Nuclear factor-κB is upregulated in colorectal cancer

doi:10.1067/msy.2001.116672

Surgery

Volume 130, Issue 2, August 2001, Pages 363-369

Presented at the 62nd Annual Meeting of the Society of University Surgeons, Chicago, Ill, February 8-10, 2001.

https://doi.org/10.1067/msy.2001.116672 Get rights and content

Abstract

Background. Chemoresistance may involve the anti-apoptotic transcriptional regulator, nuclear factor-κB (NF-κB). The purpose of this study was to determine whether chemotherapy induces NF-κB activation in a human colon cancer cell line (SW48) and whether NF-κB is constitutively activated in colorectal cancer. Methods. SW48 cells were incubated with gemcitabine hydrochloride (Gemzar) in the presence and absence of the 26s proteasome inhibitor, MG132, and NF-κB binding (electrophoretic mobility shift assay), DNA synthesis (tritiated thymidine uptake), cell viability (3-[4,5-dimethylthiazol-2-yl]-diphenyl-tetrazolium bromide assay), and apoptosis (caspase-3 activity) were measured at 24 hours. NF-κB binding (electrophoretic mobility shift assay) was also assayed in 10 colorectal cancer tumors. Results. SW48 cells demonstrated constitutive NF-κB binding that was enhanced by gemcitabine hydrochloride in a dose-dependent manner. MG132 inhibited NF-κB binding and enhanced gemcitabine hydrochloride's inhibition of DNA synthesis (gemcitabine hydrochloride = 73% ± 1.4% vs gemcitabine hydrochloride + MG132 = 6% ± 0.4%, P <.05), cell killing (gemcitabine hydrochloride = 87% ± 2.0 vs gemcitabine hydrochloride + MG132 = 25% ± 1.3%, P <.05), and caspase-3 activity (gemcitabine hydrochloride = 870 ± 17.4 vs gemcitabine hydrochloride + MG132 = 1075 ± 20.4, P <.05). NF-κB binding was increased in 8 of 10 colorectal cancer tumors compared with adjacent normal mucosa. Conclusions. Gemcitabine hydrochloride enhances NF-κB binding in a colorectal cancer cell line, whereas inhibition of NF-κB enhances gemcitabine hydrochloride's antitumor activity. NF-κB is also activated in human colorectal cancer. NF-κB may identify chemoresistant tumors, whereas inhibition of NF-κB may be a novel, biologically based therapy. (Surgery 2001;130:363-9).

Section snippets

Cell lines, cultures, and reagents

SW48 cells were purchased from American Type Culture Collection (ATCC, Rockville, Md) and were grown in complete medium (CM), which consisted of Medium 199, Ham's F-10, and Dulbecco's modified Eagle's medium and also contained 25 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), pH 7.4, 1 mmol/L sodium bicarbonate, antibiotics, and 10% fetal bovine serum. Cells were grown in 6-well microtiter plates and were used at approximately 80% confluence. Gemcitabine

Gemcitabine hydrochloride stimulates NF-κB binding

Nuclear extracts were prepared from SW48 cells exposed for 24 hours to varying doses of gemcitabine hydrochloride (0, 0.1, 1, 10, 100, 1000 μmol/L), and EMSA was performed. SW48 cells demonstrated constitutive NF-κB binding, which was increased by gemcitabine hydrochloride in a dose-dependent fashion (Fig 1).

. EMSA of nuclear extracts prepared from SW48 cells incubated for 24 hours with increasing doses of gem-citabine hydrochloride (Gemzar). Minus sign, Negative control; S, specific inhibitor; N,

Discussion

In the current study, we have demonstrated that a colorectal cancer cell line exhibits constitutive NF-κB binding, which is further stimulated by the anticancer drug, gemcitabine hydrochloride. Furthermore, inhibition of NF-κB binding by the 26s proteasome inhibitor MG132 enhanced gem-citabine hydrochloride's antitumor activity through a caspase 3-mediated mechanism.

Gemcitabine hydrochloride is a nucleoside analogue that interferes with DNA synthesis and induces tumor cell apoptosis.¹⁴

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^*: Reprint requests: D. Scott Lind, MD, Associate Professor, Department of Surgery, University of Florida College of Medicine, PO Box 100286, Gainesville, FL 32610.

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Society of University SurgeonsNuclear factor-κB is upregulated in colorectal cancer*

Abstract

Section snippets

Cell lines, cultures, and reagents

Gemcitabine hydrochloride stimulates NF-κB binding

Discussion

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Blood

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Blood

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CA Cancer J Clin

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J Clin Invest

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Society of University Surgeons
Nuclear factor-κB is upregulated in colorectal cancer*