Apoptotic action of 17beta-estradiol in raloxifene-resistant MCF-7 cells in vitro and in vivo.
BACKGROUND
Resistance to tamoxifen, a selective estrogen receptor modulator (SERM), involves changes that prevent apoptosis and enhance cell proliferation and survival. Paradoxically, estrogen treatment inhibits the growth of long-term tamoxifen-treated breast tumors. Because of the increasing use of raloxifene, another SERM, to prevent osteoporosis and potentially reduce breast cancer risk, some women will develop raloxifene-resistant breast cancer. We developed a raloxifene-resistant MCF-7 cell model (MCF-7/Ral) and investigated the nature of raloxifene-resistant breast cancer and its response to estradiol.


METHODS
Raloxifene resistance and hormone responsiveness were assessed by proliferation assays and cell cycle analysis in parental MCF-7 and MCF-7/Ral cells. Nuclear factor kappaB (NF-kappaB) activity was investigated with a transient transfection assay. Apoptosis was investigated by annexin V staining, mRNA was measured by real-time polymerase chain reaction, and protein was measured by western blotting. Tumorigenesis was studied by injecting MCF-7 or MCF-7/Ral cells into ovariectomized athymic mice (10 per group) and monitoring tumor size weekly. All statistical tests were two-sided.


RESULTS
Basal NF-kappaB activity was higher in MCF-7/Ral cells (1.6 U, 95% confidence interval [CI] = 1.2 to 2.0 U) than in MCF-7 cells (0.8 U, 95% CI = 0.4 to 1.1 U; P =.004). When cultured with 1 microM raloxifene, MCF-7/Ral cells grew statistically significantly (P<.001) faster than MCF-7 cells. Estradiol treatment of MCF-7/Ral cells arrested cells in G(2)/M phase of the cell cycle, decreased NF-kappaB activity (0.2 U, 95% CI = 0.2 to 0.3 U; P<.001), increased expression of Fas protein and mRNA (4.5-fold, 95% CI = 2.8- to 6.3-fold versus 0.5-fold, 95% CI = 0.3- to 0.8-fold for control treatment; P<.001), and induced apoptosis. Treatment with either raloxifene or tamoxifen stimulated MCF-7/Ral tumor growth, suggesting that such tumors were resistant to both drugs. When a 9-week raloxifene or tamoxifen treatment was followed by a 5-week estradiol treatment, estradiol statistically significantly reduced the size of tumors stimulated by raloxifene or tamoxifen (at week 14, P =.004 for raloxifene and P<.001 for tamoxifen).


CONCLUSIONS
Growth of raloxifene-resistant MCF-7/Ral cells in vitro and in vivo is repressed by estradiol treatment by a mechanism involving G2/M-phase arrest, decreased NF-kappaB activity, and increased Fas expression to induce apoptosis.
Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Hong
H
Liu
Eun-Sook
ES
Lee
Csaba
C
Gajdos
Sandra Timm
ST
Pearce
Bin
B
Chen
Clodia
C
Osipo
Jessica
J
Loweth
Kevin
K
McKian
Alexander
A
De Los Reyes
Laura
L
Wing
V Craig
VC
Jordan
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D016328:NF-kappa B / Q000032:analysis; D010052:Ovariectomy; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D020849:Raloxifene Hydrochloride / Q000494:pharmacology; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology
D000970:Antineoplastic Agents; D016328:NF-kappa B; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D020845:Selective Estrogen Receptor Modulators; D020849:Raloxifene Hydrochloride; D004958:Estradiol