Ultrasound microbubble-mediated delivery of the siRNAs targeting MDR1 reduces drug resistance of yolk sac carcinoma L2 cells
- Yun He†1, 2,
- Yang Bi†2,
- Yi Hua1, 2,
- Dongyao Liu1, 2,
- Sheng Wen1, 2,
- Qiang Wang1, 2,
- Mingyong Li1, 2,
- Jing Zhu2,
- Tao Lin1, 2,
- Dawei He1, 2,
- Xuliang Li1, 2,
- Zhigang Wang3 and
- Guanghui Wei1, 2Email author
© He et al; licensee BioMed Central Ltd. 2011
Received: 23 August 2011
Accepted: 28 October 2011
Published: 28 October 2011
MDR1 gene encoding P-glycoprotein is an ATP-dependent drug efflux transporter and related to drug resistance of yolk sac carcinoma. Ultrasound microbubble-mediated delivery has been used as a novel and effective gene delivery method. We hypothesize that small interfering RNA (siRNA) targeting MDR1 gene (siMDR1) delivery with microbubble and ultrasound can down-regulate MDR1 expression and improve responsiveness to chemotherapeutic drugs for yolk sac carcinoma in vitro.
Retroviral knockdown vector pSEB-siMDR1s containing specific siRNA sites targeting rat MDR1 coding region were constructed and sequence verified. The resultant pSEB-siMDR1 plasmids DNA were encapsulated with lipid microbubble and the DNA release were triggered by ultrasound when added to culture cells. GFP positive cells were counted by flow cytometry to determine transfection efficiency. Quantitative real-time PCR and western blot were performed to determine the mRNA and protein expression of MDR1. P-glycoprotein function and drug sensitivity were analyzed by Daunorubicin accumulation and MTT assays.
Transfection efficiency of pSEB-siMDR1 DNA was significantly increased by ultrasound microbubble-mediated delivery in rat yolk sac carcinoma L2 (L2-RYC) cells. Ultrasound microbubble-mediated siMDR1s delivery effectively inhibited MDR1 expression at both mRNA and protein levels and decreased P-glycoprotein function. Silencing MDR1 led to decreased cell viability and IC50 of Vincristine and Dactinomycin.
Our results demonstrated that ultrasound microbubble-mediated delivery of MDR1 siRNA was safe and effective in L2-RYC cells. MDR1 silencing led to decreased P-glycoprotein activity and drug resistance of L2-RYC cells, which may be explored as a novel approach of combined gene and chemotherapy for yolk sac carcinoma.
KeywordsYolk sac carcinoma Ultrasound therapy RNA interference Multiple drug resistance gene Transfection
Yolk sac carcinoma are the most common malignant germ cell tumors in children, which are commonly found in the ovary, testes, sacrococcygeal areas and the midline of the body [1–4]. This type of germ tumors is aggressive and highly metastatic which can rapidly spread to adjoining tissues through the lymphatic system [5–7]. Meanwhile, clinical data show that yolk sac carcinoma in children have a high recurrence rate. Most of yolk sac carcinoma are refractory to chemotherapy and require a surgical resection of primary tumors and surrounding tissues including germinative glands. While surgical treatment of yolk sac carcinoma can decrease tumor recurrence to certain extent, removal of gonadal tissues may result in long-term physiological and psychological adverse effects in the affected children. Therefore, there is an urgent need to improve the chemotherapy efficacy of yolk sac carcinoma [8–10].
Tumor drug resistance is one of the most important factors which affects the outcomes of chemotherapy [11–13]. It has been well documented that certain, genes products, such as multiple drug resistance gene (MDR1), multidrug resistance-associated protein, lung resistance protein, glutathione-S-transferase Pi, contribute to drug resistance [14–17]. Our previous studies showed that MDR1 was the most and highest expressed resistance genes in tissues of yolk sac carcinoma in children. MDR1 gene, also known as ABCB1 (ATP-binding cassette, sub-family B, member 1) gene, encodes an ATP-dependent drug transporter named permeability glycoprotein (P-glycoprotein). P-glycoprotein is an energy-dependent efflux pump that exports its substrates out of the cells. Many of chemical drugs are substrates of P-glycoprotein. P-glycoprotein plays an important role in drug kinetics, including absorption, distribution, metabolism, and excretion, which limits the accumulation of drugs inside cells and results in drug resistance [18–20]. Yolk sac carcinoma have high expression of MDR1 gene , so we hypothesize that small interfering RNA (siRNA) mediated silencing of MDR1 expression would improve the sensitivity of yolk sac carcinoma to chemotherapy drugs.
Ultrasound microbubble-mediated delivery is a novel, nonviral, effective and safe method for delivering drugs or genes to target organs or cells [22–26]. Recent studies have shown that ultrasound microbubble-mediated delivery improves the efficacy of gene transfection and reduces the side effects of other bioactive transfection agents, such as liposome, viral vectors . In this study, we constructed and characterized three effective siRNAs targeting MDR1 gene and used ultrasound microbubble-mediated gene delivery method to effectively deliver plasmid DNA into rat yolk sac carcinoma L2 (L2-RYC) cells. Our results demonstrated that the MDR1 siRNAs effectively reduced the multiple-drug resistance of L2-RYC cells. Thus, the reported approach may represent a novel and new method of combined gene silencing and chemotherapy to combat the drug resistance of yolk sac carcinoma.
Cell culture and chemicals
L2-RYC cells were purchased from ATCC (Manassas, VA), and were cultured in complete Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, Utah, USA), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2.
Construction and validation of plasmids containing siRNAs targeting MDR1
The pSEB-HUS vector (Additional file 1) containing H1 and U6 dual-promoter was used to construct the eukaryotic plasmid expressing siRNA targeting MDR1 . Four pairs of oligonucleotides specific for rat MDR1 coding region (Additional file 2) were designed by using Invitrogen Block-iT RNAi Designer software. After annealed in vitro, four double-stranded oligonucleotides cassettes with Sfi I cohesive ends were subcloned into the Sfi I sites of pSEB-HUS vector, resulting in pSEB-siMDR1 plasmids. We transfected four pSEB-siMDR1 plasmids into L2-RYC cells with Lipfectamine 2000 and detected the inhibition efficiency of each siMDR1 by quantitative real-time polymerase chain reaction (qRT-PCR), respectively. After validation, equimolar amounts of pSEB-siMDR1-1, -2 and -3 were pooled and transfected into L2-RYC cells with liposome to detect the inhibition efficiency of MDR1 by qRT-PCR.
Quantitative real-time PCR
As described previously , total RNA was extracted from L2-RYC cells after 2 days transfection using TRIZol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcripted into single-strand cDNA template with random primer and a reverse transcriptase (Takara, Japan). Primers were 18-20 mers, designed by using Primer 5 program to amplify the 3'-end of rat MDR1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (Additional file 2). Quantitative RT-PCR reaction was performed as follows: 3 min at 94°C (one cycle), 20 sec at 94°C, 20 sec at 58°C, 20 sec at 72°C, and reading plate (38 cycles). Raw data of Ct value for MDR1 in each group was normalized with GAPDH and measured as the fold change.
Preparation of the siMDR1-loaded lipid microbubble
To prepare lipid microbubble, we mixed 5 mg of dipalmitoyl phosphatidylcholine (Sigma, USA), 2 mg of distearoyl phosphatidyl ethanolamine (Sigma, USA), 1 mg of diphenyl phosphoryl azide (Sigma, USA), and 50 μl of glycerol into phosphate buffered saline (PBS) to make the 0.5 ml mixture in a tube. The tube was placed at 40°C for 30 min, then filled with perfluoropropane gas (C3F8) and mechanically shaken for 45 sec in a dental amalgamator (YJT Medical Apparatuses and Instruments, Shanghai, China). The pure lipid microbubble was PBS diluted, sterilized by Co60 and stored at -20°C. Then, the home-made lipid microbubble were mixed with poly-L-lysine (Sigma, USA), and incubated at 37°C for 30 min. Subnatant was removed and washed twice by PBS. Plasmids containing balance mixed siMDR1 plasmids were added and incubated at 37°C for 30 min, and washed by PBS twice. This procedure was repeated three times. The siMDR1-loaded lipid microbubble were obtained with an average diameter of 2.82 ± 0.76 μm, an average concentration of 8.74 × 109/ml and the average potential of -4.76 ± 0.82 mV (n = 5). The final concentration of plasmids DNA was 0.5 μg/μl.
Trypan blue staining
Cultured L2-RYC cells in 6-well plates were processed with acoustic intensity of 0.25 W/cm2, 0.5 W/cm2, 0.75 W/cm2 and 1 W/cm2 and irradiation time of 30 sec and 60 sec, respectively. Cells were washed, trypsinized and resuspended with PBS with 106 cells per milliliter. An equal volume of 0.2% trypan blue was added to a cell suspension. Then, cell suspensions were incubated at room temperature for 3 min and loaded into a hemocytometer. With an optical microscope examination, survival cells excluding trypan blue were counted in three separate fields. Survival rate = (number of survival cells/number of total cells) × 100%.
Transfection efficiency detected by flow cytometry
L2-RYC cells were seeded in each well of 24-well culture plates with 5 × 105 cell density and cultured in complete DMEM medium for 24 hrs before transfection. Then cells were treated with pSEB-siMDR1 pooled plasmids alone (group I), plasmids with ultrasound (group II), siMDR1-loaded lipid microbubble (group III), siMDR1-loaded lipid microbubble with ultrasound (group IV) and non-plasmid control (group V), respectively. We also set up a lipofection group (Lipo) for comparison of transfection efficiency. Cells in group II and IV were exposed to ultrasound with the radiation frequency of 1 MHz, pulse wave, sound intensity of 0.5 W/cm2 for 30 sec using an ultrasound treatment meter (Institute of Ultrasound Imaging, Chongqing Medical University). Since pSEB-siMDR1 plasmids express green fluorescent protein (GFP), transfected cells were collected and suspended in 1 ml of PBS/BSA buffer at 24 hrs after transfection for flow cytometry as a measurement of transfection efficiency.
Western blot analysis
Total proteins of L2-RYC cells in each group were extracted by using protein extraction kit (Beyotime, China, at 48 hrs after transfection. Approximately 20 micrograms total proteins per lane were loaded onto a 6% SDS-PAGE gel. After electrophoretic separation, proteins were transferred to an Immobilon-P membrane. The membrane was blocked with 5% fat-free skim milk in Tris buffered saline with tween-20 buffer at room temperature for 1 hr, and was incubated with anti-MDR1 or anti-β-actin primary antibody (Santa Cruz Biotechnology, USA), respective, at 4°C overnight. After being washed, the membrance was incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) (Santa Cruz Biotechnology, USA) at room temperature for 1 hr, followed by extensive wash. The protein of interest was visualized and imaged under the Syngene GBox Image Station by using Luminata Crescendo Western HRP Substrate (Millipore, USA). The expression level of MDR1 proteins was calculated using GBox Image Tools and normalized by β-actin levels.
Daunorubicin accumulation assay
Daunorubicin accumulation assay was conducted to determine P-glycoprotein activity . L2-RYC cells were treated as above mentioned in each groups, as well as a blank control. Cells were washed and changed with FBS-free DMEM. Daunorubicin was administered into culture medium at the final concentration of 7.5 μg/ml and the cells were incubated at 37°C for 30 min. Cells were then washed with FBS-free DMEM medium again, followed by incubation with Verapamil (Pharmacia Co., Italy) at the final concentration of 10 μg/ml to end the efflux function of P-glycoprotein. Subsequently, cells were washed three times with PBS and the Daunorubicin accumulation was examined under a fluorescence microscope and analyzed by flow cytometry. (FACS Calibur FCM, Becton-Dickinson, San Jose, CA)
L2-RYC cells in each treated group were seeded into 96-well culture plates with 5 × 103 cell density. After incubation in complete DMEM medium for 24 hrs, the medium was replaced with FBS-free DMEM containing Vincristine or Dactinomycin at the concentration ranges of 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8 μg/ml (for Vincristine) and 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28 μg/ml (for Dactinomycin), respectively. MTT assay was performed at 12 hrs post treatment to determine cell proliferation. Briefly, 20 μl of MTT reagent was added to each well with FBS-free DMEM medium and incubated at 37°C for 4 hrs. Medium was gently aspirated and replaced by 200 μl of DMSO. The 96-well plates were shaken for 10 min to dissolve the purple crystals and read at 520 nm in Thermo Scientific Varioskan Flash Spectral Scanning Multimode Reader. Viability of L2-RYC cells in each concentration was calculated as ODtreated/ODuntreated × 100%. The half maximal inhibitory concentration (IC50) was accounted to compare the drug sensitivity among each group.
All data were shown as mean ± standard deviation (SD). Statistical analyses were performed using SPSS 15.0 software package (SPSS, Inc, Chicago, IL). Mann-Whitney U test was performed to compare results among experimental groups. P < 0.05 was considered as statistically significant.
Construction and silencing efficiency of pSEB-siMDR1 plasmids expressing siRNAs against MDR1
Cell survival in different ultrasound parameters
Cell Viability with different ultrasound intensities and exposure time
Survival rate (%)
97.07 ± 1.14
96.03 ± 1.51
95.22 ± 1.26
70.16 ± 3.49
71.25 ± 3.22
51.75 ± 4.02
37.43 ± 3.41
23.98 ± 3.24
Transfection efficiency and silencing efficiency of different transfection groups
Analysis of P-glycoprotein activity with Daunorubicin accumulation assay
Sensitivity to chemotherapeutic drugs by MTT assay
Yolk sac carcinoma is a malignant germ cell tumor with aggressive nature in children [5, 32]. While chemotherapy is critical to control the metastasis and recurrence of this disease , it has been reported that MDR1 expression level is related to the treatment responsiveness and prognosis in chemotherapy of malignant tumors as higher expression of MDR1 maybe lead to the lower efficiency of anti-cancer chemotherapy [20, 34]. The multi-drug resistance gene MDR1 encodes an ATP-dependent efflux transporter, P-glycoprotein protein, which protects tissues or cells from environmental toxins and xenobiotics, and prevents tissues or cells from attack of anti-cancer drugs [35–37]. In this study, we investigated whether the down-regulation of MDR1 could enhance the drug sensitivity of yolk sac carcinoma in vitro.
Small interfering RNAs (siRNAs) mediated RNA interference is widely used to silence gene expression via transcript degradation in mammalian cells. We chose to use the pSEB-HUS system which was specific for constructing GFP vector containing siRNA. The expression of siRNA can be driven by dual convergent H1 and U6 promoters and GFP-positive cells post plasmid transfection were easily detected by flow cytometry. Any siRNA can also regulate the expression of unintended targets which have similar silent site of target gene and result in non-specific gene silence. This so-called off-target effect can not only disturb the effect of silence of RNAi but also induce toxic phenotype [38, 39]. The pooling strategy of multiple target sites has been used to maximize target-gene specificity and efficiency and to minimize non-specific effects [40, 41]. In this study, we first identified three effective MDR1 siRNAs from four candidate siRNA sites by qRT-PCR. The three siRNA plasmids were pooled at an equal molar concentrations and transfected into L2-RYC cells. All three siRNAs were specific for MDR1 target gene but at different mRNA degradation sites, so increased the target gene knock-down efficiency of random-designed siRNAs. The decreased concentration of individual siRNAs could reduce potential off-target effects. Our result confirmed that the pooled siRNAs have higher inhibition efficacy than that of potent individual siRNAs.
Effective siRNA DNA delivery into cells and in vivo has been a great challenge for the broad use of RNAi therapeutics. The most commonly used carriers for delivering nucleic acids into mammalian cells are non-viral and viral vectors. Liposome-mediated transfection is simple and powerful, but has cytotoxic side effects . Calcium phosphate co-precipitation has rigorous conditions of transfection and a small range of target cells [42, 43]. Virus-mediated transfection is high efficient and available to achieve sustainable transgene expression. However the biosafety for in vivo use remains a concern . Recently, ultrasound contrast agents (in a form of microbubble) have been used to deliver gene and drug in vitro and in vivo, providing a new and efficient therapeutic technique [22–25]. Ultrasound microbubble-mediated destruction has been shown to enhance cell membrane permeability and improve gene and drug delivery. It has been shown that ultrasound microbubble-mediated destruction can transfect DNA into a variety of mammalian cells [22, 24, 26, 45]. The change of cell membrane permeability is recoverable when ultrasound energy and exposure time are within a suitable range. Thus ultrasound exposure will not cause permanent damage to cells [45, 46]. We first determined the optimal ultrasound parameters of acoustic intensity and exposure time for L2-RYC cell transfection. When cultured L2-RYC cells were exposed to ultrasound with intensity of 0.75 W/cm2 and 1 W/cm2, the survival rates was too low to be used in the study. Although ultrasound with intensity of 0.25 W/cm2 did not affect cell viability, plasmids DNA delivery into cells was poor. Fortunately, we found out ultrasound with intensity of 0.5 W/cm2 for 30 s could effectively transfect plasmids into cells without causing significant amount of cell death. Our previous study on bone marrow mononuclear cells also reported gene delivery by ultrasound with intensity of 0.5 W/cm2 did not reduce cell viability and not destroy membrane of treated cells . Under the chosen condition, we found that 30% GFP-positive cells can be achieved by gene transfection using ultrasound microbubble-mediated delivery. This transfection was higher than that of lipofection group and significantly decreased the expression of MDR1 by more than 60%, suggesting that ultrasound microbubble-mediated delivery may be used as an effective gene delivery method.
We determined the effect of silencing MDR1 expression by ultrasound microbubble-mediated siRNA delivery on multidrug resistance of yolk sac carcinamo cells. P-glycoprotein encoded by MDR1 gene is in charge of decreasing drug accumulation in multidrug-resistant cells, including tumor cells. Daunorubicin is used in cancer chemotherapy and its subcellular distribution is related to multidrug resistance. Daunorubicin produces red fluorescence with laser excitation at 488 nm, which is readily detected in drug-treated tissues or cells. Thus, Daunorubicin accumulation assay was performed to detect P-glycoprotein activity. Our results indicated that ultrasound microbubble-mediated delivery effectively transferred siMDR1 into L2-RYC cells and led to an increased Daunorubicin accumulation.
Chemotherapeutic drugs are means to combat cancers clinically. However, drug-resistance of tumor cells severely limits therapeutic outcomes. Drug sensitivity can be estimated by tumor cell viability treated with anti-cancer drug. Vincristine and Dactinomycin both of which are most commonly used chemo drugs and also known as substrates of P-glycoprotein. Thus, MTT assay was carried out to detect cell viability at different concentrations of Vincristine and Dactinomycin and to determine the IC50 ratios of two drugs in each group. Our results revealed that the L2-RYC cells treated with ultrasound microbubble-mediated siMDR1 delivery became more sensitive to anti-cancer drugs. Conceivably, silencing MDR1 should achieve excellent therapeutic efficacy at lower drug dosages so that chemotherapy-associated side effects can be alleviated to certain extends.
In this study, we constructed plasmids expressing siMDR1 and confirmed their silencing efficiency in L2-RYC cells. Ultrasound microbubble-mediated delivery can effectively transfer siMDR1 into L2-RYC cells and lead to inhibition of MDR1 expression and function of P-glycoprotein. Drug sensitivity was also improved by silencing MDR1. Thus, ultrasound microbubble-mediated delivery approach is a safe and effective gene transfection method and targeted inhibition method. Our results strongly suggested that combined gene silencing and chemotherapy may be further explored as a novel and potentially efficacious treatment of yolk sac carcinoma.
rat yolk sac carcinoma L2 cells
multiple drug resistance gene
small interfering RNA
Dulbecco's modified Eagle's medium
fetal bovine serum
quantitative real-time Polymerase Chain Reaction
green fluorescent protein
phosphate buffered saline
half maximal inhibitory concentration
We thank the editors and reviewers for their valuable comments and suggestions which are helpful for improving this manuscript. This work was supported by a research grant from the National Natural Science Foundation of China (No.81001030).
- Even C, Lhommé C, Duvillard P, Morice P, Balleyguier C, Pautier P, Troalen F, de La Motte Rouge T: Ovarian yolk sac tumour: general review. Bull Cancer. 2011, 98: 963-75.PubMedGoogle Scholar
- Merchant A, Stewart RW: Sacrococcygeal yolk sac tumor presenting as subcutaneous fluid collection initially treated as abscess. South Med J. 2010, 103: 1068-1070. 10.1097/SMJ.0b013e3181efb572.View ArticlePubMedGoogle Scholar
- Pasternack T, Shaco-Levy R, Wiznitzer A, Piura B: Extraovarian pelvic yolk sac tumor: case report and review of published work. J Obstet Gynaecol Res. 2008, 4: 739-744.View ArticleGoogle Scholar
- Tsugu H, Oshiro S, Ueno Y, Abe H, Komatsu F, Sakamoto S, Matsumoto S, Nabeshima K, Fukushima T, Inoue T: Primary yolk sac tumor within the lateral ventricle. Neurol Med Chir (Tokyo). 2009, 49: 528-531. 10.2176/nmc.49.528.View ArticleGoogle Scholar
- Unal O, Beyazal M, Avcu S, Akbayram S, Akgun C: Metastasis of testicular yolk sac tumor to cauda equina. Fetal Pediatr Pathol. 2011, 30: 150-155. 10.3109/15513815.2010.547553.View ArticlePubMedGoogle Scholar
- Bayar GR, Gulses A, Sencimen M, Aydintug YS, Arpaci F, Gunhan O: Oral metastasis of the mediastinal germ cell tumor (yolk sac). J Craniofac Surg. 2010, 21: 1828-1830. 10.1097/SCS.0b013e3181f43dea.View ArticlePubMedGoogle Scholar
- Chen CJ, Hsu HT, Yen HH: An unusual cause of upper gastrointestinal bleeding: Gastric yolk sac tumor with a large retroperitoneal metastasis. Gastroenterology. 2010, 139: 1098-1427. 10.1053/j.gastro.2009.10.060.View ArticlePubMedGoogle Scholar
- Low JJ, Perrin LC, Crandon AJ, Hacker NF: Conservative surgery to preserve ovarian function in patients with malignant ovarian germ cell tumors: A review of 74 cases. Cancer. 2000, 89: 391-398. 10.1002/1097-0142(20000715)89:2<391::AID-CNCR26>3.0.CO;2-V.View ArticlePubMedGoogle Scholar
- Weinberg LE, Lurain JR, Singh DK, Schink JC: Survival and reproductive outcomes in women treated for malignant ovarian germ cell tumors. Gynecol Oncol. 2011, 121: 285-289. 10.1016/j.ygyno.2011.01.003.View ArticlePubMedGoogle Scholar
- Shibata K, Umezu T, Sakurai M, Kajiyama H, Yamamoto E, Ino K, Nawa A, Kikkawa F: Establishment of cisplatin-resistant ovarian yolk sac tumor cells and investigation of the mechanism of cisplatin resistance using this cell line. Gynecol Obstet Invest. 2011, 71: 104-111. 10.1159/000320744.View ArticlePubMedGoogle Scholar
- Garrido W, Muñoz M, San Martín R, Quezada C: FK506 confers chemosensitivity to anticancer drugs in glioblastoma multiforme cells by decreasing the expression of the multiple resistance-associated protein-1. Biochem Biophys Res Commun. 2011, 411: 62-68. 10.1016/j.bbrc.2011.06.087.View ArticlePubMedGoogle Scholar
- Carmo CR, Lyons-Lewis J, Seckl MJ, Costa-Pereira AP: A novel requirement for Janus kinases as mediators of drug resistance induced by fibroblast growth factor-2 in human cancer cells. PLoS One. 2011, 6: e19861-10.1371/journal.pone.0019861.PubMed CentralView ArticlePubMedGoogle Scholar
- Peigñan L, Garrido W, Segura R, Melo R, Rojas D, Cárcamo JG, San Martín R, Quezada C: Combined use of anticancer drugs and an inhibitor of multiple drug resistance-associated protein-1 increases sensitivity and decreases survival of glioblastoma multiforme cells in vitro. Neurochem Res. 2011, 36: 1397-1406. 10.1007/s11064-011-0464-8.View ArticlePubMedGoogle Scholar
- Shi H, Lu D, Shu Y, Shi W, Lu S, Wang K: Expression of multidrug resistance-related proteins p-glycoprotein, glutathione-s-transferases, topoisomerase-II and lung resistance protein in primary gastric cardiac adenocarcinoma. Hepatogastroenterology. 2008, 55: 1530-1536.PubMedGoogle Scholar
- He SM, Li R, Kanwar JR, Zhou SF: Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1). Curr Med Chem. 2011, 18: 439-481.View ArticlePubMedGoogle Scholar
- Zhang B, Liu M, Tang HK, Ma HB, Wang C, Chen X, Huang HZ: The expression and significance of MRP1, LRP, TOPOIIβ, and BCL2 in tongue squamous cell carcinoma. J Oral Pathol Med. published online: 28 JUL 2011
- Hu WQ, Peng CW, Li Y: The expression and significance of P-glycoprotein, lung resistance protein and multidrug resistance-associated protein in gastric cancer. J Exp Clin Cancer Res. 2009, 28: 144-10.1186/1756-9966-28-144.PubMed CentralView ArticlePubMedGoogle Scholar
- Váradi A, Szakács G, Bakos E, Sarkadi B: P glycoprotein and the mechanism of multidrug resistance. Novartis Found Symp. 2002, 243: 54-65.View ArticlePubMedGoogle Scholar
- Weinstein RS, Kuszak JR, Kluskens LF, Coon JS: P-glycoproteins in pathology: the multidrug resistance gene family in humans. Hum Pathol. 1990, 21: 34-48. 10.1016/0046-8177(90)90073-E.View ArticlePubMedGoogle Scholar
- Oshikata A, Matsushita T, Ueoka R: Enhancement of drug efflux activity via MDR1 protein by spheroid culture of human hepatic cancer cells. J Biosci Bioeng. 2011, 111: 590-593. 10.1016/j.jbiosc.2011.01.006.View ArticlePubMedGoogle Scholar
- Eid H, Bodrogi I, Csókay B, Oláh E, Bak M: Multidrug resistance of testis cancers: the study of clinical relevance of P-glycoprotein expression. Anticancer Res. 1996, 16: 3447-3452.PubMedGoogle Scholar
- Wang J, Zheng Y, Yang F, Zhao P, Li H: Survivin small interfering RNA transfected with a microbubble and ultrasound exposure inducing apoptosis in ovarian carcinoma cells. Int J Gynecol Cancer. 2010, 20: 500-506. 10.1111/IGC.0b013e3181c5ddfa.View ArticlePubMedGoogle Scholar
- Suzuki J, Ogawa M, Takayama K, Taniyama Y, Morishita R, Hirata Y, Nagai R, Isobe M: Ultrasound-microbubble-mediated intercellular adhesion molecule-1 small interfering ribonucleic acid transfection attenuates neointimal formation after arterial injury in mice. J Am Coll Cardiol. 2010, 55: 904-913. 10.1016/j.jacc.2009.09.054.View ArticlePubMedGoogle Scholar
- Luo J, Zhou X, Diao L, Wang Z: Experimental research on wild-type p53 plasmid transfected into retinoblastoma cells and tissues using an ultrasound microbubble intensifier. J Int Med Res. 2010, 38: 1005-1015.View ArticlePubMedGoogle Scholar
- Chen ZY, Liang K, Qiu RX: Targeted gene delivery in tumor xenografts by the combination of ultrasound-targeted microbubble destruction and polyethylenimine to inhibit survivin gene expression and induce apoptosis. J Exp Clin Cancer Res. 2010, 29: 152-10.1186/1756-9966-29-152.PubMed CentralView ArticlePubMedGoogle Scholar
- Dang SP, Wang RX, Qin MD, Zhang Y, Gu YZ, Wang MY, Yang QL, Li XR, Zhang XG: A novel transfection method for eukaryotic cells using polyethylenimine coated albumin microbubbles. Plasmid. 2011, 66: 19-25. 10.1016/j.plasmid.2011.03.003.View ArticlePubMedGoogle Scholar
- Wang Y, Zhou J, Zhang Y, Wang X, Chen J: Delivery of TFPI-2 using SonoVue and adenovirus results in the suppression of thrombosis and arterial re-stenosis. Exp Biol Med (Maywood). 2010, 235: 1072-1081. 10.1258/ebm.2010.010046.View ArticleGoogle Scholar
- Luo Q, Kang Q, Song WX, Luu HH, Luo X, An N, Luo J, Deng ZL, Jiang W, Yin H, Chen J, Sharff KA, Tang N, Bennett E, Haydon RC, He TC: Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing. Gene. 2007, 1-2: 160-169.View ArticleGoogle Scholar
- He Y, Zhang WY, Gong M, Huang JY, Tang N, Feng T, Wei GH, He TC, Bi Y: Low serum concentration facilitates the differentiation of hepatic progenitor cells. Saudi Med J. 2011, 32: 128-134.PubMedGoogle Scholar
- Nabekura T, Yamaki T, Hiroi T, Ueno K, Kitagawa S: Inhibition of anticancer drug efflux transporter P-glycoprotein by rosemary phytochemicals. Pharmacol Res. 2010, 61: 259-263. 10.1016/j.phrs.2009.11.010.View ArticlePubMedGoogle Scholar
- Nabekura T, Yamaki T, Kitagawa S: Effects of chemopreventive citrus phytochemicals on human P-glycoprotein and multidrug resistance protein 1. Eur J Pharmacol. 2008, 600: 45-49. 10.1016/j.ejphar.2008.10.025.View ArticlePubMedGoogle Scholar
- Shah JP, Kumar S, Bryant CS, Ali-Fehmi R, Malone JM, Deppe G, Morris RT: A population-based analysis of 788 cases of yolk sac tumors: A comparison of males and females. Int J Cancer. 2008, 123: 2671-2675. 10.1002/ijc.23792.View ArticlePubMedGoogle Scholar
- de La Motte Rouge T, Pautier P, Duvillard P, Rey A, Morice P, Haie-Meder C, Kerbrat P, Culine S, Troalen F, Lhommé C: Survival and reproductive function of 52 women treated with surgery and bleomycin, etoposide, cisplatin (BEP) chemotherapy for ovarian yolk sac tumor. Ann Oncol. 2008, 19: 1435-1441. 10.1093/annonc/mdn162.View ArticlePubMedGoogle Scholar
- Mhaidat NM, Alshogran OY, Khabour OF, Alzoubi KH, Matalka II, Haddadin WJ, Mahasneh IO, Aldaher AN: Multi-drug resistance 1 genetic polymorphism and prediction of chemotherapy response in Hodgkin's Lymphoma. J Exp Clin Cancer Res. 2011, 30: 68-10.1186/1756-9966-30-68.PubMed CentralView ArticlePubMedGoogle Scholar
- Mizutani T, Masuda M, Nakai E, Furumiya K, Togawa H, Nakamura Y, Kawai Y, Nakahira K, Shinkai S, Takahashi K: Genuine functions of P-glycoprotein (ABCB1). Curr Drug Metab. 2008, 9: 167-174. 10.2174/138920008783571756.View ArticlePubMedGoogle Scholar
- Maier P, Fleckenstein K, Li L, Laufs S, Zeller WJ, Baum C, Fruehauf S, Herskind C, Wenz F: Overexpression of MDR1 using a retroviral vector differentially regulates genes involved in detoxification and apoptosis and confers radioprotection. Radiat Res. 2006, 166: 463-473. 10.1667/RR0550.1.View ArticlePubMedGoogle Scholar
- Achard-Joris M, Bourdineaud JP: Heterologous expression of bacterial and human multidrug resistance proteins protect Escherichia coli against mercury and zinc contamination. Biometals. 2006, 19: 695-704. 10.1007/s10534-006-9006-2.View ArticlePubMedGoogle Scholar
- Jackson AL, Linsley PS: Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat Rev Drug Discov. 2010, 9: 57-67. 10.1038/nrd3010.View ArticlePubMedGoogle Scholar
- Caffrey DR, Zhao J, Song Z, Schaffer ME, Haney SA, Subramanian RR, Seymour AB, Hughes JD: siRNA Off-Target Effects Can Be Reduced at Concentrations That Match Their Individual Potency. PLoS One. 2011, 6: e21503-10.1371/journal.pone.0021503.PubMed CentralView ArticlePubMedGoogle Scholar
- Parsons BD, Schindler A, Evans DH, Foley E: A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay. PLoS One. 2009, 4: e8471-10.1371/journal.pone.0008471.PubMed CentralView ArticlePubMedGoogle Scholar
- Hsieh AC, Bo R, Manola J, Vazquez F, Bare O, Khvorova A, Scaringe S, Sellers WR: A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens. Nucleic Acids Res. 2004, 32: 893-901. 10.1093/nar/gkh238.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang X, Ito A, Li X, Sogo Y, Oyane A: Signal molecules-calcium phosphate coprecipitation and its biomedical application as a functional coating. Biofabrication. 2011, 3: 022001-10.1088/1758-5082/3/2/022001.View ArticlePubMedGoogle Scholar
- Sun B, Tran KK, Shen H: Enabling customization of non-viral gene delivery systems for individual cell types by surface-induced mineralization. Biomaterials. 2009, 30: 6386-6393. 10.1016/j.biomaterials.2009.08.006.PubMed CentralView ArticlePubMedGoogle Scholar
- Posadas I, Guerra FJ, Ceña V: Nonviral vectors for the delivery of small interfering RNAs to the CNS. Nanomedicine (Lond). 2010, 5: 1219-1236. 10.2217/nnm.10.105.View ArticleGoogle Scholar
- Guo Z, Hong S, Jin X, Luo Q, Wang Z, Wang Y: Study on the multidrug resistance 1 gene transfection efficiency using adenovirus vector enhanced by ultrasonic microbubbles in vitro. Mol Biotechnol. 2011, 48: 138-146. 10.1007/s12033-010-9354-6.View ArticlePubMedGoogle Scholar
- ter Haar GR: Ultrasonic contrast agents: safety considerations reviewed. Eur J Radiol. 2002, 41: 217-221. 10.1016/S0720-048X(01)00456-9.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.