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In vitro and in vivo multidrug resistance reversal activity by total Alkaloid of Fritillaria thunbergii in cisplatin-resistant
添加时间: 2017-9-7 11:15:04 来源: 作者: 点击数:264

In vitro and in vivo multidrug resistance reversal activity by total Alkaloid of Fritillaria thunbergii in cisplatin-resistant human ovarian carcinoma SKOV3/DDP cells

LI Ze-hui 李泽慧, AN Chao安超, TANG Min-ke唐民科, HU Kai-wen胡凯文

LI Ze-hui, TANG Min-ke, Department of Pharmacology of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China

AN Chao, HU Kai-wen, Department of Oncology, East Hospital, Beijing University of Chinese Medicine, Beijing 100078, China

Supported by Ministry of Science and Technology of the PRC: National Science and technology Major Project significant new drugs development (No. 2009ZX09103-346).


Abstract

OBJECTIVE: To explore the effect of Total Alkaloid of Fritillaria thunbergii (TAF) on multidrug resistance (MDR) of SKOV3/DDP cells.

METHODS: The cells were treated with cisplatin (DDP), DDP plus TAF, DDP plus Cyclosporine A (Cys A), DDP plus Tetrandrine (Tet), respectively. Cell viability was measured by MTT assay. The level of MDR1 mRNA and P-glycoprotein (P-gp) were detected. Tumor inhibition in Balb/c nude mice rate (%) was calculated.

RESULTS: Drug resistance reversal multiple of TAF, Cys A and Tet was 6.29±0.52, 4.87±0.93, 4.81±1.72, respectively. The relative level of MDR1 mRNA and P-gp of cells in TAF group were decreased (P<0.01). In vivo study indicated that TAF could facilitate DDP to reduce the volume and weight of SKOV3/DDP tumor.

CONCLUSIONS: We showed that TAF reversed partially MDR of SKOV3/DDP tumor cells, and the effect may be related to down-regulation of the level of MDR1 mRNA and P-gp in SKOV3/DDP cells.

Keywords:Total Alkaloid of Fritillaria thunbergii; multidrug resistance reversal; SKOV3/DDP cell; MDR1 mRNA; P- glycoprotein


INTRODUCTION

Chemotherapy is a main clinical therapy method for treating tumors. An obstacle in the process of chemotherapy is multidrug resistance (MDR). Tumor cells show drug resistance to different chemotherapeutics as the treatment extending. Over-expression of P-glycoprotein (P-gp) is one of the major mechanisms of MDR. P-gp can pump drugs to extra-cellular membranes and then compromise the efficacy of chemotherapeutics [1]. Therefore, to find some drugs which can reverse MDR of tumor cells will be helpful in the process of tumor treatment.

Studies have shown verapamil, dexniguldipine, Cyclosporine A (Cys A) et al have reversal effect on MDR, but the use of these drugs is limited because of severe side effects on heart, liver and kidney. In recent years, researchers have paid more attention to Chinese medicine because of its long history of clinical recordings. Rational use of Chinese medicine combined with chemotherapy drugs can effectively reverse MDR while causing little side effects. Some ingredients extracted from Chinese herbs such as ligustrazine [2] and Tetrandrine (Tet) [3] have been proved to reveal reversal MDR activity. These findings bring new hope to the treatment of MDR in clinic.

Isosteroidal alkaloids are the main bioactive ingredients in Fritillaria species, and they have been proved with anti-tumor activity. Our group has studied the reversal effect of Peiminine which was extracted from Fritillaria thunbergii on leukemia MDR cells in vitro, and found that it has MDR reversal activity [4]. We used Fritillaria thunbergii pulvis in clinical study for reversing acute leukemia MDR, and it demonstrated satisfactory effect [5]. This study aimed at investigating the MDR reversal effect of Total Alkaloid of Fritillaria thunbergii (TAF), a class of isosteroidal alkaloids which was extracted from Fritillaria thunbergii, on SKOV3/DDP cells. This study demonstrated that TAF can reverse the MDR of SKOV3/DDP cells both in vitro and in vivo, and the effect may relate to down-regulate P-gp and MDR1 mRNA.

MATERIALS AND METHODS

Materials

RPMI-1640 was purchased from Invitrogen (Carlsbad, USA). Fetal bovine serums (FBS), Penicillin-Streptomycin were purchased from Hyclone (Beijing, China). Trypsin was purchased from Amresco (Dallas, USA). Dimethyl sulfoxide (DMSO), 4-methyl thiazolyl tetrazolium (MTT), trypanblue, cisplatin, EDTA, Cyclosporine A (Cys A) and Tetrandrine (Tet) were purchased from Sigma-Aldrich (St. Louis, USA). M-MLV reverse transcriptase kit was purchased from TaKaRa (Dalian, China). Rabbit anti mouse P-gp polyclonal antibody was purchased from Abcam (Cambridge, England). Mouse anti mouse GAPDH monoclonal antibody was purchased from KangCheng biological engineering (Shanghai, China). Goat anti mouse IgG (H+L)/HRP was purchased from ZhongShanJinQiao (Beijing, China). TAF was generally provided by department of pharmaceutics of Beijing university of Chinese medicine (Beijing, China).

Cell culture and animal care

The human ovarian carcinoma cell lines, SKOV3 and SKOV3/DDP, were purchased from the Chinese Academy of Medical Sciences Tumor Cells Bank. The cell lines were cultured in RPMI-1640 with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in a flask in a humidified incubator with 5% CO2 at 37°C. The cells were digested with 0.25% trypsin and 0.01% EDTA and subcultured every 2-3 days.

Balb/c nude female mice (14 - 16g) were provided by National Institute for the Control of Pharmaceutical and Biological Products. The certificate number is SCXKJing2009-0017. All aspects of the animal experiment and husbandry were carried out in compliance with national regulations and were approved by the Animal Care and Use Committee of Chinese Academy of Medical Sciences Tumor Hospital.

Cytotoxicity of TAF detected by MTT assay

Parental cells SKOV3 and resistant cells SKOV3/DDP, were seeded into 96-well plate (4,000 cells/well) in RPMI-1640 with 10% FBS, respectively. TAF at different concentrations (200, 100, 50, 25, 12.5 mg L-1) and vehicle were added in the medium 24 h later, respectively. After another 72 h of incubation, 20 μl of MTT solution (5 mg ml-1) was added to each well. The plate was further incubated for 4 h and the medium was discarded. DMSO (150 μl) was added into each well and oscillated for 10 min to dissolve the formazan crystals. The absorbance value (A) was determined at 540 nm. Inhibition rate (IR) of cell viability was calculated as follows:

50% inhibitory concentration (IC50) value and 10% inhibitory concentration (IC10) value of TAF was calculated. IC10 of TAF was used as safe concentration for the following experiments. All experiments were repeated three times.

MDR reversal effect of TAF on SKOV3/DDP

The cells were seeded as mentioned above. DDP (100, 10, 1, 0.1, 0.01mg L-1), DDP (100, 10, 1, 0.1, 0.01 mg L-1) plus TAF(3 mg L-1), DDP (100, 10, 1, 0.1, 0.01 mg L-1) plus Cys A (1 mg L-1), DDP (100, 10, 1, 0.1, 0.01mg L-1) plus Tet (1 mg L-1), or vehicle were added into the medium, respectively. After 72 h of incubation, cell viability was detected by MTT assay. All experiments were repeated three times.

Inhibition rate (IR) and IC50 of DDP were calculated. Drug resistance multiple = IC50 of DDP to SKOV3/DDP cells / IC50 of DDP to SKOV3 cells. Drug resistance reversal multiple = IC50 of DDP to SKOV3/DDP cells in DDP groups / IC50 of DDP to SKOV3/DDP cells in DDP plus TAF groups.

MDR1 mRNA level detected by real time - polymerase chain reaction (RT-PCR)

SKOV3 and SKOV3/DDP cells were adjusted to 2Í104 cells mL-1 and seeded into T25 cm2 culture flasks. After treated with drugs for 72 h, cells were collected. Total RNA was isolated from cultured cells using 1 mL TRIzol reagent. Reverse transcriptase for the synthesis of complementary DNA (cDNA) was supplied by M-MLV Kit. The amount of total RNA was 3 μg and the total volume was 25 μL. The reverse conditions were as follows: 42 for 60 min, 70 for 10 min. The forward primer was 5'-AGGTTCTGGGAAGATCGCTA-3’, and the reverse primer was 5'-ATACATCATTGCCTGGGTGA-3’. PCR amplification was done under the following conditions: the initial denaturation at 94 for 15 min, then 40 cycles at 94 for 15 s, 60 for 34 s, 72 for 15 s, followed by a final extension at 72 for 15 min. The density of the bands was analyzed after electrophoresis. The relative expression was indicated by the density ratio of MDR1 mRNA to the GAPDH PCR products.

P-gp detected by western blot analysis

Cells were treated as above. Cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.5% deoxycholic acid, 0.025% NaN3, 0.1% SDS, 100 μg mL-1 PMSF (α-toluenesulphonyl fluoride), 1μg mL-1 Aprotinin, 0.1% NP-40). 40 μg protein of whole lysate were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis. After the protein were transferred to polyvinylidene fluoride membranes and blocked at room temperature 60 min, the membranes were incubated with rabbit anti mouse P-gp polyclonal antibody at 4 overnight. Then added the goat anti mouse IgG antibody and incubated at room temperature for 60 min. The protein was visualized with manual X-ray film development in the developing agent and fixation agent. The density ratio of P-pg to GAPDH was calculated.

MDR reversal effect of TAF in vivo

SKOV3/DDP cells were collected and diluted to 5 Í 107 cells mL-1 with RPMI-1640 medium and then inoculated in the right front armpits of 60 mice, 0.2 mL per mice. When the tumor size reached a diameter of 0.5 cm (7 days after inoculated), the animals were randomized into six groups: vehicle, DDP (5 mg kg-1), TAF (2 mg kg-1), DDP (5 mg kg-1) plus TAF (0.5 mg kg-1), DDP (5 mg kg-1) plus TAF (1 mg kg-1), DDP (5 mg kg-1) plus TAF (2 mg kg-1). DDP was injected intraperitoneal every two days and TAF was given intragastrically one time a day. All the mice were administrated from the seventh day to the twentieth day and the tumor volumes were measured every four days. All animals were terminated by cervical dislocation and tumors were excised and weighed on the 20 day.

The tumor volume (TV) was calculated from the following formula [6]:


Tumor inhibition rate =
W is the average tumor weight of the vehicle group, and Wt is the average tumor weight of the treatment group [7].

Statistical analysis

Statistical analysis was performed using SPSS version 17.0, and statistical differences were analyzed by one-way analysis of variance (ANOVA) and the Tukey’s test for post hoc comparison between groups. Results are presented as mean ± standard error of the mean (SEM). P<0.01 was considered statistically significant.

RESULTS

Cytoxicity of TAF on SKOV3 and SKOV3/DDP cells

After incubated with TAF for 72h, IC50 of TAF to SKOV3 and SKOV3/DDP were 384.93±85.36 mg L-1 and 156.33±13.03 mg L-1 respectively, and IC10 of TAF to SKOV3 and SKOV3/DDP were 3.00±0.04 mg L-1 and 22.97±0.81 mg L-1. TAF at the concentration of 3.00 mg L-1 and lower did not show obvious cytoxicity on both SKOV3 and SKOV3/DDP (P>0.05) compared to vehicle group. Therefore, the concentration 3.00 mg L-1 of TAF was used for the following reversal experiments.

Reversal effect of TAF on SKOV3/DDP cells

The IC50 of DDP for SKOV3 and SKOV3/DDP cells were 0.81±0.05 mg L-1 and 12.20±0.59 mg L-1, respectively. The multiple of drug resistance was 15.10±0.39. After the cell lines were treated with DDP plus TAF (3.00 mg L-1), IC50 of DDP to SKOV3 and SKOV3/DDP were 0.75±0.13 mg L-1 and 2.00±0.25 mg L-1 respectively, and the multiple of drug resistance was decreased to 2.82±0.25 mg L-1. TAF at the concentration of 3.00 mg L-1 did not significantly enhance the inhibition effect of DDP on SKOV3 cells (P>0.05). However, it could significantly improve the effect of DDP on SKOV3/DDP cells (P<0.01) (Table 1).

The drug resistance reversal multiple of TAF, CysA and Tet was 6.29±0.52, 4.87±0.93, 4.81±1.72, respectively (Table 1). The results indicated that TAF can partly reverse the resistance of SKOV3/ DDP cells to DDP, and its effect is better than CysA and Tet.

Relative MDR1 mRNA level in SKOV3/DDP cells

Compared to SKOV3/DDP cells treated with vehicle, the relative MDR1 mRNA levels of SKOV3/DDP cells treated with TAF (3.00 mg L-1), DDP (12.00 mg L-1) plus TAF (3.00 mg L-1), DDP (12.00 mg L-1) were 0.14±0.00, 0.23±0.02, 0.66±0.06 respectively. The relative level of MDR1 mRNA in SKOV3 cells was 0.04±0.00. The results suggest SKOV3/DDP cells expressed higher level of MDR1 mRNA than SKOV3 cells (P<0.01). After the treatment of TAF or DDP, the relative level of MDR1 mRNA of SKOV3/DDP cells was significantly down-regulated (P<0.01). TAF (3.00 mg L-1), when used together with DDP (12.00 mg L-1), could further decrease the MDR1 mRNA level significantly (Figure 1).

Relative P-gp level in SKOV3/DDP cells

The P-gp level in SKOV3 cells and SKOV3/DDP cells were 0.23±0.01 and 3.28±0.09 respectively. After treated with TAF (3.00 mg L-1), DDP (12.00 mg L-1) plus TAF (3.00 mg L-1), DDP (12.00 mg L-1), the P-gp level in SKOV3/DDP cells changed to 0.97±0.01, 1.48±0.03, and 1.67±0.03 respectively. Compared with SKOV3 cells, the relative level of P-gp of SKOV3/DDP cells was significantly high (P<0.01). After the treatment of TAF or DDP, the relative level of P-gp of SKOV3/DDP cells was down-regulated (P<0.01). However, the relative level of P-gp in DDP (12.00 mg L-1) plus TAF (3.00 mg L-1) group did not show significant difference with DDP (12.00 mg L-1) group (P<0.01) (Figure 2).

MDR reversal activity of TAF in vivo

The results show that tumor growth in DDP (5.00 mg kg-1), DDP (5.00 mg kg-1) plus TAF (0.50 mg kg-1), DDP (5.00 mg kg-1) plus TAF (1.00 mg kg-1), DDP (5.00 mg kg-1) plus TAF (2.00 mg kg-1) groups were significantly inhibited compared to that in vehicle group (P<0.01). DDP (5.00 mg kg-1) plus TAF (2.00 mg kg-1) revealed a remarkable inhibition effect on the growth of SKOV3/DDP tumor compared with DDP or TAF alone. The combination of TAF and DDP resulted in a decrease in the volume and weight of SKOV3/DDP tumor of mice with a dose-dependent tendency (Figure 3, Figure 4). The tumor inhibition rate of DDP was remarkably increased from 53.06 % to 73.36 % after DDP was used together with TAF (2.00 mg kg-1) (Figure 5).

 

DISCUSSION

The present study showed that TAF at the concentration of 3.00 mg L-1 did not show direct cytoxicity to SKOV3 and SKOV3/DDP cells. TAF (3.00 mg L-1) combined with DDP did not affect the cell viability of SKOV3 cells. TAF (3.00 mg L-1) can significantly decrease IC50 of DDP to SKOV3/DDP cells and thus reduce the drug resistance multiple. Drug resistance reversal multiple of TAF to SKOV3/DDP is higher than Cys A and Tet. In vivo, administration of TAF (2.00 mg kg-1) can improve the inhibiting effect of DDP to SKOV3/DDP tumor. The results suggest that TAF may be a safe MDR reversal agent.

The over-expression of P-gp takes a classical pathway of MDR. P-gp is a membrane-associated protein, which is coded by MDR1 gene, belonging to the super family of ATP – binding cassette transporters. The normal function of the protein is to secrete steroids and to metabolize toxicant [8-9]. When tumor cells are exposed to a certain antineoplastic drug for a long time, P-gp over-expresses and the cells would show resistance to the drug. Our results from RT-PCR and western blot indicate that the high level of P-gp in SKOV3/DDP cells, which is also supported by over expression of MDR1 mRNA. TAF itself can down-regulate the level of MDR1 mRNA and P-gp in SKOV3/DDP cells. TAF combined with DDP could further decrease the level of MDR1 mRNA in SKOV3/DDP cells compared to DDP alone. This combination also increased inhibition effect of DDP on the growth of SKOV3/DDP tumor in mice in vivo. The results indicated that the MDR reversal effect of TAF may be related to down-regulation of the level of MDR1 mRNA. Interestingly, the level of P-gp of SKOV3/DDP cells in TAF (3.00 mg L-1) plus DDP (12.00 mg L-1) group did not show significant difference with that in DDP (12.00 mg L-1) group, which suggest that the reversal mechanism of TAF may involve other pathways when TAF is used together with DDP.

In conclusion, the present study suggests that TAF may be a safe MDR reversal agent and the reversal activity of TAF may associate with decreasing the level of MDR1 mRNA. Nevertheless, further studies are needed to explore the effect of TAF on other signal pathways which involve in the reversing process.

TABLE AND FINGURE LEGENDS

Table 1  IC50 values calculated for DDP, when combined with reversal agents (TAF, Cys A or Tet). SKOV3 cells and SKOV3/DDP cells were treated respectively in 96-well plate with different concentrations of DDP (100, 10, 1, 0.1, 0.01mg L-1), and co-incubated with the IC10 of TAF (3 mg L-1), Cys A (1 mg L-1) or Tet (1 mg L-1). After a 72 h incubation period, cell viability was determined by MTT assay. The corresponding IC50 values were calculated. Drug resistance multiple = IC50 of DDP to SKOV3/DDP cells / IC50 of DDP to SKOV3 cells. Drug resistance reversal multiple = IC50 of DDP to SKOV3/DDP cells in DDP groups / IC50 of DDP to SKOV3/DDP cells in DDP plus reversal agent groups. Data are presented as mean ± SEM of three independent experiments. *P<0.01 vs. DDP groups.

Figure 1  Relative level of MDR1 mRNA of SKOV3/DDP cells and SKOV3 cells. SKOV3/DDP cells and SKOV3 cells were seeded into T25 cm2 culture flasks. After treated with drugs for 72 h, cells were collected and 3 μg total RNA was isolated. RNA was reversed and PCR amplification was done. The density of the bands was analyzed after electrophoresis. Analyze the RT-PCR result with the SDS 7500 software. The relative level was indicated by the density ratio of MDR1 mRNA to the GAPDH PCR products. Data are presented as mean ± SEM of three independent experiments. Significant differences between means, as specified by capped lines, are indicated by *P<0.01. NS means not significant.

 

Figure 2  Relative level of P-gp of SKOV3/DDP cells and SKOV3 cells. Typical western blot image (A). SKOV3/DDP cells and SKOV3 cells were seeded into T25 cm2 culture flasks. After treated with drugs for 72 h, cells were lysed. 40 μg protein of whole lysate were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis. Proteins was transferred to polyvinylidene fluoride membranes and blocked at room temperature 60 min. The membranes were incubated with rabbit anti mouse P-gp polyclonal antibody at 4 ℃ overnight. Then added the goat anti mouse IgG antibody and incubated at room temperature for 60 min. The proteins were visualized with manual X-ray film development in the developing agent and fixation agent. The density ratio of P-pg to GAPDH was calculated. Data are presented as mean ± SEM of three independent experiments. Significant differences between means, as specified by capped lines, are indicated by *P<0.01. NS means not significant.

Figure 3  SKOV3/DDP tumor volume in Balb/c nude mice. SKOV3/DDP cells (5 × 107 cells mL-1) were inoculated in the right front armpits of the six groups (ten mice per group). 7 days after inoculated, all groups, except the vehicle group and TAF group, were injected intraperitoneally 5mg kg-1 every other day. TAF (0.5mg kg-1, 1mg kg-1, 2mg kg-1) was given intragastrically one time a day. The tumor volume was calculated every four days. Columns represent mean ± SEM of each group (n = 10).

Figure 4  SKOV3/DDP tumor weight in Balb/c nude mice. SKOV3/DDP cells (5 × 107 cells mL-1) were inoculated in the right front armpits of the six groups (ten mice per group). 7 days after inoculated, all groups, except the vehicle group and TAF group, were injected intraperitoneally 5mg kg-1 every other day. TAF (0.5mg kg-1, 1mg kg-1, 2mg kg-1) was given intragastrically one time a day. All animals were terminated by cervical dislocation and tumors were excised and weighed on the 20 day. Columns represent tumor weight mean ± SEM of each group (n = 10). Significant differences between means, as specified by capped lines, are indicated by *P<0.01.

Figure 5  SKOV3/DDP tumor weight in Balb/c nude mice. SKOV3/DDP cells (5 × 107 cells mL-1) were inoculated in the right front armpits of the six groups (ten mice per group). 7 days after inoculated, all groups, except the vehicle group and TAF group, were injected intraperitoneally 5mg kg-1 every other day. TAF (0.5mg kg-1, 1mg kg-1, 2mg kg-1) was given intragastrically one time a day. All animals were terminated by cervical dislocation and tumors were excised and weighed on the 20 day. Tumor inhibition rate (%) was calculated from (W-Wt)/W×100%, W is the average tumor weight of the vehicle group, and Wt is the average tumor weight of the treatment group.


Table 1

Group

IC50 of DDP (mg L-1)

Drug resistance multiple

Drug resistance reversal multiple

SKOV3 cells

SKOV3/DDP cells

DDP (100, 10, 1, 0.1, 0.01mg L-1)

0.81±0.05

12.20±0.59

15.10±0.39

DDP (100, 10, 1, 0.1, 0.01mg L-1) plus TAF (3 mg L-1)

0.75±0.13

2.00±0.25*

2.82±0.25

6.29±0.52

DDP (100, 10, 1, 0.1, 0.01mg L-1) plus Cys A (1 mg L-1)

0.63±0.20

2.48±0.34*

3.92±0.11

4.87±0.93

DDP (100, 10, 1, 0.1, 0.01mg L-1) plus Tet (1 mg L-1)

0.89±0.55

2.52±0.99*

2.79±0.18

4.81±1.72

Figure 1

*

NS

*

*

*



Figure 2

A

SKOV3/DDP cells

SKOV3 cells


P-gp

          

GAPDH

-     +     +     -     -

            

-     -     +     -     -

                TAF

                 DDP

B


*

NS

*

*



Figure 3

Day from start of treatment


Figure 4

*

*

*

*

*

*


Figure 5


REFERENCES

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[2]     Mei Y, Shi YJ, Zuo GQ, Gong JP, Liu CA, Li XH, Ren MJ. Study on ligustrazine in reversing multidrug resistance of HepG2/ADM cell in vitro. Zhongguo Zhong Yao Za Zhi 2004; 29 (10): 970-3.

[3]     Fu LW, Deng ZA, Pan QC, Fan W. Screening and discovery of novel MDR modifiers from naturally occurring bisbenzylisoquinoline alkaloids. Anticancer Res 2001; 21 (4A): 2273-80.

[4]     Hu KW, Zheng HX, Qi J, Hou L, Zuo MH, Chen XY, Sun YL, Xu YF, Shao XF, Yang CZ. The study of MDR reversal effect of Peiminine for leukemia cell. In Chinese. Chin J Hematol 1999; 20 (12): 650-1.

[5]     Li W, Hu KW, Su W, Sun YL, Chen XY, Liang B. Clinical Trial of Fritillaria Thunbergii Bulb Powder for Reversing Multidrug Resistance in the Patients with Acute Leukemia. In Chinese. Journal of Beijing University of TCM 2004; 27 (1): 63-5.

[6]     Zalatnai A, Molnár J. Effect of SILA-409, a new organosilicon multidrug resistance modifier, on human pancreatic cancer xenografts. In vivo 2006; 20 (1): 137-40.

[7]     Liu Z, Ren Y, Pan L, Xu HM. In vivo Anti-Tumor Activity of Polypeptide HM-3 Modified by Different Polyethylene Glycols (PEG). Int. J. Mol. Sci. 2011; 12 (4): 2650-63.

[8]     Randolph GJ, Beaulieu S, Pope M, Sugawara I, Hoffman L, Steinman RM, Muller WA. A physiologic function for P-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels. Proc Natl Acad Sci U S A 1998; 95 (12): 6924-9.

[9]     Schinkel, AH, Mayer, U, Wagenaar, E, Mol, CA van Deemter, L, Smit, JJ, van der Valk MA, Voordouw AC, Spits H, van Tellingen O, Zijlmans JM, Fibbe WE, Borst P. Normal viability and altered pharmacokineticsin mice lacking mdr1-type (drug-transporting) P-glycoproteins. Proc Natl Acad Sci U S A. 1997; 94 (8): 4028-33.

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