Galectin-1 confers resistance to doxorubicin in hepatocellular carcinoma cells through modulation of P-glycoprotein expression

Galectin-1 (GAL1), a β-galactoside-binding protein abundantly expressed in the tumor microenvironment, has emerged as a key mechanism of chemoresistance developed by different tumors. Although increased expression of GAL1 is a hallmark of hepatocellular carcinoma (HCC) progression, aggressiveness and metastasis, limited information is available on the role of this endogenous lectin in HCC resistance to chemotherapy. Moreover, the precise mechanisms underlying this effect are uncertain. HCC has evolved different mechanisms of resistance to chemotherapy including those involving the P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, which controls intracellular drug concentration. Here, we investigated the molecular mechanism underlying GAL1-mediated chemoresistance in HCC cells, particularly the involvement of P-gp in this effect. Our results show that GAL1 protected HepG2 cells from doxorubicin (DOX)- and sorafenib-induced cell death in vitro. Accordingly, GAL1-overexpressing HepG2 cells generated DOX-resistant tumors in vivo. High expression of GAL1 in HepG2 cells reduced intracellular accumulation of DOX likely by increasing P-gp protein expression rather than altering its membrane localization. GAL1-mediated increase of P-gp expression involved activation of the phosphatidylinositol-3 kinase (PI3K) signaling pathway. Moreover, ‘loss-of-function’ experiments revealed that P-gp mediates GAL1-driven resistance to DOX, but not to sorafenib, in HepG2 cells. Conversely, in PLC/PRF/5 cells, P-gp protein expression was undetectable and GAL1 did not control resistance to DOX or sorafenib, supporting the critical role of P-gp in mediating GAL1 effects. Collectively, our findings suggest that GAL1 confers chemoresistance in HCC through mechanisms involving modulation of P-gp, thus emphasizing the role of this lectin as a potential therapeutic target in HCC.Subject terms: Glycobiology, Liver cancer     

Assay for determination of daunorubicin in cancer cells with multidrug resistance phenotype

A sensitive assay for direct determination of intracellular level of daunorubicin (DRN) in resistant leukemia cells with overexpressed P-glycoprotein has been developed. This assay is based on a rapid separation of cells from media and fast cut-off of DRN transportation by centrifugation of cells through a layer of silicone oil. Cell pellets were extracted using 1% (v/v) formic acid in 50% (v/v) ethanol in water. The cell extracts were subsequently analysed by liquid chromatography (HPLC) coupled a low-energy collision tandem mass spectrometer equipped with an electrospray ionization source (ESI-CID-MS/MS) operated in the multiple-reaction monitoring (MRM) mode. Calibration curve was linear from 0.4 to 250nM with correlation coefficient (r2) better than 0.998. The limit of quantitation (LOQ) was 0.4 nM. The assay has been successfully applied to a determination of intracellular content of daunorubicin in sensitive K562 and resistant K562/Dox and K562/HHT300 cells.     

MUC1 extracellular domain confers resistance of epithelial cancer cells to anoikis

Anoikis, a special apoptotic process occurring in response to loss of cell adhesion to the extracellular matrix, is a fundamental surveillance process for maintaining tissue homeostasis. Resistance to anoikis characterises cancer cells and is a pre-requisite for metastasis. This study shows that overexpression of the transmembrane mucin protein MUC1 prevents initiation of anoikis in epithelial cancer cells in response to loss of adhesion. We show that this effect is largely attributed to the elongated and heavily glycosylated extracellular domain of MUC1 that protrudes high above the cell membrane and hence prevents activation of the cell surface anoikis-initiating molecules such as integrins and death receptors by providing them a mechanically ‘homing'' microenvironment. As overexpression of MUC1 is a common feature of epithelial cancers and as resistance to anoikis is a hallmark of both oncogenic epithelial–mesenchymal transition and metastasis, MUC1-mediated cell resistance to anoikis may represent one of the fundamental regulatory mechanisms in tumourigenesis and metastasis.Anoikis, the apoptotic process that occurs in cells that have lost adhesion to the extracellular matrix (ECM),^1,2 is a fundamental process for maintaining tissue homeostasis. It removes displaced epithelial/endothelial cells and thus prevents them from seeding to inappropriate sites. Resistance to anoikis contributes prominently to tumourigenesis and, in particular, to metastasis by allowing survival of cancer cells that have invaded into the blood or lymphatic circulation and thus facilitating their metastatic spread to remote sites.³Initiation of anoikis starts from the cell surface through activation of the cell surface anoikis-initiating molecules, for example, integrins, cadherins and death receptors, in response to loss of cell adhesion. Loss of the integrin-mediated cell basement matrix contact,⁴ loss of the E-cadherin-mediated cell–cell contact^5,6 or ligation of the cell surface death receptors with their ligands^4,7 all induce conformational changes or oligomerization of these cell surface anoikis-initiating molecules. This triggers a series of events leading to activation of either the caspase-8-mediated extrinsic apoptotic signalling pathway or the mitochondrion-mediated intrinsic apoptotic signalling pathway.MUC1 is a large transmembrane mucin protein that is expressed exclusively on the apical side of normal epithelial and some other cell types. MUC1 consists of a large extracellular domain, a transmembrane region and a short cytoplasmic tail. The MUC1 extracellular domain contains a variable number of tandem repeats that are heavily glycosylated (up to 50% of the MUC1 molecular weight) with complex O-linked mucin-type glycans⁸ and flanked by a unique N-terminal domain and an SEA domain. In the SEA domain, autocleavage takes place resulting in a heterodimer but both moieties remain firmly attached. The cytoplasmic tail of MUC1 contains 72 amino acids and harbours several phosphorylation sites and is able to interact with various growth factor receptors and intracellular signalling proteins.^{9, 10, 11}MUC1 is overexpressed up to at least 10-fold in epithelial cancers¹² and overexpression of MUC1 is closely associated with high metastatic potential and poor prognosis in many cancer types.¹³ In epithelial cancer cells, MUC1 loses its apical membrane polarization and becomes expressed over the entire cell surface.^14,15 In epithelial cancer cells, MUC1 also shows reduced expression of complex O-glycans and increased expression of short oncofetal oligosaccharides such as GalNAc-α (Tn antigen), sialylated GalNAc-α (sialyl-Tn antigen) and Galβ1,3GalNAc-α (Thomsen–Friedenreich, TF antigen).¹⁶ Immunological targeting of cancer-associated MUC1 has been under intensive investigation as a strategy for cancer treatment.^17,18 Our recent studies have shown that interaction of TF antigen on cancer-associated MUC1 with the galactoside-binding galectins promotes metastasis by enhancing tumour cell heterotypic adhesion to the vascular endothelium and also by increasing tumour cell homotypic aggregation for the potential formation of tumour emboli.^19–21In this report, we describe a new role of MUC1 in anoikis. We show that overexpression of MUC1 in epithelial cells prevents initiation of anoikis in response to loss of cell adhesion, an effect that is found to be attributed substantially to the MUC1 extracellular domain.     

Nuclear factor of activated T cells 2 transactivation in mast cells: a novel isoform-specific transactivation domain confers unique FcepsilonRI responsiveness

Murine nuclear factor of activated T cells (NFAT)2.alpha/beta differ by 42 and 28 unique amino-terminal amino acids and are differentially expressed. Both isoforms share conserved domains that regulate DNA-binding and subcellular localization. A genetic "one-hybrid" assay was used to define two distinct transactivation (TA) domains: in addition to a conserved TAD present in both isoforms, a second, novel TAD exists within the beta-specific amino terminus. Pharmacologic inhibitors G?6976 and rottlerin demonstrate that both conventional and novel protein kinase C (PKC) family members regulate endogenous mast cell NFAT activity, and NFAT2 TA. Overexpression of dominant active PKC (which has been implicated in immune receptor signaling) induces NFAT2.alpha/beta TA. Mutations within the smallest PKC-responsive transactivation domain demonstrate that the PKC effect is at least partially indirect. Significantly, the beta-specific domain confers greater ability to TA in response to treatment with phorbol 12-myristate 13-acetate/ionomycin or lipopolysaccharide, and unique sensitivity to FcepsilonRI signaling. Accordingly, overexpression of NFAT2.beta results in significantly greater NFAT- and interleukin-4 reporter activity than NFAT2.alpha. These results suggest that whereas NFAT2 isoforms may share redundant DNA-binding preferences, there are specialized functional consequences of their isoform-specific domains.     

Reversal of multidrug resistance of vincristine-resistant gastric adenocarcinoma cells through up-regulation of DARPP-32

Here we investigated the roles of DARPP-32 in multidrug resistance (MDR) of gastric cancer cells and the possible underlying mechanisms. We constructed the eukaryotic expression vector of DARPP-32 and transfected it into human vincristine-resistant gastric adenocarcinoma cell line SGC7901/VCR. Up-regulation of DARPP-32 could significantly enhance the sensitivity of SGC7901/VCR cells towards vincristine, adriamycin, 5-fluorouracil and cisplatin, and could decrease the capacity of cells to efflux adriamycin. What's more, the results of subrenal capsule assay confirmed that DARPP-32 might play a certain role in MDR of gastric cancer. DARPP-32 could significantly down-regulate the expression of P-gp and zinc ribbon domain-containing 1 (ZNRD1), but not alter the expression of multidrug resistance-associated protein (MRP) or the glutathione S-transferase (GST). DARPP-32 could also significantly decrease the anti-apoptotic activity of SGC7901/VCR cells. Further study of the biological functions of DARPP-32 might be helpful for understanding the mechanisms of MDR in gastric cancer.     

Ceramide and glucosylceramide upregulate expression of the multidrug resistance gene MDR1 in cancer cells

In the present study we used human breast cancer cell lines to assess the influence of ceramide and glucosylceramide (GC) on expression of MDR1, the multidrug resistance gene that codes for P-glycoprotein (P-gp), because GC has been shown to be a substrate for P-gp. Acute exposure (72 h) to C8-ceramide (5 microg/ml culture medium), a cell-permeable ceramide, increased MDR1 mRNA levels by 3- and 5-fold in T47D and in MDA-MB-435 cells, respectively. Acute exposure of MCF-7 and MDA-MB-231 cells to C8-GC (10 microg/ml culture medium), a cell-permeable analog of GC, increased MDR1 expression by 2- and 4- fold, respectively. Chronic exposure of MDA-MB-231 cells to C8-ceramide for extended periods enhanced MDR1 mRNA levels 45- and 390-fold at passages 12 and 22, respectively, and also elicited expression of P-gp. High-passage C8-ceramide-grown MDA-MB-231 (MDA-MB-231/C8cer) cells were more resistant to doxorubicin and paclitaxel. Incubation with [1-(14)C]C6-ceramide showed that cells converted short-chain ceramide into GC, lactosylceramide, and sphingomyelin. When challenged with 5 mug/ml [1-(14)C]C6-ceramide, MDA-MB-231, MDA-MB-435, MCF-7, and T47D cells took up 31, 17, 21, and 13%, respectively, and converted 82, 58, 62, and 58% of that to short-chain GC. Exposing cells to the GCS inhibitor, ethylenedioxy-P4, a substituted analog of 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, prevented ceramide's enhancement of MDR1 expression. These experiments show that high levels of ceramide and GC enhance expression of the multidrug resistance phenotype in cancer cells. Therefore, ceramide's role as a messenger of cytotoxic response might be linked to the multidrug resistance pathway.     

Selective expression of high molecular weight basic fibroblast growth factor confers a unique phenotype to NIH 3T3 cells.

The phenotypes of NIH 3T3 cells transfected with basic fibroblast growth factor (bFGF) cDNAs that express only the high molecular weight (HMW) forms of bFGF, the 18-kDa form, or all forms were examined. Cells producing the 18 kDa or all forms of bFGF were transformed at high levels of growth factor expression but were nontransformed at low levels. Cell producing low levels of HMW forms of bFGF were growth impaired when compared with the parental cells. These cells tended to form multinucleated giant cells, did not grow in soft agar, were nontumorigenic, had a normal bFGF receptor number, and had a nontransformed morphology. Cells expressing high levels of HMW bFGFs had a transformed morphology and were tumorigenic. These data suggest a specific functional role for HMWbFGF.     

miR-106a confers cisplatin resistance by regulating PTEN/Akt pathway in gastric cancer cells

Recent studies have shown that microRNA-106a （miR-106a） is overexpressed in gastric cancer and contributes to tumor growth. In this study, we investigated whether miR-106a mediated resistance of the gastric cancer cell line SGC7901 to the chemotherapeutic agent cisplatin （DDP）. MiR-106a expression was up-regulated in the DDP resistant cell line SGC7901/DDP compared with its parental line SGC7901. Transfection of miR-106a induced DDP resist- ance in SGC7901, while suppression of miR-106a in SGC7901/DDP led to enhanced DDP cytotoxicity. Further study indicated that the mechanism of miR-106a-induced DDP resistance involved the expression of phosphatase and tensin homolog deleted from chromosome 10 （PTEN） protein and its downstream phosphatidylinositol 3 kinase （Pl3K）/protein kinase B （AKT） pathway. This study provides a novel mechanism of DDP resistance in gastric cancer.