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. 2010 Dec 27;16(1):101–113. doi: 10.2478/s11658-010-0043-z

Ets-1 expression and gemcitabine chemoresistance in pancreatic cancer cells

Amit Khanna 1,3, Kulandaivelu Mahalingam 3, Debarshi Chakrabarti 2, Giridharan Periyasamy 1,✉
PMCID: PMC6276009  PMID: 21225469

Abstract

Gemcitabine, a novel pyrimidine nucleoside analog, has become the standard chemotherapeutic agent for pancreatic cancer patients. The clinical impact of gemcitabine remains modest owing to the high degree of inherent and acquired resistance. There are various lines of evidence that confirm the role of Ets-1, a proto-oncoprotein, in tumor invasion, progression, and chemoresistance. This study examines a hypothesis that implicates Ets-1 in the development of gemcitabine-resistance in pancreatic cancer cells. Ets-1 protein expression was assessed in parental pancreatic cancer cells and their gemcitabine-resistant clones. Western blot analysis revealed elevated levels of Ets-1 protein expression in gemcitabine-resistant PANC1GemRes (4.8-fold increase; P < 0.05), MIA PaCa2GemRes (3.2-fold increase; P < 0.05), and Capan2GemRes (2.1-fold increase; P < 0.05) cells as compared to their parental counterparts. A time course analysis was conducted to determine the change in Ets-1 expression in the parental cells after incubation with gemcitabine. Reverse transcriptase quantitative real-time PCR (RT-qPCR) and Western blot analysis revealed a significant increase in Ets-1 expression. All the three parental cells incubated with gemcitabine showed elevated Ets-1 protein expression at 6 h. By 24 h, the expression level had decreased. Using small interfering RNA (siRNA) against Ets-1 in gemcitabine-resistant cells, we demonstrated a reversal in gemcitabine chemosensitivity and also detected a marked reduction in the expression of the Ets-1 target genes MMP1 and uPA. Our novel finding demonstrates the significance of Ets-1 in the development of gemcitabine chemoresistance in pancreatic cancer cells. Based on these results, a new siRNA-based therapeutic strategy targeting the Ets-1 genes can be designed to overcome chemoresistance.

Key words: Gemcitabine, Ets-1, MMP1, uPA, PANC1, MIA PaCa2, Capan2

Full Text

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Abbreviations used

cDNA

complementary DNA

Ct

cycle threshold

dFdCTP

2′,2′-difluorodeoxycytidine 5′-triphosphate (gemcitabine triphosphate)

DMEM

Dulbecco’s modified Eagle’s medium

DMSO

dimethyl sulfoxide

E26

avian erythroblastosis virus

Ets-1

E26 transformation specific sequence-1

FBS

fetal bovine serum

GAPDH

glyceraldehydes-3-phosphate dehydrogenase

MDR1

multiple drug resistance-1

MMP1

matrix metalloproteinase-1 (collagenase-1)

MMP3

matrix metallopeptidase-1 (stromelysin-1)

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PDAC

pancreatic ductal adenocarcinoma

RT-qPCR

reverse transcriptase real-time quantitative PCR

siRNA

small interfering RNA

uPA

urokinase-type plasminogen activator

v-ets

viral transforming gene of E26

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