scholarly journals IPEC-J2 rMdr1a, a New Cell Line with Functional Expression of Rat P-glycoprotein Encoded by Rat Mdr1a for Drug Screening Purposes

Pharmaceutics ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 673 ◽  
Author(s):  
Lasse Saaby ◽  
Josefine Trasborg ◽  
Mikkel A. Rasmussen ◽  
Bjørn Holst ◽  
Birger Brodin
Keyword(s):  
Cell Line ◽  
Efflux Pump ◽  
Cell Model ◽  
Drug Distribution ◽  
Functional Expression ◽  
Established Cell Line ◽  
Multidrug Resistance Gene ◽  
Western Blots ◽  
P Glycoprotein

The efflux pump P-glycoprotein (P-gp) affects drug distribution after absorption in humans and animals. P-gp is encoded by the multidrug resistance gene (MDR1) gene in humans, while rodents (the most common preclinical animal model) express the two isoforms Mdr1a and Mdr1b. Differences in substrate selectivity has also been reported. Our aim was to generate an in vitro cell model with tight barrier properties, expressing functional rat Mdr1a P-gp, as an in vitro tool for investigating species differences. The IPEC-J2 cell line forms extremely tight monolayers and was transfected with a plasmid carrying the rat Mdr1a gene sequence. Expression and P-gp localization at the apical membrane was demonstrated with Western blots and immunocytochemistry. Function of P-gp was shown through digoxin transport experiments in the presence and absence of the P-gp inhibitor zosuquidar. Bidirectional transport experiments across monolayers of the IPEC-J2 rMDR1a cell line and the IPEC-J2 MDR1 cell line, expressing human P-gp, showed comparable magnitude of transport in both the absorptive and efflux direction. We conclude that the newly established IPEC-J2 rMdr1a cell line, in combination with our previously established cell line IPEC-J2 MDR1, has the potential to be a strong in vitro tool to compare P-gp substrate profiles of rat and human P-gp.

Characterization of a newly established uterine carcinosarcoma cell line featuring the sarcomatous phenotype of the tumor in vitro

2008 ◽  
Vol 18 (2) ◽  
pp. 339-344 ◽  
Author(s):  
H.-J. Schulten ◽  
J. Wolf-Salgó ◽  
C. Gründker ◽  
B. Gunawan ◽  
L. FÜZESI
Keyword(s):  
Cell Line ◽  
Hormone Receptors ◽  
Tumor Biology ◽  
Growth Factor Receptor ◽  
Population Doubling ◽  
Established Cell Line ◽  
Population Doubling Time ◽  
Uterine Carcinosarcoma ◽  
Specific Staining

We describe the newly established cell line CS-99 derived from a uterine carcinosarcoma retaining features of the sarcomatous phenotype in vitro. CS-99 cells exhibit a mesenchymal morphology with predominantly spindle-shaped cells at nonconfluence turning to pleomorphic appearance at confluence. The mesenchymal phenotype was evidenced immunohistochemically by strong vimentin and moderate SM-actin, which was similar to the sarcomatous component of the primary tumor. P53 was overexpressed in a subset of CS-99 cells. Epithelial membrane antigen was moderately expressed whereas other markers including pan CK, CK 5/6, CK 34, epidermal growth factor receptor, desmin, carcinoembryonic antigen, S100, KIT, ERBB2, and the hormone receptors, estrogen receptor and progesterone receptor revealed either weak or no specific staining in CS-99 cells. High self-renewal capacity corresponded to the population doubling time of 23 h in high passage. CS-99 cells were able to develop three-dimensional tumor spheroids in vitro. Cytogenetic analysis and multicolor fluorescence in situ hybridization of CS-99 demonstrated an almost stable karyotype including numerical changes +8, +18, and +20 and translocations, amongst others der(1)t(1;2), der(1)t(1;7), der(2)t(2;19), der(5)t(5;8), and der(5)t(5;14). Taken together, the cell line CS-99 exhibits strong growths dynamics and a complex but stable karyotype in higher passages, and can be further a useful in vitro model system for studying tumor biology of carcinosarcomas.

scholarly journals The effect of strontium ranelate on titanium particle-induced periprosthetic osteolysis regulated by WNT/β-catenin signaling in vivo and in vitro

2021 ◽  
Vol 41 (1) ◽  
Author(s):  
Bolun Wang ◽  
Haohui Guo ◽  
Tianxiang Geng ◽  
Kening Sun ◽  
Liang Zhang ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Aseptic Loosening ◽  
Cell Line ◽  
Conditioned Medium ◽  
Strontium Ranelate ◽  
Cell Model ◽  
Periprosthetic Osteolysis ◽  
A Cell

Abstract Aseptic loosening following periprosthetic osteolysis is the primary complication that limits the lifetime of total joint arthroplasty (TJA). The wear particles trigger a chronic inflammation response in the periprosthetic tissue and turn over the bone balance to bone resorption. The present study aimed to investigate the possible effect and mechanism of strontium ranelate (SR), a clinically safe drug for osteoporosis, on particle-induced periprosthetic osteolysis. Thirty-six female C57BL/6j mice underwent tibial Ti-nail implantation to establish an animal model of aseptic loosening. After 12 weeks, micro-CT results showed that strontium ranelate could inhibit periprosthetic bone resorption. In vitro, Ti particles were used to stimulate RAW264.7 cell line to collect conditioned medium, and co-culture MC3T3-E1 cell line with conditioned medium to establish a cell model of aseptic loosening. The results of alkaline phosphatase (ALP) detection, immunofluorescence, and flow cytometry demonstrated that strontium ranelate could regulate the expression of OPG/RANKL, promote differentiation and mineralization, and inhibit apoptosis in osteoblasts. Moreover, we revealed that SR’s exerted its therapeutic effect by down-regulating sclerostin, thereby activating the Wnt/β-catenin signal pathway. Therefore, this research suggests that strontium ranelate could be a potential drug for the prevention and treatment of particle-induced aseptic loosening post-TJA.

Enhancement of pheomelanogenesis by L-dopa in the mouse melanocyte cell line, TM10, in vitro

1987 ◽  
Vol 87 (4) ◽  
pp. 507-512
Author(s):  
C. Sato ◽  
S. Ito ◽  
T. Takeuchi
Keyword(s):  
Cell Line ◽  
Growth Medium ◽  
Yellow Pigment ◽  
Black Pigment ◽  
Established Cell Line ◽  
Treatment Results ◽  
Common Precursor ◽  
Established Cell ◽  
The Common

Cells of TM10, an established cell line, are melanocytes that contain equal amounts of eumelanin (black pigment) and pheomelanin (yellow pigment). The content of pheomelanin drastically increased when the cells were cultured in growth medium containing 0.2mM-L-dopa (L-dihydroxyphenylalanine), which is the common precursor for both eumelanogenesis and pheomelanogenesis. After this treatment, the amount of pheomelanin was 3.7-fold greater than that of control in TM10, whereas the amount of eumelanin changed very little. In contrast, 5-S-cysteinyl-dopa, which is the specific precursor for pheomelanogenesis downstream of L-dopa, did not cause preferential increase in pheomelanogenesis. Ultrastructural observations also confirmed these results; in 0.2mM-L-dopa, an increase in the number of pheomelanosomes was observed in the cytoplasm of TM10 cells. Our results also suggest that the L-dopa treatment results in a decrease in tyrosinase activity per melanosome.

scholarly journals Characterization of a murine renal distal convoluted tubule cell line for the study of transcellular calcium transport

2004 ◽  
Vol 286 (3) ◽  
pp. F483-F489 ◽  
Author(s):  
Robin J. W. Diepens ◽  
Els den Dekker ◽  
Marcelle Bens ◽  
A. Freek Weidema ◽  
Alain Vandewalle ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Model ◽  
Collecting Duct ◽  
Primary Cultures ◽  
Ruthenium Red ◽  
Distal Convoluted Tubule ◽  
Molecular Regulation ◽  
Hek 293 ◽  
Calbindin D

To unravel the molecular regulation of renal transcellular Ca2+ transport, a murine distal convoluted tubule (mpkDCT) cell line derived from distal convoluted tubules (DCT) microdissected from a SV-PK/Tag transgenic mouse was characterized. This cell line originated from DCT only, as mRNA encoding for the DCT marker thiazide-sensitive Na+/Cl- cotransporter was expressed, whereas mRNA encoding for the connecting tubule and collecting duct marker aquaporin-2 was not detected, as determined by reverse-transcriptase PCR. mpkDCT cells expressed mRNA encoding the Ca2+ channels TRPV5 and TRPV6 and other key players necessary for transcellular Ca2+ transport, i.e., calbindin-D9k, calbindin-D28k, plasma membrane Ca2+-ATPase isoform 1b, and Na+/Ca2+ exchanger 1. Primary cultures of DCT cells exhibited net transcellular Ca2+ transport of 0.4 ± 0.1 nmol·h-1·cm-2, whereas net transcellular Ca2+ transport across mpkDCT cells was significantly higher at 2.4 ± 0.4 nmol·h-1·cm-2. Transcellular Ca2+ transport across mpkDCT cells was completely inhibited by ruthenium red, an inhibitor of TRPV5 and TRPV6, but not by the voltage-operated Ca2+ channel inhibitors felodipine and verapamil. With the use of patch-clamp analysis, the IC50 of ruthenium red on Na+ currents was between the values measured for TRPV5- and TRPV6-expressing HEK 293 cells, suggesting that TRPV5 and/or TRPV6 is possibly active in mpkDCT cells. Forskolin in combination with IBMX, 1,25-dihydroxyvitamin D3, and 1-deamino-8-d-arginine vasopressin increased transcellular Ca2+ transport, whereas PMA and parathyroid hormone had no significant effect. In conclusion, the murine mpkDCT cell line provides a unique cell model in which to study the molecular regulation of transcellular Ca2+ transport in the kidney in vitro.

scholarly journals Plasmid-Mediated Resistance to Thrombin-Induced Platelet Microbicidal Protein in Staphylococci: Role of theqacA Locus

1999 ◽  
Vol 43 (10) ◽  
pp. 2395-2399 ◽  
Author(s):  
Leon Iri Kupferwasser ◽  
Ronald A. Skurray ◽  
Melissa H. Brown ◽  
Neville Firth ◽  
Michael R. Yeaman ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Efflux Pump ◽  
Endothelial Damage ◽  
Cross Resistance ◽  
Cationic Peptide ◽  
Multidrug Resistance Gene ◽  
Phenotypic Resistance ◽  
Platelet Microbicidal Protein

ABSTRACT Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide released from rabbit platelets following thrombin stimulation. In vitro resistance to this peptide among strains of Staphylococcus aureus correlates with the survival advantage of such strains at sites of endothelial damage in humans as well as in experimental endovascular infections. The mechanisms involved in the phenotypic resistance of S. aureus to tPMP-1 are not fully delineated. The plasmid-encoded staphylococcal gene qacA mediates multidrug resistance to multiple organic cations via a proton motive force-dependent efflux pump. We studied whether the qacA gene might also confer resistance to cationic tPMP-1. Staphylococcal plasmids encoding qacA were found to confer resistance to tPMP-1 in an otherwise susceptible parental strain. Deletions which removed the region containing theqacA gene in the S. aureus multiresistance plasmid pSK1 abolished tPMP-1 resistance. Resistance to tPMP-1 in theqacA-bearing strains was inoculum independent but peptide concentration dependent, with the level of resistance decreasing at higher peptide concentrations for a given inoculum. There was no apparent cross-resistance in qacA-bearing strains to other endogenous cationic antimicrobial peptides which are structurally distinct from tPMP-1, including human neutrophil defensin 1, protamine, or the staphylococcal lantibiotics pep5 and nisin. These data demonstrate that the staphylococcal multidrug resistance geneqacA also mediates in vitro resistance to cationic tPMP-1.

Glucosylceramide Synthetase Inhibitors Sensitise B-CLL Cells to Cytotoxic Agents without Reversing P-gp Functional Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1181-1181
Author(s):  
Gareth Gerrard ◽  
Terry D. Butters ◽  
Atul B. Mehta ◽  
A. Victor Hoffbrand ◽  
Derryln Hughes ◽  
...  
Keyword(s):  
Cell Line ◽  
Functional Activity ◽  
Mononuclear Cells ◽  
Efflux Pump ◽  
Specific Inhibitor ◽  
In Vitro Cytotoxicity ◽  
Cytotoxic Drugs ◽  
Cytotoxic Agents ◽  
Peripheral Blood Mononuclear

Abstract Malignant B-cells from a high proportion of chronic lymphocytic leukaemia (B-CLL) patients over express the multidrug resistance (MDR) -1 gene encoded transmembrane efflux pump P-glycoprotein (P-gp). Inhibition of glucosylceramide synthesis has been shown to correlate with the expression and function of P-gp and sensitise cells to cytotoxic agents. We analysed the ability of glucosylceramide synthetase (GCS) inhibitors N-butyl-deoxygalactonojirimycin (OGB-1, 500μM) and N-nonyl-deoxygalactonojirimycin (OGB-2, 100μM) to sensitise B-CLL cells to conventional cytotoxic drugs 2-chlorodeoxyadenosine (CdA), chlorambucil (Chl) and fludarabine (FdR) using the in vitro cytotoxicity MTT assay. The effect on P-gp activity was also analysed using the calcein-AM accumulation assay and the results expressed as multidrug activity factor (MAF), where a MAF of >10 in the presence of a P-gp inhibitor denotes P-gp functional activity. GCS inhibitors were cultured with B-CLL cells for 24-48h before the assays were performed. The P-gp negative cell line CEM-CCRF had no MAF activity with an IC50 for vincristine (a known P-gp substrate) of <1ng/ml. The P-gp over expressing cell line CEM-VLB showed a MAF value of 96.4 with zosuquidar trihydrochloride (Z.3HCL), a specific inhibitor of P-gp, 15.7 with OGB-1 and 45.9 with OGB-2. The IC50 for vincristine was reduced from >10ug/ml to 55.5ng/ml in the presence of OGB-2. In peripheral blood mononuclear cells from 3 normal volunteers (all P-gp +ve), the mean MAF value for Z.3HCL was 23.86 and for OGB-2 was 16.2. In 9/13 B-CLL samples there was P-gp functional activity in the presence of Z.3HCL with a mean MAF value of 22.15 (range 11.27–37.3). P-gp was over expressed in10/13 B-CLL samples. However, when available samples from this cohort were assessed with OGB-1 (n=4) and OGB-2 (n=13) the MAF value was <10. Nevertheless, sensitisation of B-CLL cells was observed by a reduction in the IC50 in the presence of OGB-1 with CdA in 3/4 (to 40% in the presence of cytotoxic drug alone), Chl in 3/4 (39%), FdR in 2/4 (26%) and in the presence of OGB-2 with CdA in 8/13 (42%), Chl in 5/13 (40%) and FdR in 7/13 (34%). Although GCS inhibitors sensitize B-CLL cells to cytotoxic drugs in some B-CLL patients, they do not appear to have any effect on P-gp functional activity.

Increased susceptibility of a triclabendazole (TCBZ)-resistant isolate of Fasciola hepatica to TCBZ following co-incubation in vitro with the P-glycoprotein inhibitor, R(+)-verapamil

Parasitology ◽  
2013 ◽  
Vol 140 (10) ◽  
pp. 1287-1303 ◽  
Author(s):  
M. MEANEY ◽  
J. SAVAGE ◽  
G. P. BRENNAN ◽  
E. HOEY ◽  
A. TRUDGETT ◽  
...  
Keyword(s):  
Fasciola Hepatica ◽  
Efflux Pump ◽  
Morphological Changes ◽  
Resistant Isolate ◽  
Drug Efflux ◽  
P Glycoprotein ◽  
Efflux Pump Inhibition ◽  
Inactivity Time ◽  
Drug Efflux Pumps

SUMMARYA study was carried out to investigate whether the action of triclabendazole sulphoxide (TCBZ.SO) against the liver fluke, Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for this in vitro study and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. For experiments with the Oberon isolate, flukes were incubated for 24 h with either R(+)-VPL (1×10−4m) on its own, TCBZ.SO (15 μg mL−1) alone, a combination of R(+)-VPL (1×10−4m) plus TCBZ.SO (15 μg mL−1), TCBZ.SO (50 μg mL−1) on its own, or a combination of TCBZ.SO (50 μg mL−1) plus R(+)-VPL (1×10−4m). They were also incubated in TCBZ.SO (50 μg mL−1) alone or in combination with R(+)-VPL (1×10−4m) until they became inactive; and in TCBZ.SO (50 μg mL−1) alone for a time to match that of the combination inactivity time. Flukes from the Cullompton isolate were treated with either TCBZ.SO (50 μg mL−1) alone or in combination with R(+)-VPL (1×10−4m) until they became inactive, or with TCBZ.SO (50 μg mL−1) alone time-matched to the combination inactivity time. Morphological changes resulting from drug treatment and following Pgp inhibition were assessed by means of scanning electron microscopy. Incubation in R(+)-VPL alone had a minimal effect on either isolate. TCBZ.SO treatment had a relatively greater impact on the TCBZ-susceptible Cullompton isolate. When R(+)-VPL was combined with TCBZ.SO in the incubation medium, however, the surface disruption to both isolates was more severe than that seen after TCBZ.SO treatment alone; also, the time taken to reach inactivity was shorter. More significantly, though, the potentiation of drug activity was greater in the Oberon isolate; also, it was more distinct at the higher concentration of TCBZ.SO. So, the Oberon isolate appears to be particularly sensitive to efflux pump inhibition. The results of this study suggest that enhanced drug efflux in the Oberon isolate may be involved in the mechanism of resistance to TCBZ.

In vitro culture and cloning of Toxoplasma gondii in a newly established cell line derived from TG180

1991 ◽  
Vol 21 (1) ◽  
pp. 129-132 ◽  
Author(s):  
A. Couatarmanach ◽  
P. Andre ◽  
D. Le Minous ◽  
L. Martin ◽  
R. Robert ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
In Vitro Culture ◽  
Cell Line ◽  
Established Cell Line ◽  
Established Cell

Reversal of P-glycoprotein-medicated multidrug resistance by LBM-A5 in vitro and a study of its pharmacokinetics in vivo

2015 ◽  
Vol 93 (1) ◽  
pp. 33-38 ◽  
Author(s):  
Tianxiao Zhao ◽  
Yun Song ◽  
Baomin Liu ◽  
Qianqian Qiu ◽  
Lei Jiao ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Cancer Chemotherapy ◽  
Anticancer Agents ◽  
Efflux Pump ◽  
Therapeutic Index ◽  
Intracellular Accumulation ◽  
P Glycoprotein ◽  
Reversal Mechanism

The overexpression of P-glycoprotein (P-gp) in tumors leads to multidrug resistance (MDR), which is a significant obstacle in clinical cancer chemotherapy. The co-administration of anticancer drugs and MDR modulators is a promising strategy for overcoming this problem. Our study aimed to explore the reversal mechanism and safety of the MDR modulator LBM-A5 in vitro, and evaluate its pharmacokinetics and effects on doxorubicin metabolism in vivo. We evaluated an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay of anticancer agents mediated by LBM-A5, the effect of LBM-A5 on rhodamine123 intracellular accumulation, and the efflux in K562/DOX cells to investigate the reversal mechanisms of LBM-A5. The results showed that LBM-A5 inhibits rhodamine123 efflux and increases intracellular accumulation by inhibiting the efflux pump function of P-gp. Furthermore, the therapeutic index and CYP3A4 activity analysis in vitro suggested that LBM-A5 is reasonably safe to use. Also, LBM-A5 (10 mg/kg body mass) achieved the required plasma concentration in sufficient time to reverse MDR in vivo. Importantly, the LBM-A5 treatment group shared similar doxorubicin (DOX) pharmacokinetics with the free DOX group. Our results suggest that LBM-A5 effectively reverses MDR (EC50 = 483.6 ± 81.7 nmol·L−1) by inhibiting the function of P-gp, with relatively ideal pharmacokinetics and in a safe manner, and so may be a promising candidate for cancer chemotherapy research.

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