Kidney Epithelial LLC-PK1 Cells Research Articles

Focal adhesion kinase (FAK) inhibition induces aquaporin 2 (AQP2) plasma membrane accumulation in renal epithelial cells by remodeling the actin cytoskeleton

The actin cytoskeleton is involved in the cellular traffcking of the AQP2 water channel in kidney collecting duct (CD) principal cells (PC): this plays an important role in water homeostasis. Focal adhesion kinase (FAK) is known to modulate the actin cytoskeleton through small GTPase-mediated pathways and we, therefore, investigated the potential role of FAK in AQP2 traffcking. We and others have previously shown that AQP2 constitutively traffcs and recycles independently of vasopressin (VP) and that this involves actin depolymerization/polymerization; therefore, FAK could regulate AQP2 traffcking through actin remodeling, bypassing the VP-V2R (cAMP/PKA) signaling pathway. To examine this, we used small molecule FAK inhibitors, VS-4718, GSK2256098 (GSK), or Defactinib-Hydrochloride (DH). We used immunofluorescence staining, a transferrin endocytosis assay ± cold block treatment, a fluorometric exocytosis assay using cells expressing secreted yellow fluorescent protein (ssYFP), a RhoA activity assay, and western blotting. Inhibiting FAK with 1mM VS-4718 for 30 min increased AQP2 membrane accumulation in LLCPK1 and IMCD kidney epithelial cells expressing AQP2. VS-4718 resulted in reduced clathrin-mediated endocytosis, as well as increased vesicle exocytosis, both mechanisms contributing to increased AQP2 membrane accumulation. This effect of FAK inhibition was independent of vasopressin/cAMP signaling and did not require phosphorylation of the Serine 256 residue of AQP2, since AQP2 membrane accumulation also occurred in cells expressing the constitutive dephosphorylation AQP2 S256A mutant. The FAK inhibitor VS-4718 promoted actin depolymerization (phalloidin staining assay) which would facilitate AQP2 vesicle exocytosis into the plasma membrane. We then examined the activity of the small GTPase, RhoA, whose activity increases actin polymerization and vesicle traffcking in the exocytic and endocytic pathways. We found that FAK inhibition by VS-4718 downregulated RhoA activity using GST-RBD, a substrate that binds to active RhoA, as seen by western blotting using phospho-specific antibodies. Similar results were obtained with GSK and DH. In summary, our study revealed that the interaction between FAK and cytoskeletal architecture plays a crucial role in regulating AQP2 recycling and its subsequent entry into the endocytic pathway. Thus, targeting FAK could be a potential therapeutic strategy for water balance disorders involving defective AQP2 reabsorption, such as nephrogenic diabetes insipidus. This work was supported by NIDDK R01DK096015 (JHL) and DK096586 (DB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tumor Targeting with Bacterial Shiga Toxin B Subunit in Genetic Porcine Models for Colorectal Cancer and Osteosarcoma

The B subunit of bacterial Shiga toxin (STxB) is nontoxic and has low immunogenicity. Its receptor, the glycosphingolipid Gb3/CD77, is overexpressed on the cell surface of human colorectal cancer. We tested whether genetic porcine models, closely resembling human anatomy and pathophysiology, can be used to exploit the tumor-targeting potential of STxB. In accordance with findings on human colorectal cancer, the pig model APC1311 bound STxB in colorectal tumors, but not in normal colon or jejunum, except for putative enteroendocrine cells. In primary tumor cells from endoscopic biopsies, STxB was rapidly taken up along the retrograde intracellular route to the Golgi, whereas normal colon organoids did not bind or internalize STxB. Next, we tested a porcine model (TP53LSL-R167H) for osteosarcoma, a tumor entity with a dismal prognosis and insufficient treatment options, hitherto not known to express Gb3. Pig osteosarcoma strongly bound StxB and expressed the Gb3 synthase 1,4-galactosyltransferase (A4GALT). Primary osteosarcoma cells, but not normal osteoblasts, rapidly internalized fluorescently labeled STxB along the retrograde route to the Golgi. Importantly, six of eight human osteosarcoma cell lines expressed A4GALT mRNA and showed prominent intracellular uptake of STxB. The physiologic role of A4GALT was tested by CRISPR/Cas9 mutagenesis in porcine LLC-PK1 kidney epithelial cells and RNAi in MG-63 human osteosarcoma cells. A4GALT deficiency or knockdown abolished STxB uptake and led to significantly reduced cell migration and proliferation, hinting toward a putative tumor-promoting role of Gb3. Thus, pig models are suitable tools for STxB-based tumor targeting and may allow "reverse-translational" predictions on human tumor biology.

Similarities of Transport Characteristics of Lipophilic Cations between Porcine Kidney Epithelial LLC-PK₁ Cells and 5-Component Liposomes

Similarities of Transport Characteristics of Lipophilic Cations between Porcine Kidney Epithelial LLC-PK₁ Cells and 5-Component Liposomes

Unique Triterpenoid of Jujube Root Protects Cisplatin-induced Damage in Kidney Epithelial LLC-PK1 Cells via Autophagy Regulation.

Chronic exposure to cisplatin is associated with irreversible kidney impairment. In this present study, we explored the protective effects of 3-dehydroxyceanothetric acid 2-methyl ester (3DC2ME) isolated from roots of jujube (Ziziphus jujuba, Rhamnaceae) against cisplatin-induced damage in vitro. In kidney epithelial LLC-PK1 cells, western blotting and staining with specific autophagy epifluorescent dye CytoID were used to determine the molecular pathways involving autophagy. Treatment with 3DC2ME reduced the increased Cyto-ID-stained autophagic vesicles and reversed the protein expressions of 5’ AMP-activated protein kinase subunit β-1 (AMPK)/mammalian target of rapamycin (mTOR)-dependent signaling pathway in cisplatin-induced cell death. Additionally, treatment with autophagy inhibitor 3-methyladenine (3-MA) and with or without 3DC2ME attenuated the cisplatin-induced apoptosis. Although further research is necessary to substantiate the effects, we evaluated the potential mechanism of action of 3DC2ME as an adjuvant for cancer patients.

Multiple Targets of 3-Dehydroxyceanothetric Acid 2-Methyl Ester to Protect Against Cisplatin-Induced Cytotoxicity in Kidney Epithelial LLC-PK1 Cells

Chronic exposure to cisplatin, a potent anticancer drug, causes irreversible kidney damage. In this study, we investigated the protective effect and mechanism of nine lupane- and ceanothane-type triterpenoids isolated from jujube (Ziziphus jujuba Mill., Rhamnaceae) on cisplatin-induced damage to kidney epithelial LLC-PK1 cells via mitogen-activated protein kinase (MAPK) and apoptosis pathways. Cisplatin-induced LLC-PK1 cell death was most significantly reduced following treatment with 3-dehydroxyceanothetric acid 2-methyl ester (3DC2ME). Additionally, apoptotic cell death was significantly reduced. Expression of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 was markedly suppressed by 3DC2ME, indicating inhibition of the MAPK pathway. Treatment with 3DC2ME also significantly reduced expression of active caspase-8 and -3, Bcl-2-associated X protein (Bax), and B cell lymphoma 2 (Bcl-2), indicating the inhibition of apoptosis pathways in the kidneys. We also applied the network pharmacological analysis and identified multiple targets of 3DC2ME related to MAPK signaling pathway and apoptosis.

Evaluation of guggulsterone derivatives as novel kidney cell protective agents against cisplatin-induced nephrotoxicity

Guggulsterone derivatives were prepared using [3+2] click chemistry with aryl and alkyl acetylene. The series of derivatives were evaluated for their cellular protective effects on cisplatin-treated cultured LLC-PK1 kidney epithelial cells. Among the guggulsterone-triazole derivatives, compound 6g, which contains a hydroxyl methyl group, was the most active of all the derivatives. In an additional study, we determined that inhibition of the mitogen-activated protein kinase/caspase-3 signaling cascade by 6g mediates its protective effects against cytotoxicity in cultured LLC-PK1 cells.

Heterologous downregulation of vasopressin type 2 receptor is induced by transferrin

Vasopressin (VP) binds to the vasopressin type 2 receptor (V2R) to trigger physiological effects including body fluid homeostasis and blood pressure regulation. Signaling is terminated by receptor downregulation involving clathrin-mediated endocytosis and V2R degradation. We report here that both native and epitope-tagged V2R are internalized from the plasma membrane of LLC-PK1 kidney epithelial cells in the presence of another ligand, transferrin (Tf). The presence of iron-saturated Tf (holo-Tf; 4 h) reduced V2R binding sites at the cell surface by up to 33% while iron-free (apo-Tf) had no effect. However, no change in green fluorescent protein-tagged V2R distribution was observed in the presence of bovine serum albumin, atrial natriuretic peptide, or ANG II. Conversely, holo-Tf did not induce the internalization of another G protein-coupled receptor, the parathyroid hormone receptor. In contrast to the effect of VP, Tf did not increase intracellular cAMP or modify aquaporin-2 distribution in these cells, although addition of VP and Tf together augmented VP-induced V2R internalization. Tf receptor coimmunoprecipitated with V2R, suggesting that they interact closely, which may explain the additive effect of VP and Tf on V2R endocytosis. Furthermore, Tf-induced V2R internalization was abolished in cells expressing a dominant negative dynamin (K44A) mutant, indicating the involvement of clathrin-coated pits. We conclude that Tf can induce heterologous downregulation of the V2R and this might desensitize VP target cells without activating downstream V2R signaling events. It also provides new insights into urine-concentrating defects observed in rat models of hemochromatosis.

Abstract 843: Influx of thiosemicarbazone NSC73306 is mediated by the cisplatin and copper transporter SLC31A1 (CTR1)

Abstract Multidrug resistance (MDR) mediated via the drug efflux pump P-glycoprotein (P-gp/ABCB1) overexpression is a major obstacle to the success of chemotherapy to combat many cancers. Clinically, P-gp expression correlates with poor clinical response to chemotherapy in patients, and its presence on the blood-brain barrier (BBB) can reduce the efficacy of otherwise promising agents targeted to the brain. Attempts to reverse drug resistance by inhibiting P-gp have not been successful, and we have been investigating alternative strategies for targeting multidrug-resistant (MDR) cancer cells. One such strategy is the use of NSC73306, a thiosemicarbazone compound that is selectively toxic towards P-gp-expressing cells. Here, we investigate the cellular uptake of NSC73306, and examine its interaction with P-gp and copper transporter 1 (SLC31A1, CTR1). In porcine kidney epithelial LLC-PK1 cells, cellular accumulation of NSC73306 is saturable, and overexpression of human P-gp in LLC-PK1 cells sensitizes them to NSC73306. Accumulation of [3H]NSC73306 in LLC-PK1 cells is inhibited by cyclosporin A (IC50 = 500 μM), verapamil (IC50= 700 μM) and cisplatin (IC50 = 77 μM). Among these compounds, we tested the effect of cisplatin on NSC73306. Selective toxicity of NSC73306 to P-gp-expressing LLC-PK1 cells was reversed by cisplatin in a dose-dependent manner (up to 40 μM). In contrast, the amount of cisplatin required to kill 50% of LLC-PK1 cells was not affected by increasing the amount of NSC73306 (up to 2μM). Since cisplatin could inhibit NSC73306 uptake, we hypothesize that these two compounds share a common uptake pathway, but cisplatin has additional routes of entry into cells. Cells transiently expressing CTR1 showed significantly higher [3H]NSC73306 accumulation. In contrast, CTR1 knockdown cells had lower [3H]NSC73306 uptake. Although cisplatin is a transport substrate of CTR1, we found that CTR1 protein was overexpressed in cisplatin-resistant KB-CP20 and BEL7404-CP20 cell lines. Compared to their parental cisplatin-sensitive cells, even though these resistance cells show decreased cisplatin accumulation, suggesting that CTR1 is not rate-limited for cisplatin uptake. These cell lines also showed higher [3H]NSC73306 uptake compared to their parental cells. In contrast, MDR cell lines overexpressing P-gp had unchanged CTR1 protein levels. Incubation with NSC73306 for 24 hours down-regulated CTR1 in a dose-dependent manner. These results suggest that NSC73306 is a transport substrate of CTR1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 843. doi:1538-7445.AM2012-843

Simvastatin and lovastatin, but not pravastatin, interact with MDR1.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, pravastatin, was compared with simvastatin and lovastatin from the viewpoint of susceptibility to interaction with or via the multidrug transporter, MDR1 (P-glycoprotein). This was carried out using the MDR1-overexpressing cell line LLC-GA5-COL150, established by transfection of MDR1 cDNA into porcine kidney epithelial LLC-PK1 cells, and [3H]digoxin, which is a well-documented substrate for MDR1. Pravastatin, at 25-100 microM, had no effect on the transcellular transport of [3H]digoxin whereas simvastatin and lovastatin suppressed the basal-to-apical transport of [3H]digoxin and increased the apical-to-basal transport. It was suggested that recognition by MDR1 was due to the hydrophobicity. In conclusion, simvastatin and lovastatin are susceptible to interaction with or via MDR1, but pravastatin is not. This is important information when selecting the HMG-CoA reductase inhibitors for patients taking drugs that are MDR1 substrates.

A Pathogenic C Terminus-truncated Polycystin-2 Mutant Enhances Receptor-activated Ca2+ Entry via Association with TRPC3 and TRPC7

Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca(2+) release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca(2+) influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca(2+) influx that may lead to dysregulated cell growth in ADPKD.

Effects of α-Adrenoceptor Antagonist Doxazosin on MDR1-Mediated Multidrug Resistance and Transcellular Transport

The purpose of this study is to examine the effects of doxazosin, an alpha-adrenoceptor antagonist, on P-glycoprotein/MDR1-mediated multidrug resistance (MDR) and the transport of anticancer drugs. The effects of doxazosin, prazosin, and terazosin on MDR1-mediated MDR were assessed in human cervical carcinoma HeLa cells and the MDR1-overexpressing derivative Hvrl00-6, established by stepwise increases of the vinblastine concentration in the culture medium. The effects of doxazosin on the transcellular transport and intracellular accumulation of [3H]vinblastine, [3H]daunorubicin, and [3H]digoxin, all MDR1 substrates, were evaluated using LLC-GA5-COL150 cell monolayers, established by transfection of human MDR1 cDNA into porcine kidney epithelial LLC-PK1 cells. The sensitivity to vinblastine and paclitaxel of Hvrl00-6 cells was increased at 3.4- and 17.5-fold, respectively, by the addition of 1 microM doxazosin, whereas prazosin and terazosin had weaker or no such effects. Prazosin at 1 microM had a reversal effect on the sensitivity to vinblastine, whereas terazosin had no effect. In transport experiments, doxazosin concentration dependently increased the apical-to-basal transport of radiolabeled drugs in LLC-GA5-COL150 cells, but did not show remarkable effects on the basal-to-apical transport. In addition, doxazosin restored the intracellular accumulation in a concentration-dependent manner in LLC-GA5-COL150 cells. Doxazosin may partly reverse MDR by inhibiting MDR1-mediated transport, making it a candidate lead compound in the development of a reversing agent for MDR.

PKD2 Functions as an Epidermal Growth Factor-Activated Plasma Membrane Channel

PKD2, or polycystin 2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, belongs to the transient receptor potential channel superfamily and has been shown to function as a nonselective cation channel in the plasma membrane. However, the mechanism of PKD2 activation remains elusive. We show that PKD2 overexpression increases epidermal growth factor (EGF)-induced inward currents in LLC-PK(1) kidney epithelial cells, while the knockdown of endogenous PKD2 by RNA interference or the expression of a pathogenic missense variant, PKD2-D511V, blunts the EGF-induced response. Pharmacological experiments indicate that the EGF-induced activation of PKD2 occurs independently of store depletion but requires the activity of phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K). Pipette infusion of purified phosphatidylinositol-4,5-bisphosphate (PIP(2)) suppresses the PKD2-mediated effect on EGF-induced conductance, while pipette infusion of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) does not have any effect on this conductance. Overexpression of type Ialpha phosphatidylinositol-4-phosphate 5-kinase [PIP(5)Kalpha], which catalyzes the formation of PIP(2), suppresses EGF-induced currents. Biochemical experiments show that PKD2 physically interacts with PLC-gamma2 and EGF receptor (EGFR) in transfected HEK293T cells and colocalizes with EGFR and PIP(2) in the primary cilium of LLC-PK(1) cells. We propose that plasma membrane PKD2 is under negative regulation by PIP(2). EGF may reduce the threshold of PKD2 activation by mechanical and other stimuli by releasing it from PIP(2)-mediated inhibition.

An Organobismuth Compound that Exhibits Selective Cytotoxicity to Vascular Endothelial Cells in Vitro

Selective cytotoxicity of chemical compounds to vascular endothelial cells is often beneficial for clinical applications including antivascular cancer therapy. In this study, the cytotoxicity of 35 organic compounds containing bismuth or antimony was evaluated based on the leakage of lactate dehydrogenase and morphologic observations in cultured bovine aortic endothelial cells and other cell types. The results indicate that only tris[2-(N,N-dimethylaminomethyl)phenyl]-bismuthane (TDPBi) had potent cytotoxicity to bovine aortic endothelial cells but not to bovine aortic smooth muscle cells and porcine kidney epithelial LLC-PK1 cells; the compound exhibited moderate cytotoxicity to human fetal lung fibroblastic IMR-90 cells. Neither inorganic bismuth nor tris[2-(N,N-dimethylaminomethyl)phenyl]stibane, in which antimony substitutes for bismuth with the same organic structure as TDPBi, exhibited cytotoxicity to vascular endothelial cells, suggesting that the entire structure of TDPBi is required for selective cytotoxicity. The present data revealed that TDPBi has selective cytotoxicity to vascular endothelial cells, which may be applicable in antivascular cancer therapy.

Wound healing and angiogenic properties of supernatants from Lactobacillus cultures.

Extracts or supernatants from cultures of Lactobacilli are used for their medicinal effects, including wound healing and immune system stimulating activity. We have studied the in vivo and in vitro effects of supernatants from bacterial cultures of two strains of Lactobacillus (LS) on tissue repair and angiogenesis. Subcutaneous injection of LS into rodent ears led to proliferation of blood vessels that also exhibited strong immunostaining for Flk-1 receptor. Some inflammatory cells were scattered among the blood vessels. The continuous influx of polymorphonuclear leukocytes (PMNs) and macrophages into transcutaneous wounds in mice treated with LS resulted in prolonged inflammatory phase of wound healing and delayed wound closure, including reepithelialization. Subcutaneous injection of Matrigel impregnated with LS into the abdominal wall led to rapid and transient influx of PMNs in the vicinity of the gel. LS stimulated the proliferation of murine macrophage J774.A1 cell line and porcine lymphocytes but not that of murine fibroblast AKR-2B cells. LS also induced production of TNF-alpha by J774.A1 cells and by porcine kidney epithelial LLC-PK1 cells. LS did not appear to have an effect on collagen production. In conclusion, our study demonstrates the potential of LS to function as a stimulator of the inflammatory stage of tissue repair, TNF-alpha production, and of angiogenesis.

Interaction of imatinib mesilate with human P-glycoprotein.

The interaction of imatinib mesilate with P-glycoprotein (P-gp) was examined using pig kidney epithelial LLC-PK1 cells versus L-MDR1 cells, which overexpress human P-gp on the apical membrane. The basal-to-apical transport of imatinib mesilate in L-MDR1 cells significantly exceeded that in the parental LLCPK1 cells. The intracellular accumulation of imatinib mesilate after its basal application to LLC-PK1 and L-MDR1 cells was 35% and 15%, respectively. A P-gp modulator, cyclosporin A, inhibited the basal-to-apical transport in L-MDR1 cells. The intracellular accumulation of imatinib mesilate in L-MDR1 cells was also increased by cyclosporin A. The rhodamine 123 efflux assay showed that the efflux of rhodamine 123 in K562/DXR cells, which overexpress human P-gp, could be blocked markedly by imatinib mesilate in a dose-dependent fashion. The Ki values for the inhibition of P-gp function by cyclosporin A and imatinib mesilate were estimated to be 6.1 and 18.3 muM, respectively, using a calcein-AM efflux assay. These observations demonstrate that imatinib mesilate is a substrate as well as a modulator of human P-gp, suggesting that imatinib mesilate drug interactions may occur via P-gp. It is necessary to consider the pharmacokinetic and pharmacodynamic interactions of imatinib mesilate with other drugs via P-gp.

Up-regulation of MDR1 function and expression by cisplatin in LLC-PK1 cells.

To examine whether cisplatin affects the multidrug transporter MDR1/P-glycoprotein in the kidneys, the effects of cisplatin on cell sensitivity to an anticancer drug, MDR1 function and expression were examined by assessing the growth inhibition by the MDR1 substrate paclitaxel, the uptake and efflux of the MDR1 substrate Rhodamine123 and the level of MDR1 mRNA, respectively. Porcine kidney epithelial LLC-PK1 cells were used, as they have a structure and function similar to those of renal proximal tubular cells and physiologically express low levels of MDR1. The growth inhibitory curve of LLC-PK1 cells by paclitaxel was shifted to a higher concentration range by pretreatment with 1 micro M cisplatin for 48 h. The uptake and efflux of Rhodamine123 were significantly reduced and enhanced, respectively, by pretreatment with 1 micro M cisplatin for 48 h. This enhanced efflux was suppressed by the representative MDR1 substrate/inhibitor ciclosporin. The expression of MDR1 mRNA was increased by the existence of cisplatin for 48 h. These observations taken together suggested that the transient exposure to cisplatin could cause the up-regulation of MDR1 in LLC-PK1 cells.

Bromocriptine reverses P-glycoprotein-mediated multidrug resistance in tumor cells.

One of the most important causes of anticancer treatment failure is the development of multidrug resistance (MDR). The main characteristics of tumor cells displaying the MDR phenomena are cross‐resistance to structurally unrelated cytotoxic drugs having different mechanisms of action and the overexpression of the MDR1 gene, which encodes a transmembrane glycoprotein named P‐ glycoprotein (P‐gp). This study evaluated whether bromocriptine, a D2 dopaminergic receptor agonist, influenced anticancer drug cytotoxicity and P‐gp activity in a P‐gp‐expressing cell line compared to a non‐expressing subline. The Ki values for P‐gp of cyclosporine and verapamil were 1.09 and 540 μM, respectively, and that of bromocriptine was 6.52 μM in a calcein‐AM efflux assay using porcine kidney epithelial LLC‐PK1 and L‐MDR1 cells, overexpressing human P‐gp. Bromocriptine at 10 μM reduced the IC50 of doxorubicin (DXR) in K562‐DXR from 9000 to 270 ng/ml and that of vincristine (VCR) in K562‐VCR from 700 to 0.30 ng/ml, whereas the IC50 values of DXR and VCR in the K562 subline were only marginally affected by these drugs. Bromocriptine restored the anticancer effect of DXR, VCR, vinblastine, vinorelbine and etoposide on MDR‐tumor cells overexpressing P‐gp. These observations suggest that bromocriptine has the potential to reverse tumor MDR involving the efflux protein P‐gp in the clinical situation.

Multidrug-resistance P-glycoprotein (MDR1) secretes platelet-activating factor

The human multidrug-resistance (MDR1) P-glycoprotein (Pgp) is an ATP-binding-cassette transporter (ABCB1) that is ubiquitously expressed. Often its concentration is high in the plasma membrane of cancer cells, where it causes multidrug resistance by pumping lipophilic drugs out of the cell. In addition, MDR1 Pgp can transport analogues of membrane lipids with shortened acyl chains across the plasma membrane. We studied a role for MDR1 Pgp in transport to the cell surface of the signal-transduction molecule platelet-activating factor (PAF). PAF is the natural short-chain phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. [14C]PAF synthesized intracellularly from exogenous alkylacetylglycerol and [14C]choline became accessible to albumin in the extracellular medium of pig kidney epithelial LLC-PK1 cells in the absence of vesicular transport. Its translocation across the apical membrane was greatly stimulated by the expression of MDR1 Pgp, and inhibited by the MDR1 inhibitors PSC833 and cyclosporin A. Basolateral translocation was not stimulated by expression of the basolateral drug transporter MRP1 (ABCC1). It was insensitive to the MRP1 inhibitor indomethacin and to depletion of GSH which is required for MRP1 activity. While efficient transport of PAF across the apical plasma membrane may be physiologically relevant in MDR1-expressing epithelia, PAF secretion in multidrug-resistant tumours may stimulate angiogenesis and thereby tumour growth.

Multidrug-resistance P-glycoprotein (MDR1) secretes platelet-activating factor.

The human multidrug-resistance (MDR1) P-glycoprotein (Pgp) is an ATP-binding-cassette transporter (ABCB1) that is ubiquitously expressed. Often its concentration is high in the plasma membrane of cancer cells, where it causes multidrug resistance by pumping lipophilic drugs out of the cell. In addition, MDR1 Pgp can transport analogues of membrane lipids with shortened acyl chains across the plasma membrane. We studied a role for MDR1 Pgp in transport to the cell surface of the signal-transduction molecule platelet-activating factor (PAF). PAF is the natural short-chain phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. [(14)C]PAF synthesized intracellularly from exogenous alkylacetylglycerol and [(14)C]choline became accessible to albumin in the extracellular medium of pig kidney epithelial LLC-PK1 cells in the absence of vesicular transport. Its translocation across the apical membrane was greatly stimulated by the expression of MDR1 Pgp, and inhibited by the MDR1 inhibitors PSC833 and cyclosporin A. Basolateral translocation was not stimulated by expression of the basolateral drug transporter MRP1 (ABCC1). It was insensitive to the MRP1 inhibitor indomethacin and to depletion of GSH which is required for MRP1 activity. While efficient transport of PAF across the apical plasma membrane may be physiologically relevant in MDR1-expressing epithelia, PAF secretion in multidrug-resistant tumours may stimulate angiogenesis and thereby tumour growth.

Cytosolic phospholipase A(2) regulates golgi structure and modulates intracellular trafficking of membrane proteins.

The Golgi complex and the trans-Golgi network are critical cellular organelles involved in the endocytic and biosynthetic pathways of protein trafficking. Lipids have been implicated in the regulation of membrane-protein trafficking, vesicular fusion, and targeting. We have explored the role of cytosolic group IV phospholipase A(2) (cPLA(2)) in membrane-protein trafficking in kidney epithelial cells. Adenoviral expression of cPLA(2) in LLC-PK(1) kidney epithelial cells prevents constitutive trafficking to the plasma membrane of an aquaporin 2-green fluorescent protein chimera, with retention of the protein in the rough endoplasmic reticulum. Plasma membrane Na(+)-K(+)-ATPase alpha-subunit localization is markedly reduced in cells expressing cPLA(2), whereas the trafficking of a Cl(-)/HCO(3)(-) anion exchanger to the plasma membrane is not altered in these cells. Expression of cPLA(2) results in dispersion of giantin and beta-COP from their normal, condensed Golgi localization, and in marked disruption of the Golgi cisternae. cPLA(2) is present in Golgi fractions from noninfected LLC-PK(1) cells and rat kidney cortex. The distribution of tubulin and actin was not altered by cPLA(2), indicating that the microtubule and actin cytoskeleton remain intact. Total cellular protein synthesis is unaffected by the increase in cPLA(2) activity. Thus cPLA(2) plays an important role in determining Golgi architecture and selective control of constitutive membrane-protein trafficking in renal epithelial cells.