 mg/L Rh123 for another 1 h in the dark. The cells were rinsed twice with ice-cold PBS and subjected to Array Scan VTIHCS600 High-Contents to record the green fluorescence produced by Rh123.

Statistical Analysis

All values were expressed as means and standard deviations. The statistical significance of differences between two groups was assessed using two-tailed Student's t-test. Differences were accepted as significant at the 95% level (P < 0.05).

Results

DMAG-mediated Reversal of K562/DOX Cell Resistance to Doxorubicin

K562 cells were sensitive to doxorubicin with the IC50 of 1.07 ± 0.09 μM. In contrast, K562/DOX cells were highly resistant to doxorubicin. The IC50 of doxorubicin against K562/DOX cells was 34.93 ± 1.37 μM, 32.64 fold more resistant than parental K562 cells.

DMAG significantly enhanced the doxorubicin cytotoxicity to K562/DOX cells [Figure 1b]. In the presence of 1.0 μM of DMAG, the IC50 of doxorubicin decreased from 34.93 ± 1.37 to 12.51 ± 1.28 μM. Positive control verapamil led to only 1.21 fold decrease of IC50 of doxorubicin. DMAG did not significantly enhance the doxorubicin cytotoxicity to K562 cells (data not shown). When the K562 and K562/DOX cells were exposed to 1.0 μM DMAG alone, only wild cytotoxicity (<10%) to cells was observed [Figure 1a data of K562 cells not shown].

Figure 1

The effect of 5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG) and DOX on K562/DOX cell growth. (a) The effect of different concentrations DMAG on K562/DOX cell growth. (b) The effect of DOX alone, DOX in combination with 1.0 μM of DMAG or DOX in combination with 1.0 μM of verapamil on K562/DOX cell growth. Inhibitory rate of cells and survival rate of cells were assessed with MTT assay and expressed as mean ± standard deviations of three experiments

Enhancement of DMAG-mediated K562/DOX Cell Apoptosis

Double staining of Heochst 33342 and PI assay was used to determine doxorubicin-induced cell apoptosis in the presence of DMAG with Arrary Scan VTIHCS600 High-Contents. DMAG of 1.0 μM significantly enhanced doxorubicin-induced cell apoptosis in K562/DOX cells and the enhancement was time-dependent [Figure 2b and 3b]. DMAG did not enhance doxorubicin-induced cell apoptosis in K562 cells [Figure 2a and 3a].

Figure 2

5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG)-mediated cell apoptosis in K562 and K562/DOX cells. (a) K562 cells were treated with 0.15 μM of doxorubicin in the presence or absence of 1.0 μM of DMAG or 1.0 μM of verapamil for 24 h and 48 h, and then mean fluorescence intensity (MFI) of propidium iodide (PI) in K562 cells were detected by Array Scan VTIHCS600 High-Contents. (b) K562/DOX cells were treated with 15.0 μM of doxorubicin in the presence or absence of 1.0 μM of DMAG or 1.0 μM of verapamil for 24 h and 48 h and then MFI of PI in K562/DOX cells were detected by Arrary Scan VTIHCS600 High-Contents. *Represents P < 0.01 compared with DOX group. *Represents P < 0.05 compared with DOX + verapamil group

Figure 3

Apoptosis images of K562 and K562/DOX cells determined by double staining of Heochst 33342 and propidium iodide with Arrary Scan VTIHCS600 High-Contents. A-1~A-4: K562 cells treated with 0.15 μM of doxorubicin in the presence or absence of 1.0 μM of 5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG) or 1.0 μM of verapamil for 24 h; A-5~A-8: K562 cells treated with 0.15 μM of doxorubicin in the presence or absence of 1.0 μM of DMAG or 1.0 μM of verapamil for 48 h. B-1~B-4: K562/DOX cells treated with 15.0 μM of doxorubicin in the presence of 1.0 μM of DMAG or 1.0 μM of verapamil for 24 h; B-5~B-8: K562/DOX cells treated with 15.0 μM of doxorubicin in the presence or absence of 1.0 μM of DMAG or 1.0 μM of verapamil for 48 h. A-1, A-5, B-1, B-5: negative control; A-2, A-6, B-2, B-6: DOX treatment group; A-3, A-7, B-3, B-7: DOX in combination with DMAG group; A-4, A-8, B-4, B-8: DOX in combination with verapamil group, ×40

DMAG-mediated Intracellular Accumulation of Doxorubicin

The DMAG-mediated accumulation of doxorubicin in the K562/DOX cells was quantified with Array Scan VTIHCS600 High-Contents. The results showed a significant increase of accumulation of doxorubicin in the presence of DMAG [Figure 4]. After treatment of the K562/DOX cells for 1 h with 15.0 μM doxorubicin alone, the fluorescence intensity was 33093.12. Significantly, with the addition of 1.0 μM of DMAG, the fluorescence intensity was 2.3-fold higher. While the addition of 1.0 μM of DMAG did not increase the accumulation of doxorubicin in the K562 cells (data not shown).

Figure 4

5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG)- mediated intracellular accumulation of doxorubicin. The left showed intracellular doxorubicin fluorescence intensity. *Represents significant difference in MFI of doxorubicin between K562 cells and K562/DOX cells untreated and treated with verapamil (P < 0.01). **Represents significant difference in MFI of doxorubicin between K562/DOX cells treated with DMAG and those treated with verapamil (P < 0.01). The right shows VTIHCS600 High-Contents scanning images of intracellular doxorubicin accumulation. (a) K562 cells; (b) K562/DOX cells; (c) K562/ DOX cells treated with 1.0 μM of DMAG; (d) K562/DOX treated with 1.0 μM of verapamil, ×40

DMAG-mediated Intracellular Accumulation of Rhodamine 123

A similar test was performed with rhodamine 123 in the K562/DOX cells to validate the ability of DMAG-mediated the accumulation of doxorubicin and efflux function of P-gp. As expected, the results showed a significant increase of accumulation of rhodamine 123 in the presence of DMAG [Figure 5]. With the addition of 1.0 μM of DMAG, the fluorescence intensity was increased by 49.11% compared to control. However, DMAG showed little effect on accumulation of rhodamine 123 in K562 cells.

Figure 5

5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG)-mediated intracellular accumulation of rhodamine 123. Cells were pretreated with or without 1.0 μM of DMAG (verapamil as a positive control) for 1 h, then incubated with 5 mg/L of Hoechst 33342 for 10 min and 5 mg/L of Rh123 for another 1 h in the dark, (a) Fluorescence intensity of rhodamine 123 in the presence or absence of DMAG in K562 cells. (b) Fluorescence intensity of rhodamine 123 in the presence or absence of DMAG in K562/DOX cells.﹡Represents P < 0.05. (c) Image of fluorescence intensity of rhodamine 123. (c-1) K562 cells untreated. (c-2) K562 cells treated with 1.0 μM of 5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG). (c-3) K562 cells treated with 1.0 μM of verapamil. (c-4) K562/DOX cells untreated. (c-5) K562/DOX cells treated with 1.0 μM of DMAG. (c-6) K562/ DOX cells treated with 1.0 μM of verapamil.×40

Discussion

With the development of cancer, heart disease and other chronic diseases, western medicine alone cannot relieve the pain and death in all cases. In the “offbeat” treatments, herbal therapy in medicine is increasingly common. Development and clinical application of tradition Chinese medicine has caused widespread concern around the world, thus accelerating the spread and development of TCM in the world.

MDR, a common problem in cancer chemotherapy, means that cancer cells are simultaneously resistant to a number of structurally and functionally unrelated chemotherapeutic agents. P-gp, one of the ABC superfamily which represents one of the largest and most important families of membrane proteins, is responsible for resistance to multiple chemotherapeutic agents in many human cancers. The activity of the ABC-transporter represents a basic biological strategy to defend living cells against the cytotoxic attack of xenobiotics. A number of studies have tried to find P-gp inhibitors which increase anticancer drug accumulation in cancer cells.[1516171819] So far, no modulators have been successfully applied in clinic due to their toxicity and side-effects. The search for new drugs to overcome the limitations is in progress to satisfy an urgent need for clinical applications. Clinical practices have demonstrated that TCM can improve the effect of chemotherapy and reduce the adverse effect.[2021] Moreover, the extracts from TCM are in generally with little side-effects.[22] Various compounds from TCM, therefore, fit well with the reversal of tumor MDR.

Mahonia, a famous TCM, whose root and stem bark were popular folk medicines.[23] The plant shows biological activities including anti-bacterial, anti-fungal and anti-inflammatory effects.[242526] In addition, it has been shown that extracts from Mahonia could induce the apoptosis of cancer cells. DMAG is a purified compound extracted from Mahonia.

Safety and efficacy are the key elements in the modernization and internationalization of TCM. From a security perspective, TCM has lower toxicity than western medicine. In addition, in this study DMAG demonstrated more efficacy than verapamil on reversal of MDR. Cell viability assay demonstrated that DMAG enhanced doxorubicin cytotoxicity against resistant cells. The findings were consistent with the doxorubicin accumulation observations that the fluorescence signal of doxorubicin in the resistant cancer cells was stronger in the presence of DMAG. The results indicate that DMAG's action is mediated by its ability to promote the accumulation of doxorubicin in the resistant cancer cells.

Studies to investigate intracellular accumulation of doxorubicin in the presence or absence of DMAG may be complicated due to the cytotoxicity of doxorubicin. Therefore, further investigation of intracellular accumulation of rhodamine 123 in the presence or absence of DMAG was performed. The results showed that DMAG also enhanced the accumulation of rhodamine 123 in the resistant cells. The data with rhodamine 123 were consistent with the findings gained with doxorubicin.

In multidrug resistant K562/DOX cells, the reversal effect of DMAG on the resistance of cancer cells to doxorubicin was confirmed. The promising in vitro results suggest that DMAG may benefit MDR cancer patients when used in combination with chemotherapeutic agents. Further confirmation in animals will be performed in our laboratory, which will provide important data in human beings.