Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy

Abstract

Active cell death in hormone-dependent cells was studied using cultured human mammary carcinoma cells (MCF-7) treated with the anti-estrogens (AEs) tamoxifen (TAM), 4-hydroxy-tamoxifen (OH-TAM) or ICI 164 384 (10(-8)-10(-5) M) as a model. The following results were obtained. (i) In untreated MCF-7 cells a wave of replication occurred in the first 5 days of culture. All three AEs caused a dose-dependent inhibition of cell replication. (ii) TAM and OH-TAM at 10(-5) M, but not ICI 164 384, caused lytic cell death (necrosis) within 24 h, which was not inhibited by estradiol (10(-9)-10(-6)M). (iii) Lower concentrations of TAM or OH-TAM (up to 10(-6) M) or ICI 164 384 induced a more gradual appearance of cell death beginning at day 3. This type of cell death was inhibited by estradiol (10(-9) M), indicating its active nature. (iv) Nuclei showed two distinct patterns of alteration: (a) apoptosis-like condensation and fragmentation of chromatin to crescent masses abutting the nuclear envelope; (b) condensation of the chromatin to a single, pyknotic mass in the center of the nucleus, detached from the nuclear envelope. Quantitative histological evaluation revealed the predominance of pyknosis. (v) Biochemical DNA analysis revealed that only a relatively small amount of the total DNA was finally degraded into low molecular weight fragments (20 kb and less). (vi) Active cell death, with both apoptotic and pyknotic nuclear morphology, was associated with extensive formation of autophagic vacuoles (AV).3-Methyladenine, a known inhibitor of AV formation, partially prevented cell death as detected by nuclear changes. (vii) ICI 164 384 was about 10 times more effective than TAM or OH-TAM at inhibiting DNA synthesis, but had equal potency in inducing active cell death. It is concluded that AEs have anti-proliferative and anti-survival effects on MCF-7 human mammary cancer cells in culture. These two effects are under separate control because they differ by kinetics, dose dependence and sensitivity to the various AEs. Active cell death in MCF-7 cells seems to be initiated by autophagy, in contrast to concepts of apoptosis, and thus corresponds to autophagic/ lysosomal or type II death as previously defined. This may be important because of biochemical and molecular differences between these various subtypes of active cell death.

Artemin is estrogen regulated and mediates antiestrogen resistance in mammary carcinoma

“…However, in the presence of either tamoxifen or fulvestrant, MCF7-ARTN cells showed an increase in cell number compared with MCF7-Vec cells ( Figure 3a). As the growth inhibitory effect of antiestrogen results from both inhibition of cell-cycle progression (Doisneau-Sixou et al, 2003) and induction of cell apoptosis (Bursch et al, 1996;Mandlekar and Kong, 2001), we determined S-phase entry by bromodeoxyuridine (BrdU) incorporation and apoptosis with Hoechst staining. Both tamoxifen and fulvestrant suppressed BrdU incorporation ( Figure 3b) and increased apoptosis (Figure 3c) in MCF7-Vec cells.…”

Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells

“…In some cases, autophagy plays a key role in the elimination of cancer cells by triggering a nonapoptotic cell death program. 14,37 This type of autophagy is irreversible and is referred to type II programmed cell death or autophagic cell death, in contrast to apoptosis, which is designated as type I programmed cell death. 37,38 More recently, we have demonstrated that arsenic trioxide induces autophagic cell death in malignant glioma cells.…”

Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes

“…MCF-7 human breast cancer cells were kindly provided by Dr W Bursch, Medizinische Universität Wien, Austria; culture conditions were described in detail previously. 5 Briefly, cells were grown as a monolayer in DMEM supplemented with 10% FCS, L-glutamine (300 mg/l) and penicillin/streptomycin antibiotics at 371C in an atmosphere of 5% CO 2 . Seven days before beginning an experiment cells were plated at density of 7.5 Â 10 3 /cm 2 in DCC according to Bardon et al 23 Twenty-four hours later the cells were treated with freshly prepared TAM in dimethyl sulfoxide/ethanol (1:1, v:v) added directly to the medium.…”

“…3 Type II PCD or autophagic cell death has been observed during embryonic development. 4,5 Since the first description of autophagy in 1966, 6 numerous studies have described it as a survival mechanism under poor nutritional conditions. It is now clear this process has a dual role.…”

“…7 By contrast, autophagy is a degradative mechanism for long-lived proteins and damaged organelles through the auto-phago-lysosomal pathway and on the other, it provides possibility of self-destruction for cells. 4,5,8 In Drosophila, autophagy can be hormonally controlled by ecdysone; through inhibition of a class-I phosphatidyl-inositol 3-kinase (PI3K) pathway organs, such as the fat body, are eliminated at the end of larval stage. 8,9 MCF-7 cells can also be induced to undergo autophagy and cell death by treatment with the antiestrogen tamoxifen, which increases the intracellular ceramide level and eliminates the inhibitory effect of class-I PI3K pathway.…”