Portal de Periódicos da CAPES

Expression of GnT-III decreases chemoresistance via negatively regulating P-glycoprotein expression: Involvement of the TNFR2-NF-κB signaling pathway

2023; Elsevier BV; Volume: 299; Issue: 4 Linguagem: Inglês

10.1016/j.jbc.2023.103051

1083-351X

Wanli Song, Caixia Liang, Yuhan Sun, Sayaka Morii, Shin Yomogida, Tomoya Isaji, Tomohiko Fukuda, Qinglei Hang, Akiyoshi Hara, Miyako Nakano, Jianguo Gu,

Helicobacter pylori-related gastroenterology studies

The phenomenon of multidrug resistance (MDR) is called chemoresistance with respect to the treatment of cancer, and it continues to be a major challenge. The role of N-glycosylation in chemoresistance, however, remains poorly understood. Here, we established a traditional model for adriamycin resistance in K562 cells, which are also known as K562/adriamycin-resistant (ADR) cells. Lectin blot, mass spectrometry, and RT-PCR analysis showed that the expression levels of N-acetylglucosaminyltransferase III (GnT-III) mRNA and its products, bisected N-glycans, are significantly decreased in K562/ADR cells, compared with the levels in parent K562 cells. By contrast, the expression levels of both P-glycoprotein (P-gp) and its intracellular key regulator, NF-κB signaling, are significantly increased in K562/ADR cells. These upregulations were sufficiently suppressed by the overexpression of GnT-III in K562/ADR cells. We found that the expression of GnT-III consistently decreased chemoresistance for doxorubicin and dasatinib, as well as activation of the NF-κB pathway by tumor necrosis factor (TNF) α, which binds to two structurally distinct glycoproteins, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), on the cell surface. Interestingly, our immunoprecipitation analysis revealed that only TNFR2, but not TNFR1, contains bisected N-glycans. The lack of GnT-III strongly induced TNFR2's autotrimerization without ligand stimulation, which was rescued by the overexpression of GnT-III in K562/ADR cells. Furthermore, the deficiency of TNFR2 suppressed P-gp expression while it increased GnT-III expression. Taken together, these results clearly show that GnT-III negatively regulates chemoresistance via the suppression of P-gp expression, which is regulated by the TNFR2-NF/κB signaling pathway. The phenomenon of multidrug resistance (MDR) is called chemoresistance with respect to the treatment of cancer, and it continues to be a major challenge. The role of N-glycosylation in chemoresistance, however, remains poorly understood. Here, we established a traditional model for adriamycin resistance in K562 cells, which are also known as K562/adriamycin-resistant (ADR) cells. Lectin blot, mass spectrometry, and RT-PCR analysis showed that the expression levels of N-acetylglucosaminyltransferase III (GnT-III) mRNA and its products, bisected N-glycans, are significantly decreased in K562/ADR cells, compared with the levels in parent K562 cells. By contrast, the expression levels of both P-glycoprotein (P-gp) and its intracellular key regulator, NF-κB signaling, are significantly increased in K562/ADR cells. These upregulations were sufficiently suppressed by the overexpression of GnT-III in K562/ADR cells. We found that the expression of GnT-III consistently decreased chemoresistance for doxorubicin and dasatinib, as well as activation of the NF-κB pathway by tumor necrosis factor (TNF) α, which binds to two structurally distinct glycoproteins, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), on the cell surface. Interestingly, our immunoprecipitation analysis revealed that only TNFR2, but not TNFR1, contains bisected N-glycans. The lack of GnT-III strongly induced TNFR2's autotrimerization without ligand stimulation, which was rescued by the overexpression of GnT-III in K562/ADR cells. Furthermore, the deficiency of TNFR2 suppressed P-gp expression while it increased GnT-III expression. Taken together, these results clearly show that GnT-III negatively regulates chemoresistance via the suppression of P-gp expression, which is regulated by the TNFR2-NF/κB signaling pathway. Chronic myeloid leukemia (CML) is a myeloproliferative malignancy that arises from hematopoietic stem cells (1Fialkow P.J. Jacobson R.J. Papayannopoulou T. Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocyte/macrophage.Am. J. Med. 1977; 63: 125-130Abstract Full Text PDF PubMed Scopus (684) Google Scholar), and it accounts for 15% of adult leukemias (2Jabbour E. Kantarjian H. Chronic myeloid leukemia: 2012 update on diagnosis, monitoring, and management.Am. J. Hematol. 2012; 87: 1037-1045Crossref PubMed Scopus (112) Google Scholar). CML classically progresses through three phases (chronic phase, accelerated phase, and blast phase) and becomes more resistant to treatment in each successive phase (3Kalidas M. Kantarjian H. Talpaz M. Chronic myelogenous leukemia.JAMA. 2001; 286: 895-898Crossref PubMed Scopus (63) Google Scholar). Imatinib is a tyrosine kinase inhibitor of BCR-ABL1 that has become one of the most successful drugs yet approved for therapeutic use (4Braun T.P. Eide C.A. Druker B.J. Response and resistance to BCR-ABL1-targeted therapies.Cancer Cell. 2020; 37: 530-542Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 5Deininger M. Curing CML with imatinib—a dream come true?.Nat. Rev. Clin. Oncol. 2011; 8: 127-128Crossref PubMed Scopus (29) Google Scholar). Its broad application, however, has spurred debate regarding the efficacy of both imatinib and later generations of tyrosine kinase inhibitor, and this opposition is due mainly to the acquisition of drug resistance (6Apperley J.F. Part I: mechanisms of resistance to imatinib in chronic myeloid leukaemia.Lancet Oncol. 2007; 8: 1018-1029Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar). Therefore, overcoming multidrug resistance (MDR) is a big challenge for the successful treatment of CML or other cancers. The emergence of the MDR phenotypes is primarily involved in the overexpression of MDR-associated genes such as ATP-binding cassette (ABC) subfamily B1 genes (P-gp/MDR1/ABCB1), ABCC1 (MRP1), and ABCG2 (BCRP1). Classic MDR is caused by the overexpression of P-glycoprotein (P-gp) (7Stege A. Priebsch A. Nieth C. Lage H. Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference.Cancer Gene Ther. 2004; 11: 699-706Crossref PubMed Scopus (80) Google Scholar). P-gp is the first efflux pump to be discovered that reduces the cellular toxicities of chemotherapeutic agents by lowering the intracellular drug accumulation of taxanes (paclitaxel), vinca alkaloids (vinblastine), and anthracyclines (daunorubicin) (8Kuwazuru Y. Yoshimura A. Hanada S. Ichikawa M. Saito T. Uozumi K. et al.Expression of the multidrug transporter, P-glycoprotein, in chronic myelogenous leukaemia cells in blast crisis.Br. J. Haematol. 1990; 74: 24-29Crossref PubMed Scopus (81) Google Scholar, 9Herweijer H. Sonneveld P. Baas F. Nooter K. Expression of mdr1 and mdr3 multidrug-resistance genes in human acute and chronic leukemias and association with stimulation of drug accumulation by cyclosporine.J. Natl. Cancer Inst. 1990; 82: 1133-1140Crossref PubMed Scopus (199) Google Scholar, 10Sharom F.J. ABC multidrug transporters: structure, function and role in chemoresistance.Pharmacogenomics. 2008; 9: 105-127Crossref PubMed Scopus (810) Google Scholar). Thus, to reverse MDR by inhibiting P-gp should re-sensitize tumor cells to antineoplastic agents and allow the successful treatment of multidrug-resistant cancer cells. Promoter analyses has revealed that the human P-gp gene promoter harbors NF-κB responsive elements, which bind with NF-κB to promote P-gp gene expression (11Ogretmen B. Safa A.R. Negative regulation of MDR1 promoter activity in MCF-7, but not in multidrug resistant MCF-7/Adr, cells by cross-coupled NF-κB/p65 and c-Fos transcription factors and their interaction with the CAAT region.Biochemistry. 1999; 38: 2189-2199Crossref PubMed Scopus (89) Google Scholar, 12Bentires-Alj M. Barbu V. Fillet M. Chariot A. Relic B. Jacobs N. et al.NF-κB transcription factor induces drug resistance through MDR1 expression in cancer cells.Oncogene. 2003; 22: 90-97Crossref PubMed Scopus (406) Google Scholar) but that inhibition of the NF-κB signaling pathway decreases P-gp expression, which increases apoptotic cell death in response to daunomycin treatment in human colorectal adenocarcinoma cells (12Bentires-Alj M. Barbu V. Fillet M. Chariot A. Relic B. Jacobs N. et al.NF-κB transcription factor induces drug resistance through MDR1 expression in cancer cells.Oncogene. 2003; 22: 90-97Crossref PubMed Scopus (406) Google Scholar). Tumor necrosis factor α (TNFα) is one of the most potent activators of NF-κB signaling. Several studies have shown that the TNFα/NF-κB signaling pathway mediates P-gp upregulation in both tumor and normal cells (13Hartmann G. Kim H. Piquette-Miller M. Regulation of the hepatic multidrug resistance gene expression by endotoxin and inflammatory cytokines in mice.Int. Immunopharmacol. 2001; 1: 189-199Crossref PubMed Scopus (135) Google Scholar, 14Bauer B. Hartz A.M.S. Miller D.S. Tumor necrosis factor α and endothelin-1 increase P-glycoprotein expression and transport activity at the blood-brain barrier.Mol. Pharmacol. 2007; 71: 667-675Crossref PubMed Scopus (244) Google Scholar, 15Heemskerk S. Peters J.G.P. Louisse J. Sagar S. Russel F.G.M. Masereeuw R. Regulation of P-glycoprotein in renal proximal tubule epithelial cells by LPS and TNF-α.J. Biomed. Biotechnol. 2010; 2010: 525180Crossref PubMed Scopus (31) Google Scholar, 16Wang X. Huang S. Jiang Y. Liu Y. Song T. Li D. et al.Reactive astrocytes increase the expression of P-gp and Mrp1 via TNF-α and NF-κB signaling.Mol. Med. Rep. 2018; 17: 1198-1204PubMed Google Scholar, 17Berguetti T. Quintaes L.S.P. Hancio T. Robaina M.C. Cruz A.L.S. Maia R.C. et al.TNF-α modulates P-glycoprotein expression and contributes to cellular proliferation via extracellular vesicles.Cells. 2019; 8: 500Crossref PubMed Scopus (12) Google Scholar, 18Thévenod F. Friedmann J.M. Katsen A.D. Hauser I.A. Up-regulation of multidrug resistance P-glycoprotein via nuclear factor-κB activation protects kidney proximal tubule cells from cadmium- and reactive oxygen species-induced apoptosis.J. Biol. Chem. 2000; 275: 1887-1896Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). TNFα interacts with cells through specific receptors, TNF receptor 1 (TNFR1) or TNFR2, and triggers the downstream NF-κB pathway to regulate the expression of MDR (17Berguetti T. Quintaes L.S.P. Hancio T. Robaina M.C. Cruz A.L.S. Maia R.C. et al.TNF-α modulates P-glycoprotein expression and contributes to cellular proliferation via extracellular vesicles.Cells. 2019; 8: 500Crossref PubMed Scopus (12) Google Scholar, 19Gasparini C. Celeghini C. Monasta L. Zauli G. NF-κB pathways in hematological malignancies.Cell. Mol. Life Sci. 2014; 71: 2083-2102Crossref PubMed Scopus (130) Google Scholar). TNFR1 can be modified by N-glycosylation (20Han L. Zhang D. Tao T. Sun X. Liu X. Zhu G. et al.The role of N-Glycan modification of TNFR1 in inflammatory microglia activation.Glycoconj. J. 2015; 32: 685-693Crossref PubMed Scopus (28) Google Scholar, 21Holdbrooks A.T. Britain C.M. Bellis S.L. ST6Gal-I sialyltransferase promotes tumor necrosis factor (TNF)-mediated cancer cell survival via sialylation of the TNF receptor 1 (TNFR1) death receptor.J. Biol. Chem. 2018; 293: 1610-1622Abstract Full Text Full Text PDF PubMed Google Scholar). When TNFR1 lacks N-glycosylation, its binding to TNFα is suppressed, which thereby represses TNFα-mediated NF-κB signaling (20Han L. Zhang D. Tao T. Sun X. Liu X. Zhu G. et al.The role of N-Glycan modification of TNFR1 in inflammatory microglia activation.Glycoconj. J. 2015; 32: 685-693Crossref PubMed Scopus (28) Google Scholar). N-Glycosylation is one of the most common forms of protein postmodification and is an important process in cells (22Reily C. Stewart T.J. Renfrow M.B. Novak J. Glycosylation in health and disease.Nat. Rev. Nephrol. 2019; 15: 346-366Crossref PubMed Scopus (849) Google Scholar, 23Ohtsubo K. Marth J.D. Glycosylation in cellular mechanisms of health and disease.Cell. 2006; 126: 855-867Abstract Full Text Full Text PDF PubMed Scopus (2182) Google Scholar). Aberrant N-glycosylation has been associated with cancer chemoresistance (24Ferreira J.A. Peixoto A. Neves M. Gaiteiro C. Reis C.A. Assaraf Y.G. et al.Mechanisms of cisplatin resistance and targeting of cancer stem cells: adding glycosylation to the equation.Drug Resist. Updat. 2016; 24: 34-54Crossref PubMed Scopus (113) Google Scholar, 25Zhang Z. Zhao Y. Jiang L. Miao X. Zhou H. Jia L. Glycomic alterations are associated with multidrug resistance in human leukemia.Int. J. Biochem. Cell Biol. 2012; 44: 1244-1253Crossref PubMed Scopus (36) Google Scholar, 26Li K. Sun Z. Zheng J. Lu Y. Bian Y. Ye M. et al.In-depth research of multidrug resistance related cell surface glycoproteome in gastric cancer.J. Proteomics. 2013; 82: 130-140Crossref PubMed Scopus (25) Google Scholar, 27Zeng W. Zheng S. Mao Y. Wang S. Zhong Y. Cao W. et al.Elevated N-glycosylation contributes to the cisplatin resistance of non-small cell lung cancer cells revealed by membrane proteomic and glycoproteomic analysis.Front. Pharmacol. 2021; 12: 805499Crossref PubMed Scopus (10) Google Scholar). For example, elevating sialylated N-glycans or high mannose decreases the sensitivity of nonsmall lung cancer cells to cisplatin and contributes to the development of chemoresistance phenotypes (27Zeng W. Zheng S. Mao Y. Wang S. Zhong Y. Cao W. et al.Elevated N-glycosylation contributes to the cisplatin resistance of non-small cell lung cancer cells revealed by membrane proteomic and glycoproteomic analysis.Front. Pharmacol. 2021; 12: 805499Crossref PubMed Scopus (10) Google Scholar). The overexpression of fucosyltransferase 4 (FUT4), FUT6, or FUT8 is responsible for MDR in human hepatocellular carcinoma via activation of the PI3K/Akt signaling pathway, which induces P-gp expression (28Cheng L. Luo S. Jin C. Ma H. Zhou H. Jia L. FUT family mediates the multidrug resistance of human hepatocellular carcinoma via the PI3K/Akt signaling pathway.Cell Death Dis. 2013; 4e923Crossref Scopus (85) Google Scholar). The products of N-acetylglucosaminyltransferase V (GnT-V encoded by the Mgat5 gene), β1,6GlcNAc-branched N-glycans, have been associated with cancer growth, invasion, metastasis (29Kizuka Y. Taniguchi N. Enzymes for N-glycan branching and their genetic and nongenetic regulation in cancer.Biomolecules. 2016; 6: 25Crossref PubMed Scopus (99) Google Scholar), and drug resistance (30Kudo T. Nakagawa H. Takahashi M. Hamaguchi J. Kamiyama N. Yokoo H. et al.N-glycan alterations are associated with drug resistance in human hepatocellular carcinoma.Mol. Cancer. 2007; 6: 32Crossref PubMed Scopus (65) Google Scholar, 31Guo R. Cheng L. Zhao Y. Zhang J. Liu C. Zhou H. et al.Glycogenes mediate the invasive properties and chemosensitivity of human hepatocarcinoma cells.Int. J. Biochem. Cell Biol. 2013; 45: 347-358Crossref PubMed Scopus (17) Google Scholar). The expression levels of GnT-V are known to be higher in the adriamycin-resistant (ADR) MCF-7 cells than in parental cells, and a knockdown of GnT-V has decreased the resistance to paclitaxel, doxorubicin (DOX), and vincristine (32Ma H. Miao X. Ma Q. Zheng W. Zhou H. Jia L. Functional roles of glycogene and N-glycan in multidrug resistance of human breast cancer cells.IUBMB Life. 2013; 65: 409-422Crossref PubMed Scopus (38) Google Scholar). The expression levels of N-acetylglucosaminyltransferase IV (GnT-IV encoded by the Mgat4 gene), which is involved in β1,4GlcNAc-branched N-glycans, are also induced in the cisplatin-resistant variants of A2780 cells, compared with wildtype (WT) cells (33Lin G. Zhao R. Wang Y. Han J. Gu Y. Pan Y. et al.Dynamic analysis of N-glycomic and transcriptomic changes in the development of ovarian cancer cell line A2780 to its three cisplatin-resistant variants.Ann. Transl. Med. 2020; 8: 289Crossref PubMed Google Scholar). N-Acetylglucosaminyltransferase III (GnT-III encoded by the Mgat3 gene) is another important glycosyltransferase that has received much recent attention for its involvement in the biology of tumors (34Allam H. Johnson B.P. Zhang M. Lu Z. Cannon M.J. Abbott K.L. The glycosyltransferase GnT-III activates notch signaling and drives stem cell expansion to promote the growth and invasion of ovarian cancer.J. Biol. Chem. 2017; 292: 16351-16359Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 35Taniguchi N. Ohkawa Y. Maeda K. Harada Y. Nagae M. Kizuka Y. et al.True significance of N-acetylglucosaminyltransferases GnT-III, V and α1,6 fucosyltransferase in epithelial-mesenchymal transition and cancer.Mol. aspects Med. 2020; 79: 100905Crossref PubMed Scopus (23) Google Scholar). GnT-III plays a critical role in defining the ultimate structure of hybrid and complex N-glycans as the addition of a bisecting GlcNAc suppresses further processing and elongation of N-glycans to form branching structures that are catalyzed by GnT-IV and GnT-V, because they are not able to use the bisected oligosaccharide as a substrate (36Schachter H. Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides.Biochem. Cell Biol. 1986; 64: 163-181Crossref PubMed Scopus (493) Google Scholar). Thus, GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways (37Isaji T. Kariya Y. Xu Q. Fukuda T. Taniguchi N. Gu J. Chapter twenty - functional roles of the bisecting GlcNAc in integrin-mediated cell adhesion.in: Fukuda M. Methods in Enzymology. , Academic Press, London2010: 445-459Google Scholar). Previous studies have shown that the expression levels of GnT-III are decreased in several drug-resistant cell lines (38Zhao R. Qin W. Qin R. Han J. Li C. Wang Y. et al.Lectin array and glycogene expression analyses of ovarian cancer cell line A2780 and its cisplatin-resistant derivate cell line A2780-cp.Clin. Proteomics. 2017; 14: 20Crossref PubMed Scopus (22) Google Scholar, 39da Fonseca L.M. Calvalhan D.M. Previato J.O. Mendonça Previato L. Freire-de-Lima L. Resistance to paclitaxel induces glycophenotype changes and mesenchymal-to-epithelial transition activation in the human prostate cancer cell line PC-3.Tumour Biol. 2020; 421010428320957506Crossref PubMed Scopus (8) Google Scholar). Moreover, a knockdown of GnT-III has increased the resistance to 5-fluorouracil in human hepatocarcinoma cells (31Guo R. Cheng L. Zhao Y. Zhang J. Liu C. Zhou H. et al.Glycogenes mediate the invasive properties and chemosensitivity of human hepatocarcinoma cells.Int. J. Biochem. Cell Biol. 2013; 45: 347-358Crossref PubMed Scopus (17) Google Scholar). Taken together, these studies strongly suggest that glycosylation may play a potential role in chemoresistance as cancer cells acquire tolerance. However, only a few studies have investigated the underlying mechanisms between chemoresistance and N-glycans. In the present study, we established a traditional model of a human CML cell line, K562/ADR cells, and found that the expression levels of GnT-III and bisected N-glycans were significantly decreased in these cells, compared with the parental K562 cells. Furthermore, our studies have clearly shown that TNFR2 is modified when the GlcNAc of N-glycans are bisected, which negatively regulates trimerization of the TNFR2 and TNFα/NF-κB signaling pathways for P-gp expression and contributes to cancer chemoresistance. Elevated GnT-III expression and its products, bisected GlcNAc N-glycans, have been detected in patients with CML during blast crisis when compared with healthy controls or with patients with other hematological malignancies (40Yoshimura M. Ihara Y. Taniguchi N. Changes of beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) in patients with leukaemia.Glycoconj. J. 1995; 12: 234-240Crossref PubMed Scopus (22) Google Scholar, 41Yoshimura M. Nishikawa A. Ihara Y. Nishiura T. Nakao H. Kanayama Y. et al.High expression of UDP-N-acetylglucosamine: beta-D mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) in chronic myelogenous leukemia in blast crisis.Int. J. Cancer. 1995; 60: 443-449Crossref PubMed Scopus (29) Google Scholar). The K562/ADR cell is a traditional model for studying drug resistance in CML cells. First, we established the K562/ADR cell line using a stepwise increase in DOX concentrations, as described in the Experimental procedure section. As expected, the expression levels of P-gp were greatly increased in the K562/ADR cells compared with parent K562 cells (Fig. 1A). To compare the glycan expression patterns of K562 and K562/ADR cells, we performed lectin blotting using four different lectins: E4-PHA, ConA, SNA, DSA, and L4-PHA. These lectins preferentially recognize the bisected N-glycans catalyzed by GnT-III, α-linked mannose including high mannose–type and hybrid-type N-glycans, α2,6 sialylation on a terminal branch, β1,4-GlcNAc-branched, and β1,6-GlcNAc-branched (tri-antennary) N-glycans, respectively. The reactivities of E4-PHA were dramatically decreased in K562/ADR cells compared with WT K562 cells, as validated via lectin blot (Fig. 1B) and flow cytometry (Fig. 1G). Further lectin blotting with ConA (Fig. 1C), SNA (Fig. 1D), DSA (Fig. 1E), or L4-PHA (Fig. 1F) showed no dramatic changes between the cells. It is worth noting that both DSA and L4-PHA lectin blots tended to decrease in K562/ADR cells compared with that in K562 cells, which seems to be contradictory to previous notion since reciprocal activity is known to exist between GnT-III and GnT-V or GnT-IV (36Schachter H. Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides.Biochem. Cell Biol. 1986; 64: 163-181Crossref PubMed Scopus (493) Google Scholar, 37Isaji T. Kariya Y. Xu Q. Fukuda T. Taniguchi N. Gu J. Chapter twenty - functional roles of the bisecting GlcNAc in integrin-mediated cell adhesion.in: Fukuda M. Methods in Enzymology. , Academic Press, London2010: 445-459Google Scholar). Thus far, it is difficult to explain the phenomena, which requires further study. Consistently, the results obtained from RT-PCR showed that the expression levels of GnT-III, as opposed to the levels indicated by other GnT-Ts, were decreased in K562/ADR cells compared with the levels in WT K562 cells (Fig. 1H). Furthermore, to confirm the changes in the N-glycans shown in Figure 1, we isolated N-glycans from cell membrane proteins and desialylated them. The glycans were then labeled with aminoxy Tandem Mass Tag sixplex (TMT6) and analyzed via LC-ESI MS. Retention time, observed/theoretical mass, charge state, intensity, and composition of deduced structure of all observed N-glycans were summarized in the result file where have been deposited in GlycoPOST. As shown in Figure 2, N-glycan structures with bisecting GlcNAc were greatly reduced in K562/ADR cells, compared with the K562 cells, which is consistent with the results shown by lectin blots. These data strongly suggest that the expression levels of the bisected N-glycans are downregulated in the acquisition of drug resistance of leukemia cells. To understand the impact that the downregulation of GnT-III exerts on the drug resistance of K562/ADR cells, we carried out a transfection approach to the overexpression of GnT-III in both K562 and K562/ADR cells via viral infection encapsulating the CSIV-TRE-RfA-CMV-KT-GnT-III plasmid and used FACSAria II to establish stable K562-GnT-III and K562/ADR-GnT-III cell lines after sorting. The expression levels of GnT-III were clearly increased in K562-GnT-III and K562/ADR-GnT-III cells, compared with control cells (Fig. 3A). The expression levels of endogenous GnT-III were very low, and only a faint band was detected in K562 cells, but none was detected in the K562/ADR cells. Lectin blotting also showed that the reactive abilities with E4-PHA were significantly enhanced in the K562-GnT-III and K562/ADR-GnT-III cells, which further indicated that GnT-III was successfully overexpressed in these two cells (Fig. 3B). The resistance of cancer cells to anticancer drugs is usually due to the enhanced efflux of drugs by ABC transporters such as ABCB1 (P-gp) and others, as shown in Figure 3C. RT-PCR analysis has shown that the expression levels of P-gp, but not the other two subfamilies of the ABC transporters ABCC1 and ABCG2, were greatly enhanced in K562/ADR cells compared with that in K562 cells. It is noteworthy that the overexpression of GnT-III in K562/ADR cells substantially suppressed the expression levels of P-gp, which was also observed in the K562-GnT-III cells when compared with control cells (Fig. 3C). This tendency was also confirmed via Western blotting with anti-P-gp antibody (Fig. 3D). These results suggest that GnT-III negatively regulates P-gp expression. To further investigate the effects of GnT-III on drug efflux, a drug retention experiment was performed. Equal amounts of fluorochromes were added to indicated cells followed by incubation for 1 h, and then the intracellular accumulation of fluorescence was visualized via confocal microscopy (Fig. 4A). Clearly, a higher amount of fluorescence remained in both the K562 and K562/ADR-GnT-III cells, compared with that in K562/ADR cells. Further, the fluorescence intensities were quantified by analysis using a fluorescence plate reader. This result showed that the fluorescence intensity in K562/ADR cells was significantly lower than that in K562 cells and that the overexpression of GnT-III rescued fluorescence accumulation (Fig. 4B). In addition, the cell viabilities were examined by exposure to DOX for 4 days (Fig. 4C) and to dasatinib (Fig. 4D) for 3 days at indicated concentrations. Overexpression of GnT-III significantly decreased the chemoresistance in K562/ADR-GnT-III cells, compared with that in K562/ADR cells. Taken together, these results suggest that GnT-III negatively regulates chemoresistance by reducing P-gp expression. Next, we investigated the underlying molecular mechanisms by which GnT-III regulates P-gp expression and reverses MDR. Several studies have reported that the expression of P-gp could be regulated by the NF-κB pathway (12Bentires-Alj M. Barbu V. Fillet M. Chariot A. Relic B. Jacobs N. et al.NF-κB transcription factor induces drug resistance through MDR1 expression in cancer cells.Oncogene. 2003; 22: 90-97Crossref PubMed Scopus (406) Google Scholar). Thus, to understand whether the NF-κB pathway was involved in the regulation of P-gp in cells, we first examined cellular signaling in the NF-κB pathway. This signaling was accomplished via phospho-p65 (p-p65), active signaling, and IkBα, which is an inhibitor protein for NF-κB. As shown in Figure 5A, the p-p65 levels were obviously increased in K562/ADR cells compared with that in K562 cells, which was attenuated after the overexpression of GnT-III in these cells. By contrast, the expression patterns of IkBα were opposite that of p-p65 (Fig. 5B). Then, we examined the effects of NF-κB signaling on P-gp expression in the present model. As expected, the expression levels of P-gp and p-p65/p65 ratios were significantly suppressed in the K562/ADR cells, while the levels of IκBα were increased when using the NF-κB inhibitor BAY 11-7082 to inhibit the ubiquitination of IκBα in a concentration-dependent manner (Fig. 5C). It is well known that proinflammatory stimuli, such as TNFα can activate NF-κB signaling (42Vallabhapurapu S. Karin M. Regulation and function of NF-kappaB transcription factors in the immune system.Annu. Rev. Immunol. 2009; 27: 693-733Crossref PubMed Scopus (2127) Google Scholar). Moreover, the TNFα/NF-κB signaling pathway mediates the expression of P-gp. Then, we investigated the effects that TNFα-mediated signaling exerts on the induction of P-gp. TNFα stimulation greatly upregulated P-gp expression, which was substantially suppressed by the overexpression of GnT-III analyzed via RT-PCR (Fig. 5D) and Western blot (Fig. 5E). It is unclear why the P-gp protein in the K562 cells remained at less than detectable levels, even during TNFα stimulation. We suspect the existence of different mechanisms that could be involved with P-gp stability and/or proteasomal degradation pathways or the existence of other related factors in K562 cells and K562/ADR cells. The exact mechanism will require further study in the future. However, these results still indicate that the expression of P-gp was upregulated after stimulation by TNFα and activation of the NF-κB pathway and that GnT-III negatively regulates MDR mainly via the TNFα/NF-κB pathway. TNFα exerts its signal transduction by stimulating receptors TNFR1 and TNFR2, which contain three and two potential N-glycosylation sites, respectively. At this point, in order to understand how the expression of GnT-III could regulate P-gp expression via the TNFα/NF-κB pathway, we initially investigated whether TNFR1 and TNFR2 could be modified by GnT-III. As described in the Methods section, we constructed human pcDNA3.1-RfA-TNFR1 and pcDNA3.1-RfA-TNFR2 plasmids containing a Flag tag. After transient transfection in 293T cells, cell lysates were digested with peptide N-glycosidase F (PNGase F), which removes all forms of N-glycan. The results of Western blotting with anti-Flag antibody showed that both TNFR1 and TNFR2 have two bands, which were shifted after treatment with PNGase F, which suggests that both were modified by N-glycans (Fig. 6A). Surprisingly, the results of lectin blotting with E4-PHA, which preferentially recognizes bisected N-glycans, showed that TNFR2, but not TNFR1, was modified by GnT-III (Fig. 6B). To further confirm this observation, cell lysates were immunoprecipitated with E4-PHA-agarose, and then, the immunoprecipitates were western blotted with anti-Flag antibody. It was possible to consistently detect only TNFR2 (Fig. 6C). Furthermore, to confirm whether the specific modification also occurred in K562 cells, both K562 and K562/ADR cells were transiently transfected with pcDNA3.1-RfA-TNFR1 and pcDNA3.1-RfA-TNFR2 plasmids. And then, cell lysates were immunoprecipitate