Investigation of Calnexn Protein Expression in Imatinib Sensitive and Resistant K562 Cells

Arzu Zeynep Karabay

Research Article

İmatinib’e Duyarlı ve Dirençli K562 Hücrelerinde Kalneksinin Protein Ekspresyonunun Araștırılması

Year 2018, Volume: 71 Issue: 1, 9 - 15, 16.10.2018

Arzu Zeynep Karabay

Abstract

Amaç: Bu çalıșmada, çok sayıda kan kök hücresinin, sağlıklı beyaz kan hücrelerine dönüșemeyen anormal granülositlere dönüștüğü bir hastalık olan kronik myeloid löseminin hücre serisi modeli olan K562 hücrelerinde (K562S) ve KML tedavisinde ilk seçenek olarak uygulanan İmatinib’e karșı direnç geliștirmiș K562 hücrelerinde (K562R), endoplazmik retikulum (ER) șaperon proteini kalneksinin protein düzeylerinin belirlenmesi ve KML tedavisinde kullanılan birinci ve ikinci jenerasyon tirozin kinaz inhibitörleri İmatinib ve Nilotinib’in kalneksin protein düzeyi üzerindeki etkilerinin ve hücre canlılığı parametrelerine olan etkilerinin ortaya çıkarılması amaçlanmıștır.

Gereç ve Yöntem: Bu çalıșmada, KML’nin blast fazının hücre modeli olan K562S (İmatinib’e duyarlı) hücre serisi ve İmatinib’e (5uM) karșı dirençli hale getirilmiș K562R hücre serileri kullanılmıștır. Hücreler, paralel olarak kültür edilmișlerdir. 0.5 uM İmatinib ve 0.05 uM Nilotinib ile tedavi edilen ve edilmeyen K562S ve 20 uM İmatinib ve 0.1 uM Nilotinib K562R ile tedavi edilen ve edilmeyen K562R hücreleri tedaviden 48 saat sonra toplanarak MTT testi ile hücre canlılığı, akım sitometri ile apoptoz tayini, ıșık mikroskobisi ile hücre morfolojisi ve total hücre ekstraktlarında western blot ile kalneksin protein ekspresyonu belirlenmiștir.

Bulgular ve sonuç: Bu çalıșmada, endoplazmik retikulumda proteinlerin katlanmasını düzenleyen bir șaperon olan kalneksinin total hücre ekspresyonunun K562S ve K562R hücreleri arasında anlamlı değișiklik göstermediği belirlenmiștir, ancak, İmatinib ve Nilotinib’in duyarlı hücrelerde bu proteinin düzeyini anlamlı olarak düșürmesi ve dirençli hücrelerde anlamlı etki göstermemesi, bu ilaçların etki mekanizması arasında ER stres yolağı ve olasılıkla kalneksin proteininin yer aldığına ișaret edebilir ve duyarlı ve dirençli hücreler arasında her ne kadar kalneksin düzeyinde fark bulunmasa da ER stres yolağında farklılıklar bulunabileceğinin bir göstergesi olabilir. İleri çalıșmalar için, ilaca duyarlı ve dirençli hücrelerde kalneksin ve ilișkili bileșenlerin farklı hücre içi kompartmanlardaki değișiminin ve ilaçlarla modülasyonunun daha geniș olarak araștırılması önerilmektedir.

Keywords

Kalneksin, K562, lösemi, ilaç direnci

Ethical Statement

Supporting Institution

Project Number

Thanks

References

1) Braakman I, Hebert DN. Protein folding in the endoplasmic reticulum. Cold Spring Harb Perspect Biol. 2013; 5:a013201.
2) Schwarz DS, Blower MD. The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci. 2016; 73: 79-94.
3) Hamdan N, Kritsiligkou P, Grant CM. ERstress causes widespread protein aggregation and prion formation. J Cell Biol. 2017; 216: 2295-2304.
4) Alasiri G, Fan LY, Zona S, et al. ER stress and cancer: The FOXO forkhead transcription factor link. Mol Cell Endocrinol. 2018; 462(Pt B):67-81.
5) Gifford JB, Hill R. GRP78 Influences Chemoresistance and Prognosis in Cancer. Curr Drug Targets. 2017. doi: 10.2174/1389450118666170615100918.
6) Phelps EA, Cianciaruso C, Michael IP, et al. Aberrant Accumulation of the Diabetes Autoantigen GAD65 in Golgi Membranes in Conditions of ER Stress and Autoimmunity. Diabetes. 2016; 65: 2686-99.
7) Erpapazoglou Z, Mouton-Liger F, Corti O. From dysfunctional endoplasmic reticulum- mitochondria coupling to neurodegeneration. Neurochem Int. 2017; 109:171-183.
8) Garfinkel BP, Hotamisligil GS. ER Stress Promotes Inflammation through RewIREd Macrophages in Obesity. Mol Cell. 2017; 66:731-733.
9) Hebert DN, Molinari M. In and out of the ER: protein folding, quality control, degradation, and related human diseases. Physiol Rev. 2007; 87:1377-408.
10) Schrag JD, Bergeron JJ, Li Y, et al. The Structure of calnexin, an ER chaperone involved in quality control of protein folding. Mol Cell. 2001;3: 633-44.
11) Xie W, Nielsen ME, Pedersen C, et al. A Split-GFP Gateway Cloning System for Topology Analyses of Membrane Proteins in Plants. PLoS One. 2017; 13:e0170118.
12) Y. Shibata, T. Shemesh, W.A. Prinz, et al. Mechanisms determining the morphology of the peripheral ER Cell, 2010; 143:774- 788.
13) N. Myhill, E.M. Lynes, J.A. Nanji, et al. The subcellular distribution of calnexin is mediated by PACS-2 Mol. Biol. Cell, 2008; 19: 2777-2788.
14) Ryan D, Carberry S, Murphy ÁC, et al. Calnexin, an ER-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer. J Transl Med. 2016 ;14:196.
15) Uyy E, Suica VI, Boteanu RM, et al. Endoplasmic Reticulum Chaperones Are Potential Active Factors in Thyroid Tumorigenesis. J Proteome Res. 2016; 15:3377-87.
16) Kobayashi M, Nagashio R, Jiang SX, et al. Calnexin is a novel sero-diagnostic marker for lung cancer. Lung Cancer. 2015; 90:342-5.
17) Pavli M, Farmaki E, Merkourea S, et al. Endoplasmic Reticulum Stress-Associated Chaperones, Bip/GRP78 and Calnexin are Overexpressed in Keratocystic Odontogenic Tumours. J Oral Maxillofac Res. 2014; 5:e3
18) Seres M, Poláková E, Krizanová O, et al. Overexpression of P-glycoprotein in L1210/VCR cells is associated with changes in several endoplasmic reticulum proteins that may be partially responsible for the lack of thapsigargin sensitivity. Gen Physiol Biophys. 2008; 27:211-21.
19) Sereš M, Ditte P, Breier A, et al. Effect of thapsigargin on P-glycoprotein-negative and P-glycoprotein-positive L1210 mouse leukaemia cells. Gen Physiol Biophys. 2010; 29:396-401.
20) Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999; 17: 1330-40.
21) Frame D Chronic myeloid leukemia: standard treatment options. Am J Health Syst Pharm. 2006; 8:21-22.
22) Valent P. Imatinib-resistant chronic myeloid leukemia (CML): Current concepts on pathogenesis and new emerging pharmacologic approaches. Biologics. 2007; 4: 433-48.
23) Xia Y, Fang H, Zhang J, et al. Endoplasmic reticulum stress-mediated apoptosis in imatinib-resistant leukemic K562-r cells triggered by AMN107 combined with arsenic trioxide. Exp Biol Med (Maywood).2013; 238: 932-42
24) Kusio-Kobialka M, Podszywalow-Bartnicka P, Peidis P, et al. The PERK-eIF2α phosphorylation arm is a pro-survival pathway of BCR-ABL signaling and confers resistance to imatinib treatment in chronic myeloid leukemia cells. Cell Cycle. 2012; 21: 4069-78.
25) Salaroglio IC, Panada E, Moiso E, et al. PERK induces resistance to cell death elicited by endoplasmic reticulum stress and chemotherapy.
26) Chevet E, Hetz C, Samali A. Endoplasmic reticulum stress-activated cell reprogramming in oncogenesis. Cancer Discov. 2015;5: 586-97.
27) Shah PP, Dupre TV, Siskind LJ, et al. Common cytotoxic chemotherapeutics induce epithelial-mesenchymal transition (EMT) downstream of ER stress. Oncotarget. 2017;14: 22625-22639.
28) Podszywalow-Bartnicka P, Cmoch A, Wolczyk M, et al. Increased phosphorylation of eIF2α in chronic myeloid leukemia cells stimulates secretion of matrix modifying enzymes. Oncotarget. 2016; 48:79706-79721.
29) Ekiz HA, Can G, Gunduz U, et al. Nilotinib significantly induces apoptosis in imatinib-resistant K562 cells with wild-type BCR-ABL, as effectively as in parental sensitive counterparts. Hematology. 2010; 15:33-8.
30) Delom F, Emadali A, Cocolakis E, et al. Calnexin-dependent regulation of tunicamycin-induced apoptosis in breast carcinoma MCF-7 cells. Cell Death Differ. 2007; 3:586-96.

Investigation of Calnexn Protein Expression in Imatinib Sensitive and Resistant K562 Cells

Year 2018, Volume: 71 Issue: 1, 9 - 15, 16.10.2018

Arzu Zeynep Karabay

Abstract

Aim: The aim of this study is to determine the expression levels of endoplasmic reticulum (ER) chaperon protein calnexin in K562 cell line which is the cell line model of chronic myeloid leukemia characterised with the transformation of numerous blood stem cells into abnormal granulocytes. Calnexin levels in K562 cells and its resistant counterpart (K562R) to Imatinib, the first line therapy option for CML were examined. In addition, effects of first and second generation tyrosine kinase inhibitors, Imatinib and Nilotinib on calnexin levels and cell viability parameters in K562S and K562R cells were also determined.

Material and Method: In this study, K562S (sensitive to Imatinib) cell line, which is the cell model of the blast phase of CML, and K562R cell lines, which are resistant to imatinib (5uM), were used. Cells were cultured in parallel. K562S cells treated with or without 0.5 uM imatinib and 0.05 uM Nilotinib and K562R cells treated with or without 20 uM Imatinib and 0.1 uM Nilotinib were collected 48 hours after treatment and cell viability, apoptosis, cell morphology and calnexin protein expression were
determined with MTT assay, flow cytometry, light microscopy and western blot respectively.

Results and conclusion: In this study, it was shown that the total cellular expression of calnexin, a chaperone that regulates the folding of proteins in the endoplasmic reticulum, did not show any significant difference between the K562S and K562R cells, On the other hand, since Imatinib and Nilotinib significantly decreased calnexin protein expression in K562S cells and did not show any significant effect on K562R cells, this may indicate that the ER stress pathway and possibly calnexin are involved in the action mechanisms of these drugs. Therefore, even if no significant difference in calnexin levels was found between K562R and K562S cells, there may be differences in the ER stress pathway between the sensitive and resistant cells. For further studies, it is suggested to investigate the distribution of calnexin in different intracellular compartments as well as its modulation with drugs.

Keywords

Calnexin, K562, leukemia, drug resistance

Ethical Statement

Supporting Institution

Project Number

Thanks

References

1) Braakman I, Hebert DN. Protein folding in the endoplasmic reticulum. Cold Spring Harb Perspect Biol. 2013; 5:a013201.
2) Schwarz DS, Blower MD. The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci. 2016; 73: 79-94.
3) Hamdan N, Kritsiligkou P, Grant CM. ERstress causes widespread protein aggregation and prion formation. J Cell Biol. 2017; 216: 2295-2304.
4) Alasiri G, Fan LY, Zona S, et al. ER stress and cancer: The FOXO forkhead transcription factor link. Mol Cell Endocrinol. 2018; 462(Pt B):67-81.
5) Gifford JB, Hill R. GRP78 Influences Chemoresistance and Prognosis in Cancer. Curr Drug Targets. 2017. doi: 10.2174/1389450118666170615100918.
6) Phelps EA, Cianciaruso C, Michael IP, et al. Aberrant Accumulation of the Diabetes Autoantigen GAD65 in Golgi Membranes in Conditions of ER Stress and Autoimmunity. Diabetes. 2016; 65: 2686-99.
7) Erpapazoglou Z, Mouton-Liger F, Corti O. From dysfunctional endoplasmic reticulum- mitochondria coupling to neurodegeneration. Neurochem Int. 2017; 109:171-183.
8) Garfinkel BP, Hotamisligil GS. ER Stress Promotes Inflammation through RewIREd Macrophages in Obesity. Mol Cell. 2017; 66:731-733.
9) Hebert DN, Molinari M. In and out of the ER: protein folding, quality control, degradation, and related human diseases. Physiol Rev. 2007; 87:1377-408.
10) Schrag JD, Bergeron JJ, Li Y, et al. The Structure of calnexin, an ER chaperone involved in quality control of protein folding. Mol Cell. 2001;3: 633-44.
11) Xie W, Nielsen ME, Pedersen C, et al. A Split-GFP Gateway Cloning System for Topology Analyses of Membrane Proteins in Plants. PLoS One. 2017; 13:e0170118.
12) Y. Shibata, T. Shemesh, W.A. Prinz, et al. Mechanisms determining the morphology of the peripheral ER Cell, 2010; 143:774- 788.
13) N. Myhill, E.M. Lynes, J.A. Nanji, et al. The subcellular distribution of calnexin is mediated by PACS-2 Mol. Biol. Cell, 2008; 19: 2777-2788.
14) Ryan D, Carberry S, Murphy ÁC, et al. Calnexin, an ER-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer. J Transl Med. 2016 ;14:196.
15) Uyy E, Suica VI, Boteanu RM, et al. Endoplasmic Reticulum Chaperones Are Potential Active Factors in Thyroid Tumorigenesis. J Proteome Res. 2016; 15:3377-87.
16) Kobayashi M, Nagashio R, Jiang SX, et al. Calnexin is a novel sero-diagnostic marker for lung cancer. Lung Cancer. 2015; 90:342-5.
17) Pavli M, Farmaki E, Merkourea S, et al. Endoplasmic Reticulum Stress-Associated Chaperones, Bip/GRP78 and Calnexin are Overexpressed in Keratocystic Odontogenic Tumours. J Oral Maxillofac Res. 2014; 5:e3
18) Seres M, Poláková E, Krizanová O, et al. Overexpression of P-glycoprotein in L1210/VCR cells is associated with changes in several endoplasmic reticulum proteins that may be partially responsible for the lack of thapsigargin sensitivity. Gen Physiol Biophys. 2008; 27:211-21.
19) Sereš M, Ditte P, Breier A, et al. Effect of thapsigargin on P-glycoprotein-negative and P-glycoprotein-positive L1210 mouse leukaemia cells. Gen Physiol Biophys. 2010; 29:396-401.
20) Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999; 17: 1330-40.
21) Frame D Chronic myeloid leukemia: standard treatment options. Am J Health Syst Pharm. 2006; 8:21-22.
22) Valent P. Imatinib-resistant chronic myeloid leukemia (CML): Current concepts on pathogenesis and new emerging pharmacologic approaches. Biologics. 2007; 4: 433-48.
23) Xia Y, Fang H, Zhang J, et al. Endoplasmic reticulum stress-mediated apoptosis in imatinib-resistant leukemic K562-r cells triggered by AMN107 combined with arsenic trioxide. Exp Biol Med (Maywood).2013; 238: 932-42
24) Kusio-Kobialka M, Podszywalow-Bartnicka P, Peidis P, et al. The PERK-eIF2α phosphorylation arm is a pro-survival pathway of BCR-ABL signaling and confers resistance to imatinib treatment in chronic myeloid leukemia cells. Cell Cycle. 2012; 21: 4069-78.
25) Salaroglio IC, Panada E, Moiso E, et al. PERK induces resistance to cell death elicited by endoplasmic reticulum stress and chemotherapy.
26) Chevet E, Hetz C, Samali A. Endoplasmic reticulum stress-activated cell reprogramming in oncogenesis. Cancer Discov. 2015;5: 586-97.
27) Shah PP, Dupre TV, Siskind LJ, et al. Common cytotoxic chemotherapeutics induce epithelial-mesenchymal transition (EMT) downstream of ER stress. Oncotarget. 2017;14: 22625-22639.
28) Podszywalow-Bartnicka P, Cmoch A, Wolczyk M, et al. Increased phosphorylation of eIF2α in chronic myeloid leukemia cells stimulates secretion of matrix modifying enzymes. Oncotarget. 2016; 48:79706-79721.
29) Ekiz HA, Can G, Gunduz U, et al. Nilotinib significantly induces apoptosis in imatinib-resistant K562 cells with wild-type BCR-ABL, as effectively as in parental sensitive counterparts. Hematology. 2010; 15:33-8.
30) Delom F, Emadali A, Cocolakis E, et al. Calnexin-dependent regulation of tunicamycin-induced apoptosis in breast carcinoma MCF-7 cells. Cell Death Differ. 2007; 3:586-96.

There are 30 citations in total.

Details

Primary Language	English
Subjects	Medical Biochemistry and Metabolomics (Other)
Journal Section	Articles
Authors	Arzu Zeynep Karabay
Project Number	-
Publication Date	October 16, 2018
Published in Issue	Year 2018 Volume: 71 Issue: 1

Cite

APA	Karabay, A. Z. (2018). Investigation of Calnexn Protein Expression in Imatinib Sensitive and Resistant K562 Cells. Ankara Üniversitesi Tıp Fakültesi Mecmuası, 71(1), 9-15.
AMA	Karabay AZ. Investigation of Calnexn Protein Expression in Imatinib Sensitive and Resistant K562 Cells. Ankara Üniversitesi Tıp Fakültesi Mecmuası. October 2018;71(1):9-15.
Chicago	Karabay, Arzu Zeynep. “Investigation of Calnexn Protein Expression in Imatinib Sensitive and Resistant K562 Cells”. Ankara Üniversitesi Tıp Fakültesi Mecmuası 71, no. 1 (October 2018): 9-15.
EndNote	Karabay AZ (October 1, 2018) Investigation of Calnexn Protein Expression in Imatinib Sensitive and Resistant K562 Cells. Ankara Üniversitesi Tıp Fakültesi Mecmuası 71 1 9–15.
IEEE	A. Z. Karabay, “Investigation of Calnexn Protein Expression in Imatinib Sensitive and Resistant K562 Cells”, Ankara Üniversitesi Tıp Fakültesi Mecmuası, vol. 71, no. 1, pp. 9–15, 2018.
ISNAD	Karabay, Arzu Zeynep. “Investigation of Calnexn Protein Expression in Imatinib Sensitive and Resistant K562 Cells”. Ankara Üniversitesi Tıp Fakültesi Mecmuası 71/1 (October 2018), 9-15.
JAMA	Karabay AZ. Investigation of Calnexn Protein Expression in Imatinib Sensitive and Resistant K562 Cells. Ankara Üniversitesi Tıp Fakültesi Mecmuası. 2018;71:9–15.
MLA	Karabay, Arzu Zeynep. “Investigation of Calnexn Protein Expression in Imatinib Sensitive and Resistant K562 Cells”. Ankara Üniversitesi Tıp Fakültesi Mecmuası, vol. 71, no. 1, 2018, pp. 9-15.
Vancouver	Karabay AZ. Investigation of Calnexn Protein Expression in Imatinib Sensitive and Resistant K562 Cells. Ankara Üniversitesi Tıp Fakültesi Mecmuası. 2018;71(1):9-15.

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