Reversal effect of Tween-20 on multidrug resistance in tumor cells in vitro

Biomedicine & Pharmacotherapy 66 (2012) 187–194

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Original article

Reversal effect of Tween-20 on multidrug resistance in tumor cells in vitro Shouhui Yang, JinJuan Liu 1, Yongqiang Chen, Jihong Jiang * Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province, Xuzhou Normal University, 101, Shanghai Road, 221116 Xuzhou, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 16 August 2011 Accepted 31 October 2011

Multidrug resistance (MDR) is a major barrier for chemotherapy of many cancers. Non-ionic surfactants have great potential to reverse the MDR by preventing onset or delay progression of the carcinogenic process. However, the role of Tween-20 in the development of MDR remains unknown. The aim of this study was to explore the reversal effect and potential mechanism of Tween-20 on tumor cells in vitro. Alamar Blue assay was used to examine the reversal index of Tween-20 to vincristine (VCR), doxorubicin (DOX) and 5-fluorouracil (5-FU) in KBv200, HepG2/R and Bel-7402/5-FU, respectively. Morphological change was determined by Gimsa and Hoechst 33258 staining. The acumulation of DOX was confirmed by spectrofluorimetric assay. Cell cycle analysis was performed using flow cytometry. The mRNA and protein expression levels of MDR were assessed by semiquantitative RT-PCR and dot blot, respectively. The results showed that Tween-20 at concentrations of 0.0025%, 0.005%, 0.01% had little cytotoxicity. When combined with the cancer drugs, it significantly promoted the sensitivity of MDR cells. Fluorescence staining confirmed that the percentage of apoptotic cell increased when combined with Tween-20. This notion was further supported by the observation that Tween-20 treatment potentiated VIN-induced G2/M arrest of the cell cycle. Furthermore, Tween-20 treatment increased significantly intracellular accumulation of DOX. RT-PCR and dot blot revealed that Tween-20 could downregulate the expression of MDR and P-glycoprotein. Low concentrations of Tween-20 can efficiently reverse the multidrug resistance phenotype by enhancing accumulation of the anticancer drugs. The potential mechanism may be via inhibiting the multidrug-resistant gene expression. Crown Copyright ß 2012 Published by Elsevier Masson SAS. All rights reserved.

Keywords: Tween-20 P-glycoprotein Multidrug resistance Reversal

1. Introduction Multidrug resistance (MDR), a phenomenon of resistance of cancer cells to diverse structurally and mechanically unrelated anticancer drugs, is a primary obstacle to successful cancer chemotherapy in cancer patients [1]. Molecular alterations in drug targets, apoptotic pathways, drug metabolizing enzymes and expression of drug transporters may be involved in MDR [2], however, overexpression of P-glycoprotein, a transmembrane glycoprotein, is the most frequent event causing MDR [3–5]. The overexpression of P-gp transporters in cancer cells increase efflux, reducing intracellular drug concentrations below therapeutically effective levels, which making tumors insensitive to chemotherapy treatment, and cause the failure of cancer chemotherapy [6]. A number of natural and synthetic products have been identified for their ability to reverse MDR by blocking P-gp activity in vitro [7–11]. These products include calcium channel blockers (e.g., verapamil, nifedipine), calmodulin antagonists (e.g., trifluoperazine,

* Corresponding author. Tel.: +(86) 516 83403515; fax: +(86) 516 83403515. E-mail addresses: [email protected], [email protected] (J. Liu), [email protected] (J. Jiang). 1 Co-author.

chlorpromazine), quinolines (e.g., chloroquine, quinidin e), immunosuppressive drugs (e.g., cyclosporin A, rapamy cin) and yohimbine alkoids (e.g., reserpine, yohimbine). However, they have not been widely used in the treatment of cancer patients because these MDR reversal agents may expose the patient to unacceptable side effects or toxicity at the doses required for effectiveness and/or affect the pharmacokinetics of the anticancer drug [12,13]. These limitations have spurred efforts to search for new, more effective compounds with low toxicity and fewer side effects. Surfactants are extensively used in pharmaceutical formulations as wetting agents to improve dissolution and absorption of poorly soluble drugs. Several non-ionic surfactants, such as Solutol HS 15, Tween-80, polysorbate 80 and Cremophor EL, have been shown to modulate the sensitivity of certain antitumor agents in vitro and in vivo [14–19]. These surfactants have been shown to reverse MDR by inhibiting membrane transporters, most reports of surfactant-induced inhibition of membrane transporters have focused on P-gp. Tween-20, a non-ionic, surface-active detergent, polyethylene glycol sorbitan monolaurate, has been showed to increase absorption of epirubicin in human colon adenocarcinoma (Caco-2) cell line, and also markedly enhanced mucosal to serosal absorption of epirubicin in the rat jejunum and ileum [20,21]. However, its effect on human cancer cell lines, and its regulation on the chemosensitivity of the malignant tumor cells to conventional

0753-3322/$ – see front matter . Crown Copyright ß 2012 Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2011.10.007

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chemotherapeutic agents has poorly reported. Based on this background, the present study was designed to investigate the potential of Tween-20 to modulate the chemosensitivity of human malignant tumor cells to several conventional used chemotherapeutic agents. 2. Materials and methods 2.1. Regents and chemicals Doxorubicin (Dox), vincristine (VCR), 5-fluorouracil (5-FU), propidium iodide (PI), Alamar Blue (resazurin), Hoechst 33258 were purchased from Sigma-Aldrich (Sigma, St. Louis, MO); Tween-20 was obtained from DingGuo (Dingguo, Beijing, China). Cell culture medium RPMI-1640 was from Gibco BRL (Gibco BRL, Cergy-Pontoise, France). The fetal bovine serum (FBS) was from Invitrogen (Invitrogen, Carlsbad, CA). Trypsin, Steptomycin, Penicillin were obtained from Sunshine (Sunshine Biotechnology Co. Ltd, Nanjing, China). P-gp antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). P-Tubulin antibody was from Bioworld (Bioworld Technology, MN). All other chemicals were of the highest commercial grade available.

without Tween-20 were added for its reversal effect. Then the cells were incubated for another 2 days. Thereafter, 20 ml Alamar Blue stock solution was added to each well and cultured for an additional 4 h. The absorbance was measured using SpectraMax M2 (molecular devices) with a test wavelength of 530 nm and 590 nm. IC50 values were calculated by SPSS. IC50, the half maximal inhibitory concentration, represents the concentration of the modulators that is required for 50% inhibition of cells. The reversal fold (RF) value was calculated from dividing IC50 of the anticancer drug alone by IC50 of antidrugs in combination with Tween-20 [25]. Triplicate experiments with triplicate samples were performed. Control medium included equivalent amount of DMSO. 2.4. Giemsa staining Morphological assessment of treated cells was performed using the May-Grunwald-Giemsa (MGG) staining method [26]. Briefly, cells were seeded on to sterilized slides and incubated for 24 h. Thereafter, cells were treated for 48 h with IC50 concentrations of antidrugs alone or with Tween-20. The slides were dried and fixed with methanol for 1 min, after stained with MGG stain for 5 min followed by intense deionized water washing. After air-drying the slides were observed under light microscope (IBE2000, Nikon).

2.2. Cell lines and cell culture 2.5. Hoechst 33258 staining Human HepG2 hepatoma, and its multidrug-resistant, P-gp overexpressing, sublines HepG2/R were kind gifts from Pr F.-T. He (city university of Hong Kong, China). Human oral epidermoid carcinoma KB and the VCR-selected resistant subline KBv200, human hepatocelluar carcinoma (HCC) cell line Bel-7402 and the 5-FU selected drug-resistant Bel-7402/5-FU cells were kindly supplied by Pr G.-T. Liu (Peking Union Medical College & Chinese Academy of Medical Sciences, China) [22,23]. All cells were maintained in RPMI164- medium supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin and incubated under humidified air with 5% CO2 at 37 8C. HepG2/R cells were maintained with DOX, KBv200 cells with VCR, and Bel-7402/5-FU cells with 5-FU to retain their resistance in daily culture. The cells were cultured for 2 weeks in drug-free medium prior to their use in the experiments. The origin of the tumor cell lines used and the orders of drug resistance are listed in Table 1. 2.3. Cytotoxicity and multidrug resistance reversal assay Chemosensitivity in vitro was measured using the Alamar Blue method [24]. Briefly, cells with same number (2  103 cells/ml for KB, 5  104 cells/ml for HepG2 and Bel-7402) were inoculated into each well in 96-well plates (Costar, Charlotte, NC) with 100 ml culture medium. After an overnight incubation, various concentration of Tween-20 in 100 ml diluted with the medium were added into the wells for determine the antiproliferative effect of Tween-20. Graded concentrations of the anticancer drugs with or

Cells were seeded on to sterilized slide in 24 pore plate for 24 h, were fixed in acetic acid/methanol (1:3) solution for 5 min at 4 8C after treated with drugs for another 48 h, then washed three times with phosphate-buffered saline (PBS). Hoechst 33258 was added at a final concentration of 10 ng/ml for 5 min, and analysis was determined immediately by fluorescence microscopy after washed three times with PBS. 2.6. Cellular accumulation and retention of DOX Cellular DOX accumulation was first determined by spectrofluorometry as described previously [27]. In brief, cells were treated with 10 mM DOX for 3 h in the absence or presence of Tween-20. Then the cells were harvested by centrifugation after washed twice with ice-cold PBS. Thereafter, cell pellets were suspended with 0.3M HCl in 50% ethanol. Following centrifugation at 10,000 rpm for 15 min, the supernatant was removed and assayed spectrofluorometrically for DOX content at excitation and emission wavelengths of 470 nm and 585 nm. The amount of cellassociated DOX was derived from standard curves prepared with DOX alone and expressed as nmol ADM/106 cells. 2.7. Cell cycle analysis Approximately 1  106 KBv200 and HepG2/R cells in complete growth medium were treated with VCR and DOX, respectively

Table 1 Characteristics of tumor cell lines used in this study and the cytotoxicity of Tween-20 in these cell lines. Cell line

Origin/derivation

Level of resistancea

IC50 of Tween-20 (v/v, %)

KB KBv200 HepG2 HepG2/R Bel-7402 Bel-7402/5-FU

Human oral epidermoid carcinoma Continuous maintained in the presence of 200 nmol/mL VCR Human hepatocarcinoma cell line Continuous maintained in the presence of 1 mmol/L DOX Human hepatocellular carcinoma Continuous maintained in the presence of 10 mg/ml 5-FU

– 72.5 to VCR – 32.6 to DOX – 21.1 to 5-Fu

0.014  0.0052 0.016  0.0031 0.025  0.0030 0.021  0.0017 0.015  0.0026 0.017  0.0037

The cytotoxicity of Tween-20 to various tumor cell lines was determined by the Alamar Blue assay. Cells were exposed continuously to different concentration of Tween-20 for 48 h. The IC50 values of Tween-20 were calculated by a computer program based on the cytotoxicity curves, and the values presented are means  SD of at least three independent experiments, with each concentration tested in triplicate. a Relative resistance: IC50 of a drug in the resistant cell line divided by that of the drug in the parental drug-sensitive cell line. The data came from measurement when cell lines in well condition.

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2.8. RT-PCR analysis Total RNA was isolated from treated cells with Trizol reagent (Invitrogen) as suggested by the manufacturer. Total RNA (1 mg) was reverse-transcribed into cDNA using TAKARA RNA PCR kit. The following primers were used for RT-PCR: MDR (213 bp) sense: 50 -CACTTCAGTTACCCA TCTC-30 , antisense: 50 -TTCCCGTAGAAACCTTAC-30 ; GAPDH (250 bp) sense: 50 -GAAGGTCGG AGTCAACGGATTT-30 , antisense: 50 -CTGGAAGATGGTGATGGGATTTC-30 . The reaction conditions were set as follows: 95 8C for 5 min, 35 cycles at 95 8C for 30 s, 55 8C for 30 s, and 72 8C for 45 s, followed by a 7 min extension at 72 8C. Serial dilutions were used for each cDNA to ensure that the reaction did not reach the plateau. The PCR products were separated in 1% agarose gel. Fixed the images with gel imaging system and measure the target gene expression level with semiquantitative analysis. Relative content of mRNA = accumulative photon value of target gene band/accumulative photon value of internal standard GAPDH band. 2.9. Dot blot Cells were harvested, rinsed with PBS and lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 1% Triton X-100, 0.1% SDS, 5 mM EDTA, and freshly prepared 1 mM PMSF). Lysates were collected and cleared by centrifugation at 4 8C. The samples were heated in boiling water for 5 min and then loaded onto a hybond-P PVDF membrane (Amershan Biosciences). The membrane was blocked with 5% skimmed milk in PBS and incubated with specific rabbit anti-human antibodies to P-gp (1:200 diluted) or rabbit anti-human antibodies to tubulin (1:500 diluted), followed by 1:3000 diluted horseradish peroxidase-conjugated mouse antirabbit IgG. The dot antibody was detected using the enhanced chemilluminescent (ECL) system (Amersham, UK). Expressed protein levels were quantified by measuring the intensity of the area for each dot using ImageMaster software. These values then were normalized by that of the P-tubulin dot, and were expressed as a percentage in comparison to that of control. 2.10. Data analysis Data were expressed as mean  SD from at least three independent experiments. One-way analysis of variance and a log linear model were used to compare the mRNA and protein levels. A value of P < 0.05 was considered statistically significant, and if P < 0.01, it was noted. 3. Results 3.1. Evaluation of the cytotoxic effect of Tween-20 To detect the cytotoxicity of Tween-20 alone in each cell line, Alamar Blue assay was performed. As shown in Fig. 1, Tween-20 at 0.013–0.025% concentration exerted considerable cytotoxicity in both resistant cells and their parental cells. However, Tween-20 concentrations of less than 0.01% showed non-toxic effects in all cells with over 90% cell survival. To minimize the effect of Tween-20 itself on the cell cytotoxicity, lower concentrations of Tween-20 (0.001%, 0.0025%, 0.005%) were chosen in the reversal experiments.

120

KB KBv200 Bel7402 Bel7402 /5-FU HepG2 HepG2/R

100

Cell viability(% of control)

alone or in combination with 0.005% (v/v) Tween-20 for 24 h. Cells were harvested, washed twice with ice-cold PBS and fixed in 70% ethanol at 20 8C overnight. Thereafter, cells were washed with PBS and incubated with 100 mg/ml RNase at 37 8C for 30 min. Then stained with 100 ng/ml propidium iodide solution and analyzed with flow cytometry (Becton dickinson, USA). KB and HepG2 cell treated with VCR, DOX as control.

189

80

60

40

20

0 0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

Concentration of Tween-20(%,V/V) Fig. 1. Cytotoxicity effect of Tween-20 on the multidrug resistance cell lines KBv200, HepG2/R, Bel-7402/5-FU and their parental sensitive cells KB, HepG2, Bel-7402. The cells were cultured with a full range of concentrations of Tween-20 for 48 h. Cell viability was evaluated by Alamar Blue assay. Bars represent means  standard deviations (SD) of at least triplicate determinations.

3.2. Effect of Tween-20 on susceptibility of tumor cells to anticancer drugs The modulation of Tween-20 on the sensitivity of VCR, DOX, 5-FU against KBv200, HepG2/R, Bel-7402/5-FU and their parental cells is shown in Fig. 2 and Table 2. The three MDR cell lines showed much higher tolerance to chemotherapeutic drugs compared to their parental, drug-sensitive cell lines. The treatment of Tween-20 at non-toxic concentrations induced a significant decrease of the IC50s of cancer drugs against the resistant cells in a concentration-dependent manner. In the KBv200 cells, significant different of the IC50 (1.733, 0.343, 0.095, 0.032) were seen in dose responses (0.00%, 0.0025%, 0.005%, 0.01%) to VCR treatment. This gave a 5.09, 18.2, 54-fold reversal respectively. However, no such activity was found in KB cells. The same effect can be found in HepG2/R and HepG2 cell lines. In HepG2/R cell lines Tween-20 gave a 3.2, 10.01, 37.5-fold reversal to DOX, but had no effect on HepG2 cells (Fig. 2C and D, Table 2). In the MPR-induced MDR Bel-7402/5-FU and Bel-7402 cells, Tween-20 had no effect on the two cells to 5-FU (Fig. 2E and F). These results suggested the modulation effect of Tween-20 to cancer cells may relate to cellular phenotype or drug characteristics. These findings indicated that Tween-20 enhanced the potency of VCR, DOX against P-gp-induced KBv200, HepG2 cells, respectively, whereas, had little effects on their parental cells and MPR-induced cells, supporting the notion that Tween-20 could reverse P-gp mediated resistance of KBv200 and HepG2/R cells. In addition, the effects of Tween-20 on the relevant drugs were also examined in the drug-resistant cells. As summarized in Table 2, Tween-20 significantly increased the sensitivity of HepG2/ R cells to VIN and KBv200 cells to DOX. The IC50 values of DOX to KBv200 were lowered by 9.7- to about 52-fold, VIN to HepG2/R were lowered to 2.8- to 14.8-fold. It is very surprised to see that Tween-20 can reverse the Bel-7402/5-FU cell lines to DOX, but has little effect on the sensitivity of Bel-7402/5-FU to 5-FU. The IC50 values of DOX to Bel-7402/5-FU were lowered to 9- to 21.5-fold (Fig. 2E and F). Since DOX are known to be substrates for P-gp and MPR, we concluded that Tween-20 modulates intracellular drug levels by inhibiting P-gp.

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A

B 120 VIN VIN+0.0025% Tween-20 VIN+0.005% Tween-20 100 VIN+0.01% Tween-20

120

Cell viability(% of control)

Cell viability( % of control)

100 80 60 40 20

VIN VIN+0.0025% Tween-20 VIN+0.005% Tween-20 VIN+0.01% Tween-20

80 60 40 20 0

0 0

10

20

30

40

50

0

60

500

Concentration of VIN(ng/ml) 120

DOX DOX+0.0025% Tween-20 DOX+0.005% Tween-20 DOX+0.01% Tween-20

Cell viability(% of control)

100

80

60

40

20

D

5

10

15

20

25

60 40 20 0

30

0

20

5-FU 5-FU+0.0025% Tween-20 5-FU+0.005% Tween-20 5-FU+0.01% Tween-20

Cell viability(% of control)

100 80 60 40 20 0

40

60

80

100

120

Concentrationod DOX(umol/ml)

F

5-FU 5-FU+0.0025% Tween-20 5-FU+0.005% Tween-20 5-FU+0.01% Tween-20

120 100

Cell viability(% of control)

120

2500

80

Concentration od DOX(umol/ml)

E

2000

DOX DOX+0.0025% Tween-20 DOX+0.005% Tween-20 DOX+0.01% Tween-20

100

0 0

1500

120

Cell viability(% of control)

C

1000

Concentration of VIN(ng/ml)

80 60 40 20 0

0

2

4

6

8

10

12

14

Concentration of 5-FU(umol/ml)

0

20

40

60

80

100

120

Concentration of 5-FU(umol/ml)

Fig. 2. Effects of Tween-20 on the sensitivity of drug-resistant cells and parental cells toward cancer drugs was examined by Alarmar blue method as described in the cytotoxicity assay and multidrug resistance reversal tests. These cells were treated with varying concentrations of cancer drugs in the presence of 0.00%, 0.0025%, 0.005% and 0.01% of Tween-20. IC50 values for the drugs were calculated. Data represent means and standard errors of at least triplicate determinations: (A) KB cells; (B) KBv200 cells; (C) HepG2 cells; (D) HepG2/R cells; (E) Bel-7402 cells; (F) Bel-7402/5-FU cells.

3.3. Morphological changes To characterize morphological changes, Giemsa and Hoechst 33258 staining was performed. As displayed in Fig. 3, KBv200 and HepG2/R cells grew regularly when only treated with 95 ng/ml VIN or 7.36 mmol/ml DOX, but when co-incubated with 0.005% Tween20, the cells shrink in size, break into smaller pieces called apoptotic bodies, which are the typical of apoptosis. These results suggested that the increase inhibitory effects on KBv200 and

HepG2/R cells from the combination of Tween-20 with VCR or DOX were achieved through the action of Tween-20, which enhanced the VCR or DOX-induced apoptosis. 3.4. DOX accumulation and retention The ability of Tween-20 to increase the DOX-induced cytotoxicity towards KBv200 and Bel-7402/5-FU cells was further evaluated by the intracellular DOX-associated mean fluorescence

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Table 2 Effect of Tween-20 on the cytotoxicities of DOX, VCR and 5-FU expressed as IC50 values. Reverse agents

KBv200

HepG2/R

Bel-7402 /5-FU

IC50

RF

IC50

RF

IC50

RF

VCR alone (mg/ml) +Tween-20 0.0025% +Tween-20 0.005% +Tween-20 0.01%

1.733  0.225 0.343  0.132 0.095  0.023 0.032  0.068

– 5.09 18.2 54

1.230  0.215 0.435  0.191 0.127  0.113 0.083  0.026

– 2.8 9.7 14.8

ND ND ND ND

– – – –

Dox alone (mmol/ml) +Tween-20 0.0025% +Tween-20 0.005% +Tween-20 0.01%

17.95  0.27 1.85  0.257 0.95  0.365 0.34  0.054

– 9.7 18.9 52.8

74.32  3.025 23.25  2.34 7.36  1.061 1.98  0.69

– 3.2 10.01 37.5

13.75  0.381 1.53  0.254 0.87  0.214 0.64  0.191

– 9.0 15.8 21.5

5-Fu alone (mg/ml) +Tween-20 0.0025% +Tween-20 0.005% +Tween-20 0.01%

ND ND ND ND

– – – –

ND ND ND ND

– – – –

68.25  5.219 61.26  7.125 60.25  1.240 64.167  3.027

1.11 1.13 1.06

Tumor cells were treated with graded concentrations of DOX, VCR or 5-FU in the absence or presence of different concentration of Tween-20. After 48 h of continuous drug exposure, the cell survival rate was measured by Alamar Blue assay. The data presented are the mean  SD IC50 values from at least three independent experiments. The values in parentheses are fold reversal calculated from the expression: FR = IC50 anticancer drug alone/IC50 anticancer drug + modulator (ND: not determined).

Fig. 3. Morphology changes observed by Gimsa and Hoechst 33258 staining. KBv200 cells were treated with 100 ng/ml VIN alone or with 0.005% Tween-20 for 24 h, HepG2/R were treated with 3.125 mmol/ml DOX alone or with 0.005% Tween-20 for 24 h. All the cell lines were stained with Gimsa and Hoechst 33258 and examined by light microscope and fluorescence microscopy. Apoptotic bodies due to nuclear fragmentation, chromatin condensation and dipersed of fragmented particle-like blue fluorescence can be seen in cells co-treated with Tween-20, especially in KBv200 cells (showed with ).

intensity. As indicated in Fig. 4, the accumulation of DOX in KBv200 and Bel-7402/5-FU cells was significantly lower than that in their parental cells, which is an essential cause of decreased inhibitory effect of DOX on KBv200 and Bel-7402/5-FU cells. After treatment with 0.0025%, 0.005% and 0.01% of Tween-20 for 3 h respectively,

intracellular accumulation of DOX was completely restored in KBv200 and Bel-7402/5-FU cells. On the contrary, no such restore in their parental cells was observed. The accumulation levels of DOX in KBv200 and Bel-7402/5-FU cells were fitted well with the cytotoxicity in each data point, indicating that Tween-20 elevated

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Fig. 4. Effect of Tween-20 on DOX accumulation in KBv200, KB, Bel-7402/5-FU and Bel-7402 cells. Cells were incubated with DOX (10 mM) with or without the indicated concentrations of Tween-20 for 3 h. Intracellular DOX content was measured fluorospectrophotometrically and is expressed as nanomoles per 106 cells. (A) KB, KBv200. (B) Bel-7402, Bel-7402/5-FU. Bars present means  SD from triplicate determinations. ** and * represent P < 0.01 and 0.05 versus control group.

Fig. 5. Cell cycle analysis of KBv200 cells by flow cytometry. Cells were treated with 100 ng/ml VCR alone or Tween-20 alone or VCR and Tween-20 for 16 h. After staining with PI, cells were subjected to flow cytometry analysis to determine the cell distributions at each phase of the cell cycle. Bars present means  SD from triplicate determinations. ** represents P < 0.01 between the two groups.

their sensitivity toward DOX through increasing DOX accumulation, and simultaneously providing a strong evidence that Tween-20 could effectively reverse P-gp mediated MDR. 3.5. Restoration of specific action of vincristine in MDR cells If Tween-20 main action is to maintain the intracellular concentration of cancer drugs, it should also restore the original action of respective drugs. This was demonstrated by the effect on the specific action of vincristine. Vincristine is a microtubule inhibitor impeding mitotic spindle formation and inducing G2/M phase arrest during cell cycle progression [24]. As displayed in Fig. 5, the treatment of VCR or Tween-20 alone showed no effect on the cell cycle of KBv200 cells, while the treatment of these two together increased dramatically the number of cells at the G2/M phase to 85.56% compared with 18.27% treated with vincristine alone. Similar results were observed in the HepG2/R cells (data not shown). 3.6. Tween-20 decreasing the expression of P-gp The MDR gene encodes the drug efflux pump P-gp, whose overexpression is associated with the MDR phenotype in many selected drug-resistant cell lines. To further confirm whether Tween-20 could down regulate MDR/P-gp expression, the RT-PCR

and dot blot technique were performed. As indicated by the results, HepG2/DR cells showed a marked up-regulation of MDR/P-gp gene expression (Fig. 6A). After 48-h incubation with 0.005% of Tween-20, the expression level of MDR was decreased in comparison with untreated HepG2/R cells. The expression of Pgp showed consistent trend with the mRNA level of MDR (P < 0.05) (Fig. 6B). The results indicated that Tween-20 downregulated expression of P-glycoprotein. 4. Discussion Chemotherapy is the most effective treatment for patients who suffer from metastatic cancers. The effectiveness of chemotherapy, however, is seriously limited by MDR which is mainly due to the overexpression of P-gp and MRP, they function as a drug efflux pump which actively transports drugs from the inside to the outside of cancer cells and prevents the intracellular accumulation of anticancer drugs inside cancer cells necessary for cytotoxic activity. Therefore, agents which can inhibit the drug transporter function of P-gp and MRP, or their expression have the potential to overcome the MDR phenotype by enhancing intracellular accumulation of anticancer drugs. The present study shows that treatment with nontoxic concentration of Tween-20 can lead to a reversal of the MDR phenotype by enhancing the intracellular accumulation of

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Fig. 6. Effect of Tween-20 in HepG2/R cells on the expression of MDR. RT-PCR analysis and dot blot were performed after treating with Tween-20 for 24 h and 36 h respectively. The results showed that Tween-20 could downregulate the mRNA abundance of MDR and the expression of P-gp. Measurements were plotted as the mean of at least three biological replicates  SD. Changes are labeled as significant (*) if P < 0.05.

anticancer drugs and decreasing the expression of MDR in mRNA level and protein level. Tween-20 has been showed to increase absorption of epirubicin in human colon adenocarcinoma (Caco-2) cell line, and also markedly enhanced absorption of epirubicin in the rat jejunum and ileum [21,28]. To investigate its effect of chemosensitivity on other human cancer cell lines to cytotoxic drugs, we selected three couples of cell line (Table 1). KBv200 and HepG2/R which expressed p-gp, have shown resistance to anthracyclines, taxanes, vinca alkaloids [29,30], and Bel-7402/5-FU, which was established by in vitro continuous stepwise exposure of human hepatocellular carcinoma (HCC) cell line Bel-7402 to 5-FU, has shown multidrug resisance to 5-FU and DOX [22]. So, we selected VIN, DOX, and 5-FU as the xenobiotic, and to determine the effects of Tween-20 on cell killing by these drugs in the three couples of human carcinoma cells. Our experimental results indicated Tween-20 itself exerted similar cytotoxicity in both sensitive and resistant cell lines, indicating that Tween-20 itself may not be a substrate of P-gp or MRP. In an attempt to discriminate the synergic cytotoxicity from the chemosensitivity-enhancing effect of Tween-20, noncytotoxic concentrations of Tween-20 were chosen. Tween-20 at amount of 0.0025%, 0.005%, 0.01% potentiated the sensitivities of resistant cells KBv200, HepG2/R to DOX, VCR, However, no similar effects were observed in non-P-gp or MPR-overexpressing parental cell lines. This suggests that Tween-20 is a potent MDR modulator. Similar results can be viewed in Bel-7402/5-FUcells, although Tween-20 had little effect on cell killing ability of 5-FU, this may be resulted from different drug binding site of the MRP to 5-FU, or the different properties of the chemical itself. It is thought that the decreased cellular accumulation of anticancer drug is due to an increase in the extrusion of the drug mediated by P-gp or MRP [6]. Thus, it is paramount that the accumulation of anticancer drugs in MDR cells is improved if MDR is to be reversed. In this study, Tween-20 significantly increased DOX accumulation in KBv200 and Bel-7402/5-FUcells in a dosedependent manner (Fig. 4). This demonstrates that the circumvention of MDR mediated by Tween-20 is associated with an increased accumulation of the anticancer drug. Furthermore, the increased accumulation of anticancer drugs in response to Tween-20 dramatically enhances apoptotic potential, as evidenced

by the observations that co-treatment with Tween-20 increases fragmented punctuate blue nuclear fluorescence by vincristine or DOX (Fig. 3). Altogether, these findings indicate that Tween-20 may reverse the MDR phenotype in drug-resistant cells by inhibiting the drug efflux function of P-gp. This notion is further confirmed by our observation that Tween-20 treatment enhances the ability of VIN to induce G2/M arrest of KBv200 cells (Fig. 5). There may be several possible mechanisms that Tween-20 increases the cellular concentration of anticancer drugs in MDR cells. It had been reported that the functional of P-glycoprotein is dependent on maintenance of a lipid-protein interface [31]. Surfactants may disrupt the lipid arrangements in cell membranes and increase the water content of the membrane proteins [32,33], which can inhibit the function of membrane transporters. Several reports demonstrated that plasma membrane lipid fluidity is related to the functional of P-gp. Tetsuo Yamazaki [16] and Ichiro Tsujino [34] suggested that lipophilic VP-16 is transported more readily at the cell membrane following a change in the character of the membrane caused by Tween-80. However Bhagwant D. Rege [35] argued that surfactants can inhibit multiple transporters but that changes in membrane fluidity may not be a generalized mechanism to reduce transporter activity. Yu-li Lo [28] demonstrated pharmaceutical excipients, such as Tween-20, Tween-80, enhanced efficacy of epirubicin by fluidizing membranes and thus promoting the diffusion of amphiphilic epirubicin across Caco-2 cells, these reverse effects owed to the lipophilic and hydrophilic structural properties. There are other reports suggest that low concentrations of Triton X-100 reverse the MDR phenotype by inhibiting P-glycoprotein drug binding. However, our results showed that Tween-20 decreased the expression of P-gp, therefore increasing cellular concentratrion of antidrugs. Taken together, we concluded that Tween-20 enhanced the cytotoxicity of anticancer agents by increased accumulation of the anticancer drugs through inhibiting the expression of P-pg. The study of reversal effect of Tween-20 in vivo pharmacokinetics will be further investigated. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.

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Acknowledgements [18]

This study was supported by grants from the Major Project of Educational Commission of Jiangsu province (NOs. 08KJA350001) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, Gottesman MM. P-glycoprotein: from genomics to mechanism. Oncogene 2003;22:7468–85 [PubMed: 14576852]. [2] Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATPdependent transporters. Nat Rev Cancer 2002;2:48–58 [PubMed: 11902585]. [3] Nielsen D, Skovsgaard T. P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines. Biochim Biophys Acta 1992;1139:169–83 [PubMed: 1392705]. [4] Deeley RG, Cole SP. Substrate recognition and transport by multidrug resistance protein 1 (ABCC1). FEBS Lett 2006;580:1103–11 [PubMed: 16387301]. [5] Bradley G, Juranka PF, Ling V. Mechanism of multidrug resistance. Biochim Biophys Acta 1988;948:87–128 [PubMed: 2899442]. [6] Krishna R, Mayer LD. Multidrug resistance (MDR) in cancer. Mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs. Eur J Pharm Sci 2000;11:265–83 [PubMed: 11033070]. [7] Tan B, Piwnica-Worms D, Ratner L. Multidrug resistance transporters and modulation. Curr Opin Oncol 2000;12:450–8 [PubMed: 10975553]. [8] Yang L, Wei DD, Chen Z, Wang JS, Kong LY. Reversal of multidrug resistance in human breast cancer cells by Curcuma wenyujin and Chrysanthemum indicum. Phytomedicine 2011 [PubMed: 21306883]. [9] Xiang W, Gao A, Liang H, Li C, Gao J, Wang Q, et al. Reversal of P-glycoproteinmediated multidrug resistance in vitro by milbemycin compounds in adriamycin-resistant human breast carcinoma (MCF-7/adr) cells. Toxicol In Vitro 2010;24:1474–81 [PubMed: 20656007]. [10] Loe DW, Deeley RG, Cole SP. Verapamil stimulates glutathione transport by the 190-kDa multidrug resistance protein 1 (MRP1). J Pharmacol Exp Ther 2000;293:530–8 [PubMed: 10773025]. [11] Barancik M, Bohacova V, Sedlak J, Sulova Z, Breier A. LY294,002, a specific inhibitor of PI3K/Akt kinase pathway, antagonizes P-glycoprotein-mediated multidrug resistance. Eur J Pharm Sci 2006;29:426–34 [PubMed: 17010577]. [12] Bellamy WT, Dalton WS, Dorr RT. The clinical relevance of multidrug resistance. Cancer Invest 1990;8:547–62 [PubMed: 1979932]. [13] Kerb R, Hoffmeyer S, Brinkmann U. ABC drug transporters: hereditary polymorphisms and pharmacological impact in MDR1, MRP1 and MRP2. Pharmacogenomics 2001;2:51–64 [PubMed: 11258197]. [14] Coon JS, Knudson W, Clodfelter K, Lu B, Weinstein RS. Solutol HS 15, nontoxic polyoxyethylene esters of 12-hydroxystearic acid, reverses multidrug resistance. Cancer Res 1991;51:897–902 [PubMed: 1988130]. [15] Friche E, Jensen PB, Sehested M, Demant EJ, Nissen NN. The solvents cremophor EL and Tween-80 modulate daunorubicin resistance in the multidrug resistant Ehrlich ascites tumor. Cancer Commun 1990;2:297–303 [PubMed: 1976341]. [16] Yamazaki T, Sato Y, Hanai M, Mochimaru J, Tsujino I, Sawada U, et al. Non-ionic detergent Tween-80 modulates VP-16 resistance in classical multidrug resistant K562 cells via enhancement of VP-16 influx. Cancer Lett 2000;149:153– 61 [PubMed: 10737719]. [17] Tahara K, Kato Y, Yamamoto H, Kreuter J, Kawashima Y. Intracellular drug delivery using polysorbate 80-modified poly(D,L-lactide-co-glycolide)

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

nanospheres to glioblastoma cells. J Microencapsul 2011;28:29–36 [PubMed: 21171814]. Bhattacharjee J, Verma G, Aswal VK, Date AA, Nagarsenker MS, Hassan PA. Tween-80-sodium deoxycholate mixed micelles: structural characterization and application in doxorubicin delivery. J Phys Chem B 2010;114:16414–21 [PubMed: 21080698]. Omokawa Y, Miyazaki T, Walde P, Akiyama K, Sugahara T, Masuda S, et al. In vitro and in vivo anti-tumor effects of novel Span 80 vesicles containing immobilized Eucheuma serra agglutinin. Int J Pharm 2010;389:157–67 [PubMed: 20100554]. Lo YL. Relationships between the hydrophilic-lipophilic balance values of pharmaceutical excipients and their multidrug resistance modulating effect in Caco-2 cells and rat intestines. J Control Release 2003;90:37–48 [PubMed: 12767705]. Lo YL, Hsu CY, Huang JD. Comparison of effects of surfactants with other MDR reversing agents on intracellular uptake of epirubicin in Caco-2 cell line. Anticancer Res 1998;18:3005–9 [PubMed: 9713500]. Jin J, Huang M, Wei HL, Liu GT. Mechanism of 5-fluorouracil required resistance in human hepatocellular carcinoma cell line Bel (7402). World J Gastroenterol 2002;8:1029–34 [PubMed: 12439919]. Jin J, Wang FP, Wei H, Liu G. Reversal of multidrug resistance of cancer through inhibition of P-glycoprotein by 5-bromotetrandrine. Cancer Chemother Pharmacol 2005;55:179–88 [PubMed: 15378274]. Nakayama GR, Caton MC, Nova MP, Parandoosh Z. Assessment of the Alamar Blue assay for cellular growth and viability in vitro. J Immunol Methods 1997;204:205–8 [PubMed: 9212838]. Labeed FH, Coley HM, Thomas H, Hughes MP. Assessment of multidrug resistance reversal using dielectrophoresis and flow cytometry. Biophys J 2003;85:2028–34 [PubMed: 12944315]. Tolis C, Peters GJ, Ferreira CG, Pinedo HM, Giaccone G. Cell cycle disturbances and apoptosis induced by topotecan and gemcitabine on human lung cancer cell lines. Eur J Cancer 1999;35:796–807 [PubMed: 10505042]. Huang M, Liu G. The study of innate drug resistance of human hepatocellular carcinoma Bel7402 cell line. Cancer Lett 1999;135:97–105 [PubMed: 10077227]. Terzis G, Georgiadis G, Vassiliadou E, Manta P. Relationship between shot put performance and triceps brachii fiber type composition and power production. Eur J Appl Physiol 2003;90(1–2):10–5. Gottesman MM, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem 1993;62:385–427 [PubMed: 8102521]. Quan F, Pan CE, Zhang SQ, Yan LY, Yu L. Reversal effect of resveratrol on chemotherapy resistance in KBv200 cell line and underlying mechanisms. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2009;44:63–8 [PubMed: 19484992]. Callaghan R, Berridge G, Ferry DR, Higgins CF. The functional purification of P-glycoprotein is dependent on maintenance of a lipid-protein interface. Biochim Biophys Acta 1997;1328:109–24 [PubMed: 9315609]. Dudeja PK, Anderson KM, Harris JS, Buckingham L, Coon JS. Reversal of multidrug resistance phenotype by surfactants: relationship to membrane lipid fluidity. Arch Biochem Biophys 1995;319:309–15 [PubMed: 7771801]. Sakai M, Imai T, Ohtake H, Azuma H, Otagiri M. Effects of absorption enhancers on the transport of model compounds in Caco-2 cell monolayers: assessment by confocal laser scanning microscopy. J Pharm Sci 1997;86:779–85 [PubMed: 9232516]. Tsujino I, Yamazaki T, Masutani M, Sawada U, Horie T. Effect of Tween-80 on cell killing by etoposide in human lung adenocarcinoma cells. Cancer Chemother Pharmacol 1999;43:29–34 [PubMed: 9923538]. Rege BD, Kao JP, Polli JE. Effects of nonionic surfactants on membrane transporters in Caco-2 cell monolayers. Eur J Pharm Sci 2002;16:237–46 [PubMed: 12208453].