NF-κB–mediated chemoresistance in breast cancer cells

doi:10.1067/msy.2001.115512

Surgery

Volume 130, Issue 2, August 2001, Pages 143-150

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

https://doi.org/10.1067/msy.2001.115512 Get rights and content

Abstract

Background. Nuclear factor-kappa B (NF-κB) is a known survival pathway, and it may explain differential sensitivity to tumor necrosis factor-alpha (TNF-α) and chemotherapeutic-induced apoptosis in apoptotically sensitive (APO+) and apoptotically resistant (APO−) Michigan Cancer Foundation-7 breast cancer cells. Methods. Crystal violet viability and luciferase reporter gene assays were used to determine the inhibitory concentration of viability at 50% (IC₅₀) and the inhibitory concentration of activity at 50% (EC₅₀) values in APO− and APO+ cells with the selective NF-κB inhibitor, BAY 11-7082 (BAY). The apoptotic reporter assay was used to determine the effects of the transfection of the inhibitory kappa B-dominant negative (IκB-DN) construct in conjunction with TNF, paclitaxel, or doxorubicin treatments in these cells. Results. The concentrations at which 50% of cell viability is inhibited (IC₅₀) and at which 50% of NF-κB activity is inhibited (EC₅₀) for BAY in APO− and APO+ cells were 95.24 μmol/L and 1.53 μmol/L, respectively, and 7.62 μmol/L and 2.64 μmol/L, respectively. The IC₅₀ and the EC₅₀ values were equivalent for the APO+ cells (P =.665), but not for the APO− cells (P =.025). IκB-DN-transfection alone, or with TNF, doxorubicin, or paclitaxel treatments resulted in cell death of both APO− and APO+ cells as compared with vector-control; however, greater cytotoxicity was seen in the APO+ cells. Direct comparison of the APO+ cells versus the APO− cells revealed that these differences were significant (P =.05). Conclusions. Pharmacologic or molecular inhibition of the NF-κB pathway blocked cell survival in MCF-7 APO+ cells, while only molecular inhibition induced cytotoxicity in the APO− cells. Selective manipulation of the NF-κB pathway in combination with standard chemotherapeutic agents may lead to an increased potency and efficacy of these agents. (Surgery 2001;130:143-50.)

Section snippets

Cell culture

The MCF-7 cell variant APO− (previously designated “M,” passage 180) and the MCF-7 APO+ cell variant (previously designated “N,” passage 50) have been previously described13, 14; and these cells were maintained as outlined by Burow et al.¹³ Cells were plated in 25 cm² (T-25) and 75 cm² (T-75) Falcon (BD Biosciences, Bedford, Mass) tissue culture flasks, 6-well plates, 12-well plates, or 96-well plates.

Reagents

The following chemicals were used: TNF (R&D Systems, Minneapolis, Minn), PAC (Biomol, Plymouth

Determination of NF-κB/LUC dose response

Given the role of NF-κB in mediating cell survival despite chemotherapeutic-induced apoptosis, we initially attempted to determine whether differences in basal NF-κB activity exist among the previously described APO− and APO+ MCF-7 cell variants. Basal NF-κB activity was determined in the APO+ and APO− cells by using an NF-κB/LUC reporter assay (Fig 1).

. NF-κB/LUC transient transfection dose response in APO+ and APO− MCF-7 cells. The 2.5 × 10⁵ cells were plated in 12-well plates and transfected

Discussion

It has been implicated that the transcription factor NF-κB is responsible for regulating a host of molecular determinants that have been shown to activate cellular-survival machinery and gene expression.⁸ The survival cascades initiated by this transcription factor have been shown to be a key component of cellular apoptotic resistance.3, 4, 5, 6, 7 Our previous research efforts have partly concentrated on the identification and characterization of phenotypically different MCF-7 breast

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^*: Supported by National Institutes of Health grant 1-T32-CA65436-01A3 (B. M. J.), U.S. Department of Defense Army Breast Cancer Research fellowship DAMD17-97-1-7024 (M. E. B.), the Cancer Association of Greater New Orleans (M. E. B., B. S. B.), the Tulane Cancer Center (B. S. B.), and the Center for Bioenvironmental Research at Tulane and Xavier Universities (B. S. B., M. E. B.).

^**: Reprint requests: Bernard M. Jaffe, MD, Department of Surgery, Tulane University School of Medicine, Tulane University Health Sciences Center, 1430 Tulane Ave, SL-22, New Orleans, LA 70112.

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Society of University SurgeonsNF-κB–mediated chemoresistance in breast cancer cells*,**

Abstract

Section snippets

Cell culture

Reagents

Determination of NF-κB/LUC dose response

Discussion

Exp Cell Res

Surg Oncol

Surgery

Cell

J Biol Chem

Hum Pathol

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

Therapeutic potential of inhibition of the NF-κB pathway in the treatment of inflammation and cancer

J Clin Invest

Inhibition of NF-κB activity enhances TRAIL mediated apoptosis in breast cancer cell lines

Breast Cancer Res Treat

Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth

Mol Cell Biol

Society of University Surgeons
NF-κB–mediated chemoresistance in breast cancer cells*,**