Tgp chi Cdng ngh^ Sinh hgc 11(2): 211-217, 2013
G E N E R A T I O N A N D C H A R A C T E R I Z A T I O N O F G E M C I T A B I N E ( G E M ) - R E S I S T A N T P A N C R E A T I C C A N C E R M I A P a C a - 2 C E L L S
Duong Hong Quan'"'', Insoo Bae^ Yeon-Sun Seong', Dinh Duy Khang^, Dong Van Quyen^
'Dankook University, Cheonan, Korea
Insthule of Biotechnology, Vietnam Academy of Science and Technology Georgetown University, USA
''Duy Tan University, Vietnam
SUMMARY
Gemcitabine (GEM) is fir^t-line standard chemotherapy used for the treatment of pancreatic cancer;
however resistance of pancreatic cancer to GEM remains the major obstacle to the successful control of this disease. Toward understanding the molecular mechanisms associated with GEM-induced resistance, here we developed and investigated the GEM-resistant pancreauc cancer MIA PaCa-2 cells (MIA PaCa-2/GR). We showed that MIA PaCa-2/GR cells underwent distinct morphological changes including spindle-shaped morphology, appearance of pseudopodia and reduced adhesion characteristic of transformed fibloblasts We also found that Vimentin was up-regulated, while E-Cadherin was down-regulated in MIA PaCa-2/GR cells;
these are the typical characteristics of epithelial-to-mescnchymal transition (EMT) phenotype. In addition, MIA PaCa-2/GR cells induced the expression of anti-apoptotic proteins including Survivin. XIAP and Bcl2, and activated the phosphorylation and expression of c-MET, Moreover, MIA PaCa-2/GR cells displayed higher expression level and activity of ALDHl Al, the molecular maker of cancer stem cells, emphasizing that MIA PaCa-2/GR cells increased the population of pancreatic cancer stem cells These novel findings may lead to development of an effective therapeutic regimen to overcome the resistance of pancreatic cancer to GEM Keywords: Gemcilahine .EMT. c-MET. Cancel cell, puiicn -. MIA PaCa-2 cells INTRODUCTION
Pancreatic adenocarcinoma is the fourth leading cause of cancer-related deaths in the Western world (Jemal et ai. 2010). The overall prognosis for patients with pancreatic cancer is extremely poor: the five-year survival rate is less than 5% (Vincent el ai, 2011), More than 85% of patients with pancreatic cancer show distant metastatic characteristics at the time of diagnosis which render them unsuitable for surgery (Vincent et ai, 2011; Stathis and Moore (2010); Vaccaro el ai, 2011).. Current therapy for pancreatic cancer involved surgery and chemotherapy; however metastatic disease is very resistant lo most chemotherapeutic agents and to novel targeted therapies (Mattaios et ai. 2011)
Gemcitabine (2",2'-difluoro-2-deoxycylidme) (GEM), a pyrimidme-based antimetabolite, remains the front-line standard drug for the treatment of advanced human pancreatic cancer cither alone or in combination with other chemotherapeutic agents (Burris et al. 1997; Conroy et ai. 2011, Cunninqham et ai, 2009). GEM is metabolized lo gemcitabine triphosphate (dFdCTP) in cells and
incorporated into the DNA resulting in the increase in apoptotic cell death due to the inhibition of DNA synthesis (Ueno el ai. 2007) , However, the intrinsic and/or acquired resistance of pancreatic cancer to GEM shows a major challenge. Pancreatic tumor cells acquire resislance to GEM by various mechanisms, including alterations in transport, drug targets and metabolism or in the genes regulating eel! survival (Galmarim et al. 2011) Therefore, a belter understanding of how resistance arises and what molecular alterations cause or correlate with resistance is very much helpful to develop the novel therapeutic strategy to improve the treatment of pancreatic cancer with GEM and survival of patients with metastatic disease.
In this sludy, we isolated GEM-resistant pancreatic cancer MIA PaCa-2 cells (MIA PaCa- 2/GR) from well-characterized parental pancreatic cancer MIA PaCa-2 cells (MIA PaCa-2/P) and studied the molecular mechanism of resistance associated with GEM. We demonstrate that aside from emergence of fibroblastic, specific activation and expression of c-MET, expression of anli- apoplotic proteins, significant increase of cancer
211
Duong Hong Quan et ai stem cell population consistently occurs as cells
acquire resistance of GEM. Our results provide new msights into phenolypic changes associated with molecular mechanism of resistance of pancreatic cancer to GEM,
MATERIALS AND METHODS Cell culture and reagents
MIA PaCa-2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA), MIA PaCa-2 cells were cultured in Dulbecco's Modified Eagles" Medium (DMEM) containing 10% PES, 2,5% horse serum (HS), IOO units/ml penicillin, and IOO ng/ml streptomycin Cell culture reagents were purchased from BioWhillaker (Walkersville, MD) and Invitrogen (Carlsbad, CA). Gemcitabine was obtained from Sigma (St. Louis, MO).
Morphologic analysis
MIA PaCa-2/P and MIA PaCa-2/GR cells were grown to 70% confluence in the appropriate GEM- free media. Cell morphologies were visualized via light microscopy. Digital pictures were taken from a camera mounted to the microscopy.
3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazoHum bromide (MTT) assay
A total of 2000 either MIA PaCa-2/P or MIA PaCa-2/GR cells were plated in 96-well flat bottom plates and then exposed lo test the sensitivity of GEM at various concentrations. Al the indicated times, 10 pi of 1 mg/ml MTT (Sigma, St. Louis, MO) in PBS was added lo each well for 4 h. After cenlrifugation and removal ofthe medium, 150 \x\ of DMSO (Sigma, Sl Louis, MO) was added lo each well to dissolve the formazan crystals. The absorbance was measured at 560 nm using an ELx808 Absorbance Microplate Reader (BioTek Instruments, I n c , Winooski, VT). Absorbance of untreated cells was designated as 100% and cell survival was expressed as a percentage of this value.
Triplicate wells were assayed for each condition and standard deviation (SD) was determined (Duong et
^/., 2012).
Western blot (WB) analysis
Cells were grown to - 7 0 % confluence and reagents were added at the indicated concentrations Afler 48 h. MIA PaCa-2/P and/or MIA PaCa-2/GR 212
cells were lysed in cell lysis buffer containing 20 mM Tris-HCL 0.5 M NaCL 0.25% Triton X-100, 1 mM EDTA, I mM EGTA, 10 mM P-glycophosphate, 10 mM NaF, 300 p M NajV04, 1 mM benzamidine, 2 pM PMSF, and 1 mM DTT. The prolein concentration was determined by a BCA protein assay kit (Thermo Scientific, Rockford, IL). Proteins were separated on SDS-PAGE, transferred on to PVDF membrane, blocked in IX blocking buffer (Sigma, Sl Louis, MO) and probed with the following primary antibodies: p-c-MET, c-MET, E-cadherin, Vimentin, j3-Catenin and Snail (Cell Signaling Technology, Danvers, MA), A L D H l A l (Abeam, Cambridge, UK), Survivm, Bcl2 and XIAP (BD Biosciences, Franklin, NJ), and a-lubulin (Sigma, St, Louis, MO). Then, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma, Sl Louis, MO) and visualized with a chemiluminescence kit (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturers recommended protocol and exposed with X-ray film (American X-ray& Medical Supply, Jackson, CA) (Duong e^fl/.. 2012)
A L D E F L U O R assay
Active ALDHIA1 was identified with the ALDEFLUOR assay according lo manufacturer s instructions (StemCell Technologies, Durham, NC).
The ALDHlAl-posilive population was defined by cells with increased FITC signal, with gates determined by diethylaminobenzaldehyde (DEAB)- ireated cells (DEAB being an inhibitor of ALDHlAl activity). The ALDEFLUOR-positive cell population was measured by FACSCalibur flow cylometer and analyzed as previously described in the flow cytometry section (Duong et ai, 2012)
RESULTS AND DISCUSSION
Generation of G E M - r e s i s t a n t M I A PaCa-2 cells (MIA P a C a - 2 / G R cells)
Pancreatic adenocarcinoma is the fourth leading cause of cancer-related deaths in the Western world (Jemal et ai, 2010) The overall prognosis of pancreatic cancer patients is extremely poor: ihe five-year survival rate is estimated less than 5-6%
(Vincent et al, 2011). Current therapy for pancreatic cancer involved surgery and chemoriierapy; however pancreatic cancer is a notorious disease with resistant lo many key chemotherapeutic agents and novel targeted therapies (Mattaios ei ai. 2011). The first-
Tap chi Cdng nghi Sinh hgc 11(2) 211 -217, 2013 line standard drug, GEM, is used for the treatment of advanced human pancreatic cancer However, the intnnsic and/or acquired resislance of GEM in pancreatic cancer shows a major problem in treatment of pancreatic cancer patients. Therefore, improved therapeutic treatment will require a better understanding ofthe molecular mechanism by which these tumors become chemoresistant and development of strategies to overcome this resistance. To further study the molecular mechanism of GEM resistance, we generated the stable pancreatic cancer MIA PaCa-2 cell line chronically resistant tp GEM, MIA PaCa-2 cells were used as the parental cell line (MIA PaCa-2/P).
The MIA PaCa-2/P cells were serially subcultured through incrementally increasing GEM concentrations, starting with 0.1 pM for six months.
MIA PaCa-2/GR cells retained the capacity for proliferation when returned to medium containing GEM (0 5 |iM). To confirm that the MIA PaCa- 2/GR cell line accquired resistance against GEM, MIA PaCa-2/P and MIA PaCa-2/GR cells were treated with GEM in a dose-dependent manner (0, 0.01, 0 1, 1 and/or 10 pM) for 72 h and cell viability was evaluated by the MTT assay and the half maximal inhibitory concentration (IC^,,) was calculated by using CompuSyn software (ComboSyn, Inc., Paramus, NJ) (Chou and Talalay
(1984)). The results showed that MIA PaCa-2.GR cells were relatively resistant to GEM with an ICso value of 4.43 pM, whereas MIA PaCa-2/P cells were relatively sensitive to GEM with an ICso value of 0.11 nM (Fig. 1). The MIA PaCa-2/GR cells demonstrated a 40 3-fold higher resistant index towards GEM than that of MIA PaCa-2/P cells.
GEM (MM)
Figure 1 Generation of GEM-resislant MIA PaCa-2 cells (MIA PaCa-2/GR) The resuHs of MTT assay of MIA PaCa- 2/P and MIA PaCa-2'GR cells, which were treated with GEM in a dose-dependent manner (0. 0 01, 0 1, 1 and/or 10 pM) for 72 h Error bars represent the standard deviation '"p< 0 001 represents significant difference between GEM groups m MIA PaCa-2/GR cells and GEM groups in MIA PaCa-2/P cells
MIAPaCa-2/P
Figure 2 MIA PaCa-2/GR cells showed the change ot cell morphology. Cells were grown lo 60% confluency and then photographed under 40X magnification
CKM-restsliinl .MU PaCa-2 cells sigruficantly expressed EM T m a r k e r s
Solid tumor cells have been shown to undergo epithelial-lo-mesenchymal (EMT)-like transition when exposed to anticancer drugs as oxaliplalin in colon carcinoma cells and tamoxifen in breast cancer cells (Yang et id.. 2006; Hiscox el ai.
2006). It IS also well known that the morphologic
changes of other GEM-resistant pancreatic cancer L3 6pl and AsPC-l cell lines with demonstration of a loss of cell-cell adhesion, spindle shaped morphology and increased formation of pseudopodia (Shah et at. 2007) In this study, we showed the same morphologically distinct character of MIA PaCa-2/GR cell line like other GE-resislanl pancreatic cancer L3.6pl and AsPC-I cell lines (Fig. 2). Western bipl analysis was
Duong Hong Quan et ai performed to examine the expression of E-
Cadherin and Vimentin, the two known E M T markers. As expected, in MIA PaCa-2/GR cells, the expression of E-Cadherin was significantly down-regulated, whereas the expression of Vimentin was significantly increased (Fig 3A).
Moreover, the EMT regulator Snail expression was also markedly increased in MIA PaCa-2/GR cells as compared lo MIA PaCa-2/P cells (Fig 3B). Taken together, our results suggest thai EMT regulators are importanl lo GEM-induced EMT in pancreatic cancer cells.
MIAP F
aCa-2 GR M M :
—
1 ""^ —»
B
MIAPP
——
aCa-2 GR
^ ^
f —
« • • » — •
Figure 3 MIA PaCa-2/GR cells showed mesenchymal-epithelial transition (EMT) phenotype. (A) WB analysis of exponentially growing MIA PaCa-2/P and MIA PaCa-2/GR cells was used to determine an expression level of E-cadhenn and Vimentin. Anti-o-tubulin antibody was used for a loading and transfer control (B) WB analysis of exponentially growing MIA PaCa-2/P and MIA PaCa-2/GR cells was used to determine an expression level of Snail and p-catenin, Anti-a-tubulin antibody was used for a loading and transfer control
GEM-resistant MIA PaCa-2 cells significantly increased expression of anti-apoptotic proteins
p GR
- .
—— —
— ^
^HIMflHB^
Suivivin XIAP
Bcl2
a-tLibulin
Figure 4 The expression level of anti-apoptotic proteins is Eigmricantly increased in MIA PaCa-2/GR cells WB analysis of exponentially growing MIA PaCa-2/P and MIA PaCa-2/GR cells was used to determine an expression level of anti-apoptotic proteins such as Survivin. XIAP, Bci2. Anti- a-tubulin antibody was used for a loading and transfer control.
Increasing evidences show that anli-apoptotic proteins such as Survivin, XIAP and Bcl2 are potential therapeutic targets for pancreatic cancer and knockdown of anti-apoptotic proteins is a promising strategy lo enhance the sensitivity of cancer cells lo chemotherapeutic agents (Trabulo el al.. 2011) To determine the relationship between 214
anti-apoptotic proteins and resistance of GEM, we evaluated the expression levels of anti-apoptolic proteins in MIA PaCa-2/GR and MIA PaCa-2/P cells by Western blotting. The results indicated that expression levels of Survivin, XIAP and Bcl2 were significantly higher in MIA PaCa-2/GR cells than in MIA PaCa-2/T' cells (Fig 4), suggesting that Survivin, XIAP and Bcl2 are involved in GEM resistance in pancreatic cancer cells,
GEM-resistant MIA PaCa-2 cells significantly increased the expression and phosphorylation of c-MET
The activity of EMT was known to be driven by activafion of c-MET in several cancer cell lines and the constitutive phosphorylation of c-MET was markedly increased, while the expression of c-MET was not detected in GEM-resistanl pancreatic cancer in GEM-resislant pancreatic cancer L3.6pl and AsPC-I cell lines as compared to those in parental cell lines (Shah et ai. 2007; Bladt et ai, 1995, Herynk el cd.. 2003). We here examined the activity and expression of c-MET in MIA PaCa-2/GR and MIA PaCa-2/P cell lines by Western blot analysis As expected, the constitutive phosphorylation of c- MET was significantly higher in MIA PaCA-2/GR cells than in MIA PaCa-2/P cells (Fig. 5).
Interestingly, we found that the constitutive expression of c-MET was also markedly increased in MIA PaCa-2/GR cells than in MIA PaCa-2/P cells (Fig. 5). It is well known that activation of c-MET
Tgp chi Cdng nghe Sinh hoc 11(2): 211-217, 2013 signaling pathway leads lo a wide array of biological activities including mobility, migration, growth, angiogenesis and invasion (Bladt ei ai. 1995;
Herynk et ai. 2003). Activation of c-MET also significantly enhances the metastatic potential of tumor cells. Therefore, our finding suggests that c- MET plays a role in the resistance of pancreatic cancer to GEM
IVllAPaCa-2
Figure 5. The expression level of total and phosphorylaied form of c-MET is significantly increased in MIA PaCa-2/GR cells WB analysis of exponentially growing MIA PaCa-2/P and MIA PaCa-2/GR cells was used to detennine an expression level of total and phosphorylaied form of c-MET Anti-a-lubulin antibody was used for a loading and transfer control,
GEM-rcsistant MIA PaCa-2 cells highly expressed cancer stem cell m a r k e r s
A line of evidence has indicated that one ofthe intrinsic properties of cancer stem cells was anticancer drug resistance and the expression and activity of ALDHlAl are imporlanl features of luinor-iniliating and/or cancer stem cell properties in
multiple t>'pes of human cancer (Duong ei ai, 2012).
Previously, our study firstly demonstrated that comparing with MIA PaCa-2/P cells, MIA PaCa- 2/GR significantly expressed higher levels of A L D H l A l (Duong et al.. 2012), To confirm the significant higher expression of ALDHlAl in ML^
PaCa-2/GR ceils, we performed an AiderFluor assay, a functional flow cytometric assay that identifies cells with active A L D H l A l , As expected, the AldeFluor positive cell population was markedly induced from 18 1% in MIA PaCa-2/P cells to 29.2% in MIA PaCa-2/GR cells (Fig, 6), The data obtained fi-om our previous sludy and this study indicated that the expression and activity of ALDHlAl were significantly higher in the GEM- resistant MIA PaCa-2 cell line (MIA PaCa-2/GR)- Since we knew a side population of pancreatic cancer ceils that are posirive expression of CD24, CD44 and ESA are highly tumorigenic and cancer slem cells (Li et al.. 2007; Shah et ai. 2007), our sludy aim to to investigate the new marker of pancreatic cancer stem cells. Cancer stem cells may have a plastic morphology that allows them to undergo EMT in order to invade and metastasize and cause resistance of pancreatic cancer lo GEM, Taken together, our results suggest that cancer stem ceil population significantly increased in GEM-resislant pancreatic cancer cell lines I.VIIA PaCa-2/GR) as compared to their parental pancreatic cancer cell lines (MIA PaCa-2/P) Further study on animal model will be necessary to investigate the close relationship between the molecular mechanism of resistance of GEM and characteristic of pancreatic cancer stem cells.
MIA PaCa-2/GR
Figure 6 The expression level and activity of A L D H l A l is significantly higher in MIA PaCa-2/GR cells ALDEFLUOR assay of exponentially growing MIA PaCa-2/P and MIA PaCa-2/GR cells was used to determine the activity of A L D H l A l The ALDH1A1 inhibitor, DEAB was added lo ensure accurate identification of A L D H l A l positive and negative cells
Duong Hong Quan et ai CONCLUSION
Numerous studies have been carried out lo elucidate diverse biological effects of GEM in pancreatic cancer. Understanding the molecular mechanisms of how pancreatic cancer cells resistant to GEM are critical for development of a successful drug/therapy to control this disease We here investigated the molecular mechanism associated with GEM-resistant pancreatic cancer MIA PaCa-2 cells. Our data suggest that EMT, anti-apoplotic proteins, c-MET, and molecular markers of cancer stem cells may regulate the intrinsic and/or acquired resistance of GEM in pancreatic cancer. Further study need to be carried out to understand the significant role of each molecule to overcome the resistance of pancreatic cancer to GEM resistance
Acknowledgments: This work was supponed by National Institutes of Health (1R03CAI52530). the National Research Foundation of Korea [R3J- 10069; World Class University (WCU) program}
and the Georgetown University Lombardi Comprehensive Cancer Center (P30-CA05J008).
REFERENCES
Bladt F, Riethmacher D, Isenmann S, Aguzzi A, Birchmeier C (1995) Essential role for the c-MET receptor in the migration of myogenic precursor cells into the limb bud,/Varu/-f 376, 768-771
Bums HA y^, Moore MJ, Andersen J, Green MR, Rothenberg ML, Modiano MR, Cripps MC, Portenoy RK, Siomiolo AM. Tarassoff P, Nelson R, Dorr FA, Stephens CD, Von Hoff DD (1997) Improvements m survival and clinical benefit with gemcitabine as first-line therapy for patients nith advanced pancreas cancer; a randomized trial JC/mOHC(j/15:2403-2413.
Chou TC, Talalay P (1984) Quantitative analysis of dose- effect relationships the combined effects of multiple drugs and enzyme inhibitors, Adv Enz Regul 22: 27-55.
Conroy T, Desseiqne F. Ychou M, Bouche O, Gmmbaud R, Becouam Y, Adems A. Raoul JL, Gourgou-Bourgade S, delaFouchardicre C, Bennouna J, Bachet JB, Khemissa- Akouz F, Pere-Verqe D, Delbaldo C, Assenat E, Chauffert B. Michel P, Monloto-Grillot C, Ducreux M (2011) FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer N Engl J Med \2: 1817-1825 Cunninqham D, Chau 1, Stocken DD, Valle JW, Smith D, Steward W, Harper PG, Dunn J, Tudur-Smidi C, West J, Talk S, Crellin A, Adab F, Thompson J, Leonard P, Osirowski J. Eatock M, Scheiihauer W, Herrmann R,
Neopiolemos JP (2009) Phase III randomized companson of gemcitabine versus gemcitabine plus capecitabine in patients with advanced pancreatic cancer. J Chn Oncol 21- 5513-5518,
Duong HQ, Hwang JS, Kim HJ, Kang HJ, Seong YS, Bae I (2012) Aldehyde dehydrogenase lAl confers intrinsic and acquired resistance to gemcitabine in human pancreatic adenocarcinoma MIA PaCa-2 cells. Ini J Oncol 41 855-861
Duong HQ, Kim HJ, Kang HJ, Seong YS, Bae I (2012) ZSTK474, a PI3K inhibitor, suppresses proliferation and sensitizes human pancreatic adenocarcinoma cells to gemcitabine OncolRep21- 182-188,
Galmarim CM, Mackey JR, Dumontet C (2011) Nucleoside analogous: mechanisms of dmg resistance and reversal strategies Leukemia \5: 875-890.
Herynk MH, Tsan R, Radinsky R, Gallick GE (2003) Acnvation of c-MET in colorectal carcinoma cells leads to constitutive association of tyrosme-pbosphorylated beta- catenin Chn Exp Metastasis 20: 291-300,
Hiscox S, Jiang WG, Obermeier K, Taylor K, Morgan L, Bumn R, Barrow D, Nicholson RI (2006) Tamoxifen resistance in MCF7 cells promotes EMT-like behavior and involves modulation of beta-catenin phosphorylation hit J Cancer 118 290-301.
Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010 CA Cancer J Chn 60: 277-300
Li C, Heidt DG, Dalerba P, Biirant CF, Zhang L, Adsay V, Wicha M, Clarke MF, Simeone DM (2007), Cancer Res 67- 1030-1037
Matthaios D, Zaroqouhdis P, Balgouranidou I, Chatzaki £, Kakolyris S (2011) Molecular pathogenesis of pancreatic cancer and clinical perspectives. Oncology 81: 259-272.
Shah AN, Summy JM, Zhang J, Park SI, Parikh NU, Galhck GE (2007) Development and characterization of gemcitabine-resislant pancreatic mmor cells, Ann Surg Oncol 14: 3629-3637,
Stathis A, Moore MJ (2010) Advanced pancreatic carcinoma current treatment and future challenges Nat RevClmOncoll: 163-172,
Trabuio S, Cardoso AM, Santos-Ferreira T, Cardoso AL, Simoes S, Pedroso de Lima MC (2011) Survivin silencing as a promising strategy to enhance the sensitivity of cancer cells to chemotherapeutic agents.
Mol PharmS. 1120-1131.
Ueno H, Kiyosawa K, Kaniwa N (2007) Pharmacogenomics of gemcitabine' can genetic studies lead to tailor-made therapy. Br J Cancer 97: 145-151.
Vaccaro V, SperduH l, Mileila M (2011) FOLFINOX versus gemcitabine for metastatic pancreaUc cancer N EnglJMed365. 76Z-769.
Tgp chi Cong nghi Sinh hgc 11(2): 211-2I7, 2013
Vincent A. Herman J, Schulick R, Hruban RH, Goggms M R, Gray MJ, Cheng H, Hoff PM, Ellis LM (2006) Chrome (2011) Pancreatic cancer La/itef 378'607-620. oxaliplatm resistance mduces epithelial-to-mesenchymal ,, _ transition in colorectal cancer cell lines. Clin cancer Res Yang AD. Fan F, Camp ER, van Buren G, Liu W, Somcio ij- 4147-4153
N G H I E N C U U T A O D O N G T E B A G U N G T H U T U V M I A P a C a - 2 KH.ANG G E M C I T A B I N E ( G E M ) V A C O C H E P H A N TLT L I E N Q U . 4 N D £ N T J N H K H . i N G G E M Du-oTig H6ng Q u a n ' ' " ' , Bae l n s o o ^ Seong Yeon-Sun', Dmh Du\ K h a n g ^ D6ng Van Quyfn^
'Tnr&ng Dgi hoc Dankook, Cheonan, Hdn Quoc
Vi^n Cong nghe Sinh hgc. Viin Hdn lain Khoa boc vd Cong nghe Viei Nam Tnrdng Dgi hgc Georgetown, Washington DC, M\
Trudng Dgi hgc Duy Tdn, Viet Nam
TOM TAT
Gemcitabine (GEM) la thii6c hoa tn lieu hieu qua duy nhat duoc su dung de dieu tri ung thu tuy, luy nhien linh khang thuoc GEM la tro ngai Ion nhat den sir thanh cong trong dieu tri benh De danh gia co che phan ni lien quan den tinh khang GEM, chung toi da tao ra cac dong te bao ung thu tuy MIA PaCa-2 khang GEM (MIA PaCa-2/GR) va nghien cuu su bien dong lien quan den GEM trong dong te bao nay Ket qua nghien cim cho thay, so vdi cac dong te bao MIA PaCa-2 nguon ban dau (MIA Paca-2/P), te bao MIA PaCa- 2/GR CO su thay doi Ion ve mat hinh thai le bao bao gom hinh thai te bao dang thoi. sir xual hien hinh thai tua va dac biet giam kha nang bam dinh ciia cac nguyen bao soi. Te bao MIA PaCa-2/GR cung gia tang su bieu hien Vimentin va giam su' bieu hien ciia E-cadherin, hai dau an (marker) chuan cua qua trinh chuyen lii te bao bieu mo toi trung mo. Ngoai ra, te bao MIA PaCa-2/GR bieu hien rat manh cac protein co vai tro khang lal qua trinh te bao chet theo chirong trinh nhir Survivin, XIAP va Bcl2. va kich boat sir bieu hien va qua trinh phosphoryl hoa ciia c-MET. Hon the nira, su bieu hien va hoat dong cua ALDHlAl- dau an chuan cua te ban ung thu, gia tang m^nh me trong te bao MIA PaCa-2/GR nhan manh rang le bjo MIA PaCa-2/GR ihiic day su phat tricn ciia le bao ung ihu goc tuy Ket qua nghien ciru nay gop phan lam sang to co che phan tii cua tinh khang thuoc GEM va lit do phat tnen cac phac do dieu in hieu qua va toi uu trong dieu in ung thu tuy.
Tirkhoa: Sir Uuiiii; thudc i>cmciuibiiu: EMT. c -MET. Te bao fiik am; ibir I in; iliir mv. Te bao Ml.\ PuCa-2
' Author for correspondence: E-mail: tiuanvspt^ aniail com
