LncRNA FENDRR represses proliferation,migration and invasion through suppression of survivin in cholangiocarcinoma cells

This study was to investigate the biological function and underlying mechanisms of FENDRR in cholangiocarcinoma (CCA) cell proliferation, migration and invasion. FENDRR and survivin expression in CCA tissues or cell lines were measured by qRT-PCR. In QBC939 and HuCCTl cells, cell proliferation was detected by CCK-8, cell migration and invasion were using transwell assay. RNA pull-down and RIP assay were performed to determine whether FENDRR can combine with SETDB1 in CCA cell. The effect of SETDB1 on survivin and H3K9me1 expression in CCA cells were determined by western blotting. ChIP analysis was performed to analyze the combination of SETDB1 with survivin promoter in CCA cell. The effect of SETDB1 knockdown on survivin and H3K9me1 expression in CCA cells after transfection with FENDRR were determined by western blotting. The results showed that lncRNA FENDRR was downregulated in CCA tissues and cells, and was negatively correlated with survivin expression. Further investigation demonstrated that FENDRR represses CCA cell proliferation, migration and invasion through regulating survivin expression. FENDRR associated with SETDB1 and H3K9 to epigenetically silence survivin and then regulated cell proliferation, migration and invasion. These findings indicate an important role for FENDRR–survivin axis in CCA cell proliferation, migration and invasion, and reveal a novel epigenetic mechanism for survivin silencing. Our data indicated that FENDRR silences survivin via SETDB1-mediated H3K9 methylation, thereby represses CCA cell proliferation, migration and invasion.     

CXCR7 contributes to the aggressive phenotype of cholangiocarcinoma cells

Development of cholangiocarcinoma (CCA) is dependent on a cross-talk with stromal cells, which release different chemokines including CXCL12, that interacts with two different receptors, CXCR4 and CXCR7. The aim of the present study was to investigate the role of CXCR7 in CCA cells. CXCR7 is overexpressed by different CCA cell lines and in human CCA specimens. Knock-down of CXCR7 in HuCCT-1 cells reduced migration, invasion, and CXCL12-induced adhesion to collagen I. Survival of CCA was also reduced in CXCR7-silenced cells. The ability of CXCL12 to induce cell migration and survival was also blocked by CCX733, a CXCR7 antagonist. Similar effects of CXCR7 activation were observed in CCLP-1 cells and in primary iCCA cells. Enrichment of tumor stem-like cells by a 3D culture system resulted in increased CXCR7 expression compared to cells grown in monolayers, and genetic knockdown of CXCR7 robustly reduced sphere formation both in HuCCT-1 and in CCLP-1 cells. In HuCCT-1 cells CXCR7 was found to interact with β-arrestin 2, which was necessary to mediate CXCL12-induced migration, but not survival. In conclusion, CXCR7 is widely expressed in CCA, and contributes to the aggressive phenotype of CCA cells, inducing cell migration, invasion, adhesion, survival, growth and stem cell-like features. Cell migration induced by CXCR7 requires interaction with β-arrestin 2.     

4F2hc stabilizes GLUT1 protein and increases glucose transport activity

Glucose transporter 1 (GLUT1) is widely distributed throughout various tissues and contributes to insulin-independent basal glucose uptake. Using a split-ubiquitin membrane yeast two-hybrid system, we newly identified 4F2 heavy chain (4F2hc) as a membrane protein interacting with GLUT1. Though 4F2hc reportedly forms heterodimeric complexes between amino acid transporters, such as LAT1 and LAT2, and regulates amino acid uptake, we investigated the effects of 4F2hc on GLUT1 expression and the associated glucose uptake. First, FLAG-tagged 4F2hc and hemagglutinin-tagged GLUT1 were overexpressed in human embryonic kidney 293 cells and their association was confirmed by coimmunoprecipitation. The green fluorescent protein-tagged 4F2hc and DsRed-tagged GLUT1 showed significant, but incomplete, colocalization at the plasma membrane. In addition, an endogenous association between GLUT1 and 4F2hc was demonstrated using mouse brain tissue and HeLa cells. Interestingly, overexpression of 4F2hc increased the amount of GLUT1 protein in HeLa and HepG2 cells with increased glucose uptake. In contrast, small interfering RNA (siRNA)-mediated 4F2hc gene suppression markedly reduced GLUT1 protein in both cell types, with reduced glucose uptake. While GLUT1 mRNA levels were not affected by overexpression or gene silencing of 4F2hc, GLUT1 degradation after the addition of cycloheximide was significantly suppressed by 4F2hc overexpression and increased by 4F2hc siRNA treatment. Taken together, these observations indicate that 4F2hc is likely to be involved in GLUT1 stabilization and to contribute to the regulation of not only amino acid but also glucose metabolism.     

MicroRNA-451 regulates AMPK/mTORC1 signaling and fascin1 expression in HT-29 colorectal cancer

The earlier studies have shown that Fascin1 (FSCN1), the actin bundling protein, is over-expressed in colorectal cancers, and is associated with cancer cell progression. Here, we aimed to understand the molecular mechanisms regulating FSCN1 expression by focusing on mammalian target of rapamycin (mTOR) signaling and its regulator microRNA-451. We found that microRNA-451 was over-expressed in multiple colorectal cancer tissues, and its expression was correlated with mTOR complex 1 (mTORC1) activity and FSCN1 expression. In cultured colorectal cancer HT-29 cells, knockdown of FSCN1 by RNAi inhibited cell migration and proliferation. Activation of mTORC1 was required for FSCN1 expression, HT-29 cell migration and proliferation, as RAD001 and rapamycin, two mTORC1 inhibitors, suppressed FSCN1 expression, HT-29 cell migration and proliferation. Meanwhile, forced activation of AMP-activated protein kinase (AMPK), the negative regulator of mTORC1, by its activators or by the genetic mutation, inhibited mTORC1 activation, FSCN1 expression, cell migration and proliferation. In HT-29 cells, we found that over-expression of microRNA-451 inhibited AMPK activation, causing mTORC1 over-activation and FSCN1 up-regulation, cells were with high migration ability and proliferation rate. Significantly, these effects by microRNA-451 were largely inhibited by mTORC1 inhibitors or the AMPK activator AICAR. On the other hand, knockdown of miRNA-451 by the treatment of HT-29 cells with miRNA-451 antagomir inhibited mTORC1 activation and FSCN1 expression. The proliferation and migration of HT-29 cells after miRNA-45 knockdown were also inhibited. Our results suggested that the over-expressed microRNA-451 in colon cancer cells might inhibit AMPK to activate mTORC1, which mediates FSCN1 expression and cancer cell progression.     

Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines

System L is a major nutrient transport system responsible for the transport of large neutral amino acids including several essential amino acids. We previously identified a transporter (L-type amino acid transporter 1: LAT1) subserving system L in C6 rat glioma cells and demonstrated that LAT1 requires 4F2 heavy chain (4F2hc) for its functional expression. Since its oncofetal expression was suggested in the rat liver, it has been proposed that LAT1 plays a critical role in cell growth and proliferation. In the present study, we have examined the function of human LAT1 (hLAT1) and its expression in human tissues and tumor cell lines. When expressed in Xenopus oocytes with human 4F2hc (h4F2hc), hLAT1 transports large neutral amino acids with high affinity (K(m)= approximately 15- approximately 50 microM) and L-glutamine and L-asparagine with low affinity (K(m)= approximately 1.5- approximately 2 mM). hLAT1 also transports D-amino acids such as D-leucine and D-phenylalanine. In addition, we show that hLAT1 accepts an amino acid-related anti-cancer agent melphalan. When loaded intracellularly, L-leucine and L-glutamine but not L-alanine are effluxed by extracellular substrates, confirming that hLAT1 mediates an amino acid exchange. hLAT1 mRNA is highly expressed in the human fetal liver, bone marrow, placenta, testis and brain. We have found that, while all the tumor cell lines examined express hLAT1 messages, the expression of h4F2hc is varied particularly in leukemia cell lines. In Western blot analysis, hLAT1 and h4F2hc have been confirmed to be linked to each other via a disulfide bond in T24 human bladder carcinoma cells. Finally, in in vitro translation, we show that hLAT1 is not a glycosylated protein even though an N-glycosylation site has been predicted in its extracellular loop, consistent with the property of the classical 4F2 light chain. The properties of the hLAT1/h4F2hc complex would support the roles of this transporter in providing cells with essential amino acids for cell growth and cellular responses, and in distributing amino acid-related compounds.     

Aldehyde dehydrogenase 3B2 promotes the proliferation and invasion of cholangiocarcinoma by increasing Integrin Beta 1 expression

Aldehyde dehydrogenases (ALDHs) play an essential role in regulating malignant tumor progression; however, their role in cholangiocarcinoma (CCA) has not been elucidated. We analyzed the expression of ALDHs in 8 paired tumor and peritumor perihilar cholangiocarcinoma (pCCA) tissues and found that ALDH3B1 and ALDH3B2 were upregulated in tumor tissues. Further survival analysis in intrahepatic cholangiocarcinoma (iCCA, n = 27), pCCA (n = 87) and distal cholangiocarcinoma (dCCA, n = 80) cohorts have revealed that ALDH3B2 was a prognostic factor of CCA and was an independent prognostic factor of iCCA and pCCA. ALDH3B2 expression was associated with serum CEA in iCCA and dCCA, associated with tumor T stage, M stage, neural invasion and serum CA19-9 in pCCA. In two cholangiocarcinoma cell lines, overexpression of ALDH3B2 promoted cell proliferation and clone formation by promoting the G1/S phase transition. Knockdown of ALDH3B2 inhibited cell migration, invasion, and EMT in vitro, and restrained tumor metastasis in vivo. Patients with high expression of ALDH3B2 also have high expression of ITGB1 in iCCA, pCCA, and dCCA at both mRNA and protein levels. Knockdown of ALDH3B2 downregulated the expression of ITGB1 and inhibited the phosphorylation level of c-Jun, p38, and ERK. Meanwhile, knockdown of ITGB1 inhibited the promoting effect of ALDH3B2 overexpression on cell proliferation, migration, and invasion. ITGB1 is also a prognostic factor of iCCA, pCCA, and dCCA and double-positive expression of ITGB1 and ALDH3B2 exhibits better performance in predicting patient prognosis. In conclusion, ALDH3B2 promotes tumor proliferation and metastasis in CCA by regulating the expression of ITGB1 and upregulating its downstream signaling pathway. The double-positive expression of ITGB1 and ALDH3B2 serves as a better prognostic biomarker of CCA.Subject terms: Prognostic markers, Bile duct cancer     

Cloning and functional characterization of a Na(+)-independent, broad-specific neutral amino acid transporter from mammalian intestine

We have isolated a cDNA from a rabbit intestinal cDNA library which, when co-expressed with the heavy chain of the human 4F2 antigen (4F2hc) in mammalian cells, induces system L-like amino acid transport activity. This protein, called LAT2, consists of 535 amino acids and is distinct from LAT1 which also interacts with 4F2hc to induce system L-like amino acid transport activity. LAT2 does not interact with rBAT, a protein with a significant structural similarity to 4F2hc. The 4F2hc/LAT2-mediated transport process differs from the 4F2hc/LAT1-mediated transport in substrate specificity, substrate affinity, tissue distribution, interaction with D-amino acids, and pH-dependence. The 4F2hc/LAT2-associated transport process has a broad specificity towards neutral amino acids with K(t) values in the range of 100-1000 microM, does not interact with D-amino acids to any significant extent, and is stimulated by acidic pH. In contrast, the 4F2hc/LAT1-associated transport process has a narrower specificity towards neutral amino acids, but with comparatively higher affinity (K(t) values in the range of 10-20 microM), interacts with some D-amino acids with high affinity, and is not influenced by pH. LAT2 is expressed primarily in the small intestine and kidney, whereas LAT1 exhibits a much broader tissue distribution.     

Overexpression of non-functional LAT1/4F2hc in renal proximal tubular epithelial cells from the spontaneous hypertensive rat.

The present study examined the expression of type 1 L-amino acid transporter (LAT1) and its associated glycoprotein 4F2hc in freshly isolated renal proximal tubules and immortalized renal proximal tubular epithelial (PTE) cells from spontaneously hypertensive (SHR) and normotensive (WKY) rats. The study also examined the inward and outward transport of [(14)C]-L-leucine, the preferred substrate of LAT1. The abundance of LAT1 and 4F2hc was greater in SHR than in WKY, both in freshly isolated renal proximal tubules and immortalized renal proximal tubular cells. In the absence of extracellular Na(+) the BCH (2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid)-sensitive [(14)C]-L-leucine uptake in SHR PTE cells was approximately 50% that observed in WKY PTE cells (77+/-4 vs 164+/-7 pmol/mg protein). In the absence of extracellular Na(+) the affinity of the transporter for the substrate in WKY PTE cells was 7.7-fold that in SHR cells, as evidenced by lower K(0.5) values. Gene silencing with a LAT1 siRNA and a 4F2hc siRNA significantly reduced LAT1 and 4F2hc expression, which was accompanied by a marked reduction in Na(+)-independent [(14)C]-L-leucine uptake in both SHR and WKY PTE cells. The spontaneous and L-leucine-stimulated outward transfer of [(14)C]-L-leucine was Na(+)-independent in both SHR and WKY PTE cells. The spontaneous [(14)C]-L-leucine efflux was higher in WKY than in SHR PTE cells and the potency of L-leucine to stimulate [(14)C]-L-leucine efflux in WKY (EC(50) = 9 microM) was greater than in SHR PTE cells (EC(50) = 41 microM). It is concluded that the SHR kidney overexpress LAT1/4F2hc units which display low affinity for L-leucine transport.     

Activation of system L heterodimeric amino acid exchangers by intracellular substrates

System L-type transport of large neutral amino acids is mediated by ubiquitous LAT1-4F2hc and epithelial LAT2-4F2hc. These heterodimers are thought to function as obligatory exchangers, but only influx properties have been studied in some detail up until now. Here we measured their intracellular substrate selectivity, affinity and exchange stoichiometry using the Xenopus oocyte expression system. Quantification of amino acid influx and efflux by HPLC demonstrated an obligatory amino acid exchange with 1:1 stoichiometry. Strong, differential trans-stimulations of amino acid influx by injected amino acids showed that the intracellular substrate availability limits the transport rate and that the efflux selectivity range resembles that of influx. Compared with high extracellular apparent affinities, LAT1- and LAT2-4F2hc displayed much lower intracellular apparent affinities (apparent K(m) in the millimolar range). Thus, the two system L amino acid transporters that are implicated in cell growth (LAT1-4F2hc) and transcellular transport (LAT2-4F2hc) are obligatory exchangers with relatively symmetrical substrate selectivities but strongly asymmetrical substrate affinities such that the intracellular amino acid concentration controls their activity.     

The role of CD98 in astrocytic neoplasms

The high expression of CD98 was reported in some normal tissues, including blood brain barrier, activated lymphocytes, the basal layer of skin, proximal tubles of kidney, placenta, testis and a wide variety of tumors. The CD98 complex consists of an 80-85kD heavy chain (4F2hc/FRP-1) and a 40-45kD light chain. CD98hc, 4F2hc, and FRP-1 are the same glycosylated protein each other and define antigenicity of CD98. LAT1, the sodium-independent L-type amino acid transporter 1, has been identified as a light chain of the CD98 heterodimer from C6 glioma cells. LAT1 also corresponds to TA1, an oncofetal antigen that is expressed primarily in fetal tissues and cancer cells such as glioma cells. Increased LAT1 expression was found in various malignancies including human gliomas. Several studies implicated the important role of LAT1 and 4F2hc in malignant transformation and carcinogenesis. The LAT1-CD98 pathway may represent a unique therapeutic target for cancer intervention.     

Functional cooperation of epithelial heteromeric amino acid transporters expressed in madin-darby canine kidney cells

The heteromeric amino acid transporters b(0,+)AT-rBAT (apical), y(+)LAT1-4F2hc, and possibly LAT2-4F2hc (basolateral) participate to the (re)absorption of cationic and neutral amino acids in the small intestine and kidney proximal tubule. We show now by immunofluorescence that their expression levels follow the same axial gradient along the kidney proximal tubule (S1>S2S3). We reconstituted their co-expression in MDCK cell epithelia and verified their polarized localization by immunofluorescence. Expression of b(0,+)AT-rBAT alone led to a net reabsorption of l-Arg (given together with l-Leu). Coexpression of basolateral y(+)LAT1-4F2hc increased l-Arg reabsorption and reversed l-Leu transport from (re)absorption to secretion. Similarly, l-cystine was (re)absorbed when b(0,+)AT-rBAT was expressed alone. This net transport was further increased by the coexpression of 4F2hc, due to the mobilization of LAT2 (exogenous and/or endogenous) to the basolateral membrane. In summary, apical b(0,+)AT-rBAT cooperates with y(+)LAT1-4F2hc or LAT2-4F2hc for the transepithelial reabsorption of cationic amino acids and cystine, respectively. The fact that the reabsorption of l-Arg led to the secretion of l-Leu demonstrates that the implicated heteromeric amino acid transporters function in epithelia as exchangers coupled in series and supports the notion that the parallel activity of unidirectional neutral amino acid transporters is required to drive net amino acid reabsorption.     

Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris

Human heteromeric amino acid transporters (HATs) play key roles in renal and intestinal re-absorption, cell redox balance and tumor growth. These transporters are composed of a heavy and a light subunit, which are connected by a disulphide bridge. Heavy subunits are the two type II membrane N-glycoproteins rBAT and 4F2hc, while L-type amino acid transporters (LATs) are the light and catalytic subunits of HATs. We tested the expression of human 4F2hc and rBAT as well as seven light subunits in the methylotrophic yeast Pichia pastoris. 4F2hc and the light subunit LAT2 showed the highest expression levels and yields after detergent solubilization. Co-transformation of both subunits in Pichia cells resulted in overexpression of the disulphide bridge-linked 4F2hc/LAT2 heterodimer. Two sequential affinity chromatography steps were applied to purify detergent-solubilized heterodimers yielding ～1 mg of HAT from 2 l of cell culture. Our results indicate that P. pastoris is a convenient system for the expression and purification of human 4F2hc/LAT2 for structural studies.     

Apical and basolateral 4F2hc and the amino acid exchange of L-DOPA in renal LLC-PK1 cells

Summary. The present study aimed to examine the presence and define the role of 4F2hc, a glycoprotein associated with the LAT2 amino acid transporter, in L-DOPA handling by LLC-PK₁ cells. For this purpose we have measured the activity of the apical and basolateral inward and outward transport of [¹⁴C] L-DOPA in cell monolayers and examined the influence of 4F2hc antisense oligonucleotides on [¹⁴C] L-DOPA handling. The basal-to-apical transepithelial flux of [¹⁴C] L-DOPA progressively increased with incubation time and was similar to the apical-to-basal transepithelial flux. The spontaneous and the L-DOPA-stimulated apical fractional outflow of [¹⁴C] L-DOPA were identical to that through the basal cell side. The L-DOPA-induced fractional outflow of [¹⁴C] L-DOPA through the apical or basal cell side was accompanied by marked decreases in intracellular levels of [¹⁴C] L-DOPA. In cells treated with an antisense oligonucleotide complementary to 4F2hc mRNA for 72 h, [¹⁴C] L-DOPA inward transport and 4F2hc expression were markedly reduced. Treatment with the 4F2hc antisense oligonucleotide markedly decreased the spontaneous fractional outflow of [¹⁴C] L-DOPA through the apical or the basal cell side. It is likely that the Na⁺-independent and pH-sensitive uptake of L-DOPA include the hetero amino acid exchanger LAT2/4F2hc, which facilitates the trans-stimulation of L-DOPA and its outward transfer at both the apical and basal cell sides.     

LAT2, a new basolateral 4F2hc/CD98-associated amino acid transporter of kidney and intestine

Glycoprotein-associated amino acid transporters (gpaAT) are permease-related proteins that require heterodimerization to express their function. So far, four vertebrate gpaATs have been shown to associate with 4F2hc/CD98 for functional expression, whereas one gpaAT specifically associates with rBAT. In this study, we characterized a novel gpaAT, LAT2, for which mouse and human cDNAs were identified by expressed sequence tag data base searches. The encoded ortholog proteins are 531 and 535 amino acids long and 92% identical. They share 52 and 48% residues with the gpaATs LAT1 and y(+)LAT1, respectively. When mouse LAT2 and human 4F2hc cRNAs were co-injected into Xenopus oocytes, disulfide-linked heterodimers were formed, and an L-type amino acid uptake was induced, which differed slightly from that produced by LAT1-4F2hc: the apparent affinity for L-phenylalanine was higher, and L-alanine was transported at physiological concentrations. In the presence of an external amino acid substrate, LAT2-4F2hc also mediated amino acid efflux. LAT2 mRNA is expressed mainly in kidney and intestine, whereas LAT1 mRNA is expressed widely. Immunofluorescence experiments showed colocalization of 4F2hc and LAT2 at the basolateral membrane of kidney proximal tubules and small intestine epithelia. In conclusion, LAT2 forms with LAT1 a subfamily of L-type gpaATs. We propose that LAT1 is involved in cellular amino acid uptake, whereas LAT2 plays a role in epithelial amino acid (re)absorption.     

Oncogenic Activity of Retinoic Acid Receptor γ Is Exhibited through Activation of the Akt/NF-κB and Wnt/β-Catenin Pathways in Cholangiocarcinoma

Aberrant expression and function of retinoic acid receptor γ (RARγ) are often involved in the progression of several cancers. However, the role of RARγ in cholangiocarcinoma (CCA), chemoresistant bile duct carcinoma with a poor prognosis, remains unclear. In the present study, we found that RARγ was frequently overexpressed in human CCA specimens. Its overexpression was associated with poor differentiation, lymph node metastasis, high serum carbohydrate antigen 19-9 level, and poor prognosis of CCA. Downregulation of RARγ reduced CCA cell proliferation, migration, invasion, and colony formation ability in vitro and tumorigenic potential in nude mice. RARγ knockdown resulted in upregulation of cell cycle inhibitor P21, as well as downregulation of cyclin D1, proliferating cell nuclear antigen, and matrix metallopeptidase 9, in parallel with suppression of the Akt/NF-κB pathway. Furthermore, overexpression of RARγ contributed to the multidrug chemoresistance of CCA cells, at least in part due to upregulation of P glycoprotein via activation of the Wnt/β-catenin pathway. Molecular mechanism studies revealed that RARγ interacted with β-catenin and led to β-catenin nuclear translocation. Taken together, our results suggested that RARγ plays an important role in the proliferation, metastasis, and chemoresistance of CCA through simultaneous activation of the Akt/NF-κB and Wnt/β-catenin pathways, serving as a potential molecular target for CCA treatment.     

Up‐regulation of ZFAS1 indicates dismal prognosis for cholangiocarcinoma and promotes proliferation and metastasis by modulating USF1 via miR‐296‐5p

LncRNAs has been demonstrated to modulate neoplastic development by modulating downstream miRNAs and functional genes. In this study, we aimed to detect the interaction among lncRNA ZFAS1 miR‐296‐5p and USF1. We explored the proliferation, migration and invasion of cholangiocarcinoma. The differentially expressed ZFAS1 was discovered in both tissues and cell lines by qRT‐PCR. The targeting relationship between miR‐296‐5p and ZFAS1 or USF1 was validated by dual‐luciferase assay. The impact of ZFAS1 on CCA cell proliferation was observed by CCK‐8 assay. The protein expression of USF1 was determined by Western blot. The effects of ZFAS1, miR‐296‐5p and USF1 on tumour growth were further confirmed using xenograft model. LncRNA ZFAS1 expression was relatively up‐regulated in tumour tissues and cells while miR‐296‐5p was significantly down‐regulated. Knockdown of ZFAS1 significantly suppressed tumour proliferation, migration, invasion and USF1 expression. Overexpressed miR‐296‐5p suppressed cell proliferation and metastasis. Knockdown of USF1 inhibited cell proliferation and metastasis and xenograft tumour growth. In conclusion, ZFAS1 might promote cholangiocarcinoma proliferation and metastasis by modulating USF1 via miR‐296‐5p.     

Homologues of amino acid permeases: cloning and tissue expression of XAT1 and XAT2

The L-type (LAT) family of amino acid transporters is composed of exchangers for neutral, cationic, and anionic amino acids. They form functional heterodimers with membrane glycoproteins, rBAT or 4F2hc/CD98, to which they are linked by a disulphide bond. We report the molecular cloning and tissue expression of new mouse and human homologues of the LAT family, termed mXAT1, mXAT2 and hXAT2. The latter two proteins may correspond to ortholog genes in mouse and human. The hXAT2 gene is located on chromosome 8q21.3. The cloned X amino acid transporter (XAT) cDNAs are predicted to encode proteins of about 50 kDa. From a phylogenetic point of view, the three XAT proteins cluster together, but sequence comparison and secondary structure prediction show that they are also related to the members of the LAT family. Like these transporters, the XAT proteins show 12 transmembrane domains and a conserved cysteine residue, located in the second extracellular loop. This conserved cysteine is involved in the disulphide bond formed between the known members of the LAT family and 4F2hc or rBAT. The mXAT1 and hXAT2 mRNAs are expressed in the kidney but they are not detectable in a variety of other tissues. The corresponding proteins were efficiently translated following transfection of their cDNAs in Chinese hamster ovary (CHO) cells. However, cDNA transfection in CHO cells did not induce amino acid uptake, even when cotransfected with vectors expressing 4F2hc or rBAT. This could be related to the fact that mXAT1 and hXAT2 did not form detectable disulphide-linked heterodimers with 4F2hc or rBAT when they were co-expressed in CHO cells. Identification of other putative partner(s) of these LAT family-related transporters may be necessary to understand their role in renal physiology.     

Crucial role of heme oxygenase-1 on the sensitivity of cholangiocarcinoma cells to chemotherapeutic agents

Cancer cells acquire drug resistance via various mechanisms including enhanced cellular cytoprotective and antioxidant activities. Heme oxygenase-1 (HO-1) is a key enzyme exerting potent cytoprotection, cell proliferation and drug resistance. We aimed to investigate roles of HO-1 in human cholangiocarcinoma (CCA) cells for cytoprotection against chemotherapeutic agents. KKU-100 and KKU-M214 CCA cell lines with high and low HO-1 expression levels, respectively, were used to evaluate the sensitivity to chemotherapeutic agents, gemcitabine (Gem) and doxorubicin. Inhibition of HO-1 by zinc protoporphyrin IX (ZnPP) sensitized both cell types to the cytotoxicity of chemotherapeutic agents. HO-1 gene silencing by siRNA validated the cytoprotective effect of HO-1 on CCA cells against Gem. Induction of HO-1 protein expression by stannous chloride enhanced the cytoprotection and suppression of apoptosis caused by anticancer agents. The sensitizing effect of ZnPP was associated with increased ROS formation and loss of mitochondrial transmembrane potential, while Gem alone did not show any effects. A ROS scavenger, Tempol, abolished the sensitizing effect of ZnPP on Gem. Combination of ZnPP and Gem enhanced the release of cytochrome c and increased p21 levels. The results show that HO-1 played a critical role in cytoprotection in CCA cells against chemotherapeutic agents. Targeted inhibition of HO-1 may be a strategy to overcome drug resistance in chemotherapy of bile duct cancer.