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Year : 2015  |  Volume : 11  |  Issue : 1  |  Page : 18-23

The overexpression of MRP4 is related to multidrug resistance in osteosarcoma cells

1 Department of Oncology, Shanghai Fengxian Central Hospital, Shanghai 200233, China
2 Department of Oncology, 6th People's Hospital Affiliated to Shanghai Jiaotong University, Shanghai 200233, China

Date of Web Publication16-Apr-2015

Correspondence Address:
Feng Lin
Department of Oncology, 6th People's hospital affiliated to Shanghai Jiaotong university, 600, Yishan Road, Shanghai
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Source of Support: This work was supported by grants from Shanghai Health Bureau (Grant No 2011171) and National Natural Science Foundation of China (Grant No 81001192), Conflict of Interest: None

DOI: 10.4103/0973-1482.143334

Clinical trial registration NA

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 > Abstract 

Doxorubicin (Adriamycin, ADM) is an antimitotic drug used in the treatment of a wide range of malignant tumors, including acute leukemia, lymphoma, osteosarcoma, breast cancer, and lung cancer. Multidrug resistance-associated proteins (MRPs) are members of a superfamily of ATP-binding cassette (ABC) transporters, which can transport various molecules across extra- and intra-cellular membranes. The aim of this study was to investigate whether there was a correlation between MRP4 and primary ADM resistance in osteosarcoma cells. In this paper, we chose the human osteosarcoma cell line MG63, ADM resistant cell line MG63/DOX, and the patient's primary cell GSF-0686. We checked the ADM sensitivity and cytotoxicity of all the three cells by cell proliferation assay. The intracellular drug concentrations were measured by using LC-MS/MS. We also examined MRP4 gene expression by RT-PCR and Western Blot. We found that the intracellular ADM concentration of the parent osteosarcoma cell line MG63 was higher than the ADM resistant osteosarcoma MG63/DOX cell line or the GSF-0686 cell after ADM treatment (P < 0.05). In addition, MRP4 mRNA and protein levels in ADM resistant osteosarcoma cells were higher than in MG63 cell (P < 0.05). Taking together, this work suggests that overexpression of MRP4 may confer ADM resistance in osteosarcoma cells.

Keywords: Osteosarcoma, MRP4, Doxorubicin

How to cite this article:
He Z, Hu B, Tang L, Zheng S, Sun Y, Sheng Z, Yao Y, Lin F. The overexpression of MRP4 is related to multidrug resistance in osteosarcoma cells. J Can Res Ther 2015;11:18-23

How to cite this URL:
He Z, Hu B, Tang L, Zheng S, Sun Y, Sheng Z, Yao Y, Lin F. The overexpression of MRP4 is related to multidrug resistance in osteosarcoma cells. J Can Res Ther [serial online] 2015 [cited 2020 Jan 23];11:18-23. Available from: http://www.cancerjournal.net/text.asp?2015/11/1/18/143334

 > Introduction Top

The existence of osteosarcoma can be traced back to over 5000 years ago to mummified corpses from ancient Egypt. [1] Osteosarcoma is the most common primary malignant bone tumor occurring in children and adolescents between the ages of 10 and 20. Osteosarcomas account for about 60% of bone cancers in patients younger than 20 years old. Osteosarcoma also exist in elderly patients.

Great advances have been made in the treatment of osteosarcoma during the past several decades, including multi-agent chemotherapy that is given before and after surgery. However, there are two major problems that affect the treatment of osteosarcoma: The distant metastasis and multidrug resistance (MDR). [2] Multidrug resistance in cancer is the principle mechanism by which cancer cells develop resistance not only to single cytotoxic drugs, but to a whole range of drugs with different structures and cellular targets.

One of the mechanisms of drug resistance is the overexpression of MDR proteins. MDR proteins can work as a molecular pump, decreasing the intracellular drug concentration. P-glycoprotein (P-gp) is the first reported MDR protein (MDR1), which is encoded by the ATP-binding cassette sub-family B member 1 (ABCB1) gene. [3],[ 4] It has previously been shown that MDR1 suppress caspase-3 and caspase-8 activities, inhibiting cell apoptosis induced by anti-tumor drugs. In addition to P-gp, other multidrug resistance-associated proteins (MRPs) [5] and breast cancer resistance protein (BCRP) have been discovered, and all of these proteins belong to ATP-binding cassette (ABC) transporter proteins.

There are nine MRPs identified in humans so far: ABCC1-6 (ATP-binding cassette sub-family C) and ABCC10-12, respectively (www.gene.ucl.ac.uk/nomenclalure/genefamily/abc.html). In polarized cells, MRPs localize to either the basolateral membrane (which may increase drug absorption) [6],[7] or apical membrane (which enhance drug elimination). In non-polarized cells, such as transporters, MRPs pump intracellular drugs out, resulting in a decrease in drug concentration. [8],[9],[10] MRP1 specifically localizes to the basolateral membrane of the choroid plexus and intestine, whereas MRP2 was found on the apical membranes in the intestine, kidney, liver, [11],[ 12] and the carboxy-proximal region containing a specific protein interaction domain. However, MRP4, which distinguishes itself from the other C subfamily members, is localized to both the apical (kidney, the brain capillaries endothelial cells) or basolateral membrane (prostate, choroid plexus). [13],[14],[15],[16] Most of MRPs play roles in cellular detoxification as pumps for their substrate, i.e. organic anions. Transport of non-anionic compounds such as vincristine and doxorubicin has also been reported. However, this requires glutathione co-transport. [17],[ 18]

MRP4/ABCC4 is expressed in many tissues and cells, including ovary, testis, kidney, lung, intestine, liver, brain, pancreas, prostate (highly), and various of blood cells (lowly). [13],[ 14],[ 19] MRP4 was first described in 1999 in a human T-lymphoid cell line, in which overexpression of MRP4 was directly linked to impaired antiviral efficacy and enhanced efflux of antiviral nucleoside based drugs [20] . Markos proved that the MRP4 deficient mice could enhance accumulation of the anticancer agent topotecan in specific tissues [20] , while MRP4 was overexpressed in the topotecan resistant cells. Further studies showed that MRP4-overexpressing cells could become resistant to anticancer agents. MRP4 is known to transport a diverse array of compounds, [17],[ 21],[ 22] including pravastatin, tetracycline, beta-lactamsa, fluoroquinolonesa, adefovir, ganciclovir, zidovudine (AZT), cladribine, methotrexate, irinotecan, topotecan, gimatecan, mefloquine, and cetirizine. MRP4 also has the outstanding ability to transport nucleotides and nucleotide analogs.

Doxorubicin is a drug used in cancer chemotherapy. It is an anthracycline antibiotic, and is commonly used in the treatment of a wide spectrum of cancers, including hematological malignancies, many types of carcinoma, and soft tissue sarcomas. [23] It interacts with DNA by intercalation and inhibition of macromolecular biosynthesis. This inhibits the progression of the enzyme topoisomerase II, which relaxes supercoils in DNA leading to transcription. [6],[ 16]

A series of cancer cell lines have been used to study doxorubicin resistance. However, there is no data regarding osteosarcoma cells. This paper investigates the pharmacological role of MRP4 in osteosarcoma cells and provides insights into putative pathophysiological function in signaling processes as a potential new target for drug development.

In our preliminary clinical trails, we found a osteosarcoma patient, who was diagnosed primary ADM drug resistant. In addition, our hospital had the human osteosarcoma cell line MG63 and ADM resistant cell line MG63/DOX. So we used the patient›s primary cell GSF-0686 to identify the relation between the MRP gene expression and the osteosarcoma ADM resistance.

 > Materials and methods Top


Doxorubicin (ADM) was obtained from Pfizer Pharma (New York, USA). It was dissolved with normal saline to make a 1000x peak plasma concentration (PPC) stock solution at 1μg/ml. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan).

Cell lines and cell culture

The Human osteosarcoma cell line MG63 and MG63/DOX were obtained from Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai of China. The human MDR osteosarcoma cell line MG63/DOX, in which P-glycoprotein (P-gp) was overexpressed, was selected in a step-wise manner by exposing drug-sensitive MG63 cells to increasing doses of ADM. The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Hyclone, UT, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS; Si Ji Qing, Hangzhou, China), 100 units/mL penicillin and 100 mg/mL streptomycin (Gibco, NY, USA) in a humidified atmosphere at 37°C with 5% CO 2 .

The primary cell GSF-0686 was obtained from the osteosarcoma tissue of a patient who had multi-drug resistance. The tissue was cut into 2-4 millimeter pieces and incubated with 0.5% trypsin at 37°C for 20 minutes. The cells were gently dispersed by pipetting, and then filtered through fine mesh. After centrifugation, the cells were resuspended and seeded into tissue culture dishes in DMEM containing 10% fetal bovine serum (FBS).

Cell proliferation assay

Cells were seeded into 24-well plates, where each treatment group was plated in replicates of three. The cell number was checked from day one through day seven. The doubling time was calculated using the Patterson formula: Td = Tln2/ln (N T /N O ), where Td is defined as doubling time (h); T is defined as the time taken for the numbers of cells growing from N O to N T , and N is cell number.

Drug sensitivity and cytotoxicity assays

The effects of ADM to the proliferation of MG63, MG63/DOX, and GSF-0686 cells were measured using the Cell Counting Kit-8 (CCK-8). Briefly, the cells were seeded into a 96-well plate at 5 × 10 4 cells/ml (100 μl). After a 24 hour incubation with fresh medium, 10 μl of the various chemical dilutions at the indicated concentrations of ADM was added to the plates, and the cells were incubated for an additional 24, 48, and 72 hours. At the end of drug treatment, 10 μl of CCK-8 was added to each well, and the cells were incubated for 4 hours at 37 0 C. Absorbance (A) was analyzed using Multiskan MK3 microplate reader (Thermo Labsystems, Beverly, MA, USA) at 450 nm. The half maximal inhibitory concentration (IC50) and 30% inhibitory concentration (IC30) were calculated. The resistance factor of multidrug-resistant cell line MG63/DOX for a particular drug is defined as the ratio of IC50 of MG63/DOX cell to IC50 of MG63 cell.

Intracellular drug concentration measurement by Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

ADM treated MG63, MG63/DOX, and GSF-0686 cells were collected at different time points. The cell lysates and the cell culture media were subjected to LC-MS/MS analysis. Non-treated cells were used as control. The conditions of liquid chromatography were as follows: Zorbax XDB-C18 column (2.1 mm × 150 mm i.d., 3.5 μm) (Agilent Co., MA, USA), the mobile phase A (0.1% formic acid, contained 5 mM Ammonium formate) and the mobile phase B (acetonitrile, flow rate was 1.0 ml/min, 35). The positive electrospray ionization (ESI) was performed under ion multiple reaction monitored (MRM) mode. The precursor-to-product ion transitions of m/z 544.2 → 397.2 and m/z 528.2 → 321.3 were used to measure and quantify ADM. 1 mg/ml ADM was used as standard, while 100 μg/ml daunorubicin (DNR) as internal control.

RNA extraction and real-time PCR

Total RNA of cells was isolated using the RNeasy Mini Kit (Qiagen, Germany), and 2 μg of RNA was subjected to reverse transcription-PCR with M-MLV Reverse Transcriptase (Promega, USA). Real time-PCR amplification mixtures (20 μl) contained 150 ng template cDNA, 2x SYBR Green I Master Mix buffer (10 μl) (Applied Biosystems, USA), and 200 nM forward and reverse primers. Reactions were run on a Sequence Detection System (ABI Prism 7900HT, Applied Biosystems, USA). The ABCC4 forward primer was: 5'-GTT CTT CTG GTG GCT CAA TCC-3', reverse primer was: 5'- GGC TTC TGT GCG TCA TTC TC-3'; the β-actin forward primer was: 5'- GTG GAC ATC CGC AAA GAC-3', reverse primer was: 5'-AAA GGG TGT AAC GCA ACT A-3'. The specificity and quantity were validated via melting curve analysis. Relative expression levels of ABCC4 gene were normalized to β-actin.

Statistical analysis

The statistical analyses were assessed using the unpaired one-tailed Student's t-tests. All comparisons were considered significant at P <0.05.

Western blotting

Total cell proteins were extracted using a Total Protein Extraction Kit (Applygen Technologies Inc., China) according to the manufacturer's instructions. Protein was quantified using the BCA Assay Kit (Beyotime, China). Protein samples were separated by electrophoresis on 10% SDS/PAGE gels and transferred to polyvinyl difluoride (PVDF) membranes (Thermo, USA). Antibodies were used as indicated by the manufacturer: anti-MRP4 (Abcam, USA), anti-β-actin (Beijing CoWin Biotech Co., Ltd., China).

 > Results Top

Comparing the growth rate and doubling time between human osteosarcoma cell lines

To investigate the cell proliferation of several human osteosarcoma cell lines, viable cells were counted at the indicated days. Compared to the non-resistant MG63 cell line, the drug-resistant cell line MG63/DOX has a reduced growth rate and a smaller slope, while the primary GSF-0686 cell line has a minimum growth rate and the smallest slope [Figure 1]. The doubling times of MG63/DOX (46.9 hours) and GSF-0686 (61.3 hours) are significantly different from that of MG63 (38.0 hours) (P < 0.05).
Figure 1: Growth curve of MG63, MG63/DOX, and GSF-0686 cells. Osteosarcoma cells were seeded into 24-well plates, and the total numbers of cells were counted at the indicated days. Data are representative of the mean value of three independent experiments. *means P < 0.05 relative to MG63. (Student's t test)

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Detection of sensitivity of MG63/DOX and GSF0686 cells to ADM

The CCK-8 assay shows that the IC 50 of ADM in MG63, MG63/DOX, and GSF-0686 is 0.004 mM, 0.025 mM, and 1.5 mM, respectively, when treated with ADM for 72 hours [Figure 2]. The IC 30 of ADM is less than 10 times of its peak plasma drug concentration (PPC) in MG63 cell, indicating that MG63 is drug sensitive. Moreover, the IC 30 of ADM in MG63/DOX and GSF-0686 cells are more than 10 times of PPC, suggesting that they are drug resistant.
Figure 2: Cytotoxicity of ADM to osteosarcoma cells. Osteosarcoma cells MG63, MG63/DOX, and GSF-0686 were treated by serial concentration of ADM for 72 hours. Cell viability was measured by CCK8 assay. A, MG63. B, MG63/DOX. C, GSF-0686. Each point is an average value (n = 3)

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ADM has inhibitory effects in the osteosarcoma cell MG63, the multidrug-resistant cell line MG63/DOX and the primary drug-resistant cell line GSF-0686, but has lesser effects in MG63/DOX and GSF-0686 cells.

Determination of intra- and extra-cellular drug concentration by LC-MS/MS

LC-MS/MS revealed that when incubated with 50 ng/ml ADM for 6 hours, the drug concentration inside MG63 cells was significantly higher than in MG63/DOX cells (1.7 ± 1.1 times) and GSF-0686 cells (8 ± 0.9 times) (P < 0.05) [Figure 3]a. Meanwhile, the extracellular concentration of ADM at 8 hours was 1.8 ± 0.7 times higher in MG63/DOX cells, and 2.1 ± 0.5 times higher in GSF-0686 cells when compared with that in MG63 cells [Figure 3]b.
Figure 3: Intra- (a) and extra- (b) cellular ADM concentration in osteosarcoma cells. MG63, MG63/DOX, and GSF-0686 cells were treated with 50 ng/ml ADM. The intra- and extra- cellular ADM concentration was measured by LS-MS/MS at indicated time points

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When these three cell lines were treated with the same concentration of ADM, the intracellular concentration of ADM in the drug resistant MG63/DOX and GSF-0686 cells was lower than in MG63 cells, while the extracellular concentration in the drug-resistant cells was higher than in MG63 cells. This suggests that ADM is pumped out of the drug-resistant cells and may explain why they fail in osteosarcoma chemotherapy.

The expression of ABCC4 in MG63, MG63/DOX and GSF0686 cells

We performed Q-PCR in MG63, MG63/DOX, and GSF-0686 cells to detect the expression level of ABCC4 mRNA. The results were normalized to the expression of β-actin. Compared to the expression of ABCC4 in MG63 cells (100%), the expression of ABCC4 in GSF-0686 and MG63/DOX cells were 129% and 119%, respectively [Figure 4]a. In other words, the expression levels of ABCC4 in those three cell lines were: GSF-0686 > MG63/DOX > MG63.
Figure 4: The expressional levels of the MRP4/ABCC4 in osteosarcoma cells. The MRP4 mRNA and protein levels were detected in osteosarcoma cells. A, ABCC4 mRNA levels in GSF-0686, MG63-DOX, and MG63 cells. B, MRP4 protein levels in MG63DOX, GSF-0686, and MG63. β-actin was used as a loading control.* means P < 0.05 relative to MG63. (Student's t test)

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Western blotting showed that the expression of the ABCC4 protein in these cell lines was consistent with the expression of ABCC4 mRNA [Figure 4].

mRNA expression in ADM-treated MG63, MG63-DOX cells

We found that the efficiency of ADM uptake was the highest when cells were incubated for 10 minutes. In order to detect the expression level of ABCC4 mRNA in both osteosarcoma and drug-resistant cells, we treated MG63, MG63/DOX, and GSF-0686 cells with indicated concentration of ADM for 10 min, respectively, harvested all the cells and performed Q-PCR. The results were compared to the expression of ABCC4 of untreated MG63 cells, and normalized to the expression levels of β-actin. The expression of ABCC4 was the lowest when treated with 10μM ADM, which indicated that this dose was least likely to induce drug resistance [Figure 5]a.
Figure 5: The expressional levels of the ABCC4 after treatment with ADM in osteosarcoma cells. A: ABCC4 mRNA expressional levels in MG63 and MG63-DOX cell lines at 10 mins after treatment with a series of concentrations of ADM; B: ABCC4 mRNA expressional levels in MG63 and MG63-DOX cell lines at different times after treating with 10 μM ADM.* means P < 0.05 relative to MG63. (Student's t test)

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We treated MG63, MG63/DOX, and GSF-0686 cells with 10 μM ADM, and harvested the cells at the indicated time points, to detect the expression of ABCC4. When treated with the same concentration of drug, the expression of ABCC4 in both cell lines underwent a down-regulation first, and then peaked at 2-hour treatment [Figure 5]b. However, when compared to control group, GSF-0686 cells had a much higher peak than the control (121:96), whereas MG63 cells showed no significant difference.

Whether exposed to the concentration gradient or to the same concentration, the expression of ABCC4 in MG63, MG63/DOX, and GSF-0686 cells increased first and then decreased. In addition, the expression of ABCC4 in GSF-0686 cells was higher than in MG63 cells at the most of time points. At the lower concentrations (2 μM and 5 μM), there was no significant difference of ABCC4 expression between these two cell lines. However, at higher concentrations (10 μM, 15 μM, and 30 μM), the expression of ABCC4 in GSF-0686 cells was higher than in MG63 cells.

 > Discussion Top

HPLC was widely used in related research before. But in this study, we used LC-MS/MS to check the drug concentration because of the simpler sample extraction, better repeatability and higher sensitivity than HPLC

The occurrence of resistance against cytotoxic agents is a major problem in cancer chemotherapy. Various pathological types of osteosarcomas have different reactions to chemotherapy drug. Different chemosensitivities are even seen in patients with the same pathological tumors.

In our study, we investigated ADM resistance in the osteosarcoma cell line MG63, drug-resistant cell line MG63/DOX, and primary cell GSF-0686.

The overexpression of P-glycoprotein (Pgp) in tumor tissues is the main mechanism of multidrug resistance (MDR), but it is not the only mechanism. Many studies showed that MDR-associated proteins (MRPs), which are members of the ABC transporter superfamily, play important roles in cellular multidrug resistance. Like Pgp, MRP seems work as a drug pump that transport drugs from the cell. It is mainly present in the plasma membrane and is able to decrease cellular drug levels.

Here, we found MG63 cells were much more sensitive to ADM than the resistant cells MG63/DOX and GSF-0686. After the same treatment of ADM, the drug resistant cells MG63/DOX and GSF-0686 have a lower intracellular drug concentration than the non-resistant MG63 cells. In other words, the extracellular drug concentration of drug resistant cells is higher than in MG63 cells. This suggests that drug-resistant proteins are involved in the transport of ADM from intracellular to extracellular, which leads to the resistance to ADM in osteosarcoma cells and may explain why they fail in osteosarcoma chemotherapy.

MRP4/ABCC4 is one of the proteins that are capable of transporting a wide spectrum of organic compounds out of the cell. MRP4 has been reported to be involved in the resistance against mercaptopurines and 2-amino-6-purinethiol. [3],[4],[5] In vitro experiments showed that the anti-cancer drug Celebrex can induce the overexpression of the MRP4 protein in the colon cancer cell line colo-205 [3] , resulting in drug resistance. MRP4 also affected the sensitivity of human pancreatic cancer cells to 5-fluorouracil (5-FU) and was expressed specifically in 5-FU resistant cells.

We found that MRP4 was highly expressed in MG63/DOX cells when compared to MG63 cells, indicating that MRP4 plays an important role in drug resistance in osteosarcoma cells. After the same treatment with ADM, MRP4 mRNA levels first decreased and then increased in all the three cells. The change of expression is time and dose-dependent. The decrease in ABCC4 expression by ADM may induce cell apoptosis. The higher expression of ABCC4 in GSF-0686 and MG63/DOX cells than in MG63 cells suggests that drug-resistant cell lines are more resistant to cell growth.

In conclusion, this study provides an analysis of ADM resistance in osteosarcoma cells, and it aids in the understanding of how MRP4 confers resistance to ADM. However, the relationship between MRP4 and ADM still needs further investigation.

 > References Top

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