Annonacin induces cell cycle-dependent growth arrest and apoptosis in estrogen receptor-α-related pathways in MCF-7 cells

doi:10.1016/j.jep.2011.07.056

Journal of Ethnopharmacology

Volume 137, Issue 3, 11 October 2011, Pages 1283-1290

https://doi.org/10.1016/j.jep.2011.07.056 Get rights and content

Abstract

Ethnopharmacological relevance

Tamoxifen resistance is common in estrogen receptor-α (ERα)-positive breast cancers. Pawpaw and soursop are anticancer annonaceous plants in complementary medicine. Thus, we studied the effects of annonacin, an annonaceous acetogenin, in breast cancer cells.

Materials and Methods

Cell growth and ERα-related pathways were studied. The effects of annonacin were tested in MCF-7 xenografts in nude mice.

Results

In ERα-positive MCF-7 cells, annonacin (half-effective dose ED₅₀ = 0.31 μM) and 4-hydroxytamoxifen (ED₅₀ = 1.13 μM) decreased cell survival whereas annonacin (0.5-1 μM) increased cell death at 48 h. Annonacin and 4-hydroxytamoxifen were additive in inhibiting cell survival. Annonacin (0.1 μM) induced G₀/G₁ growth arrest while increasing p21^WAF1 and p27^kip1 and decreasing cyclin D1 protein expression. Annonacin (0.1 μM) decreased cyclin D1 protein expression more than 4-hydroxytamoxifen (1 μM). Annonacin (0.1 μM) increased apoptosis while decreasing Bcl-2 protein expression. The combination of annonacin (0.1 μM) and 4-hydroxytamoxifen (1 μM) decreased Bcl-2 protein expression and ERα transcriptional activity more than annonacin (0.1 μM) did alone. Annonacin, but not 4-hydroxytamoxifen, decreased ERα protein expression. Moreover, annonacin decreased phosphorylation of ERK1/2, JNK and STAT3. In nude mice, annonacin decreased MCF-7 xenograft tumor size at 7–22 days. Moreover, annonacin decreased ERα, cyclin D1 and Bcl-2 protein expression in the xenograft at 22 days.

Conclusions

Annonacin induced growth arrest and apoptosis in ERα-related pathways in MCF-7 cells. Annonacin and 4-hydroxytamoxifen were additive in inhibiting cell survival and ERα transcriptional activity. Moreover, annonacin attenuated MCF-7 xenograft tumor growth while inhibiting ERα, cyclin D1 and Bcl-2 protein expressions in nude mice.

Graphical abstract

Introduction

The pathogenesis of breast cancer includes estrogen and estrogen receptor-α (ERα)-related pathways (Osborne and Schiff, 2011) whereby nuclear ERα activates target genes via the estrogen-response elements (ERE) (Osborne and Schiff, 2011). Additionally, nonnuclear ERα rapidly activates growth factor downstream signals such as extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinase (JNK) and signal transducers and activators of transcription 3 (STAT3) (Musgrove and Sutherland, 2009, Osborne and Schiff, 2011). Interestingly, STAT integrates cytoplasmic and nuclear estrogen actions (Fox et al., 2009).

Selective estrogen receptor modulators (SERM, such as tamoxifen) or selective estrogen receptor down-regulators (SERD, such as fulvestrant) are effective treatments for ERα-positive breast cancers commonly limited by resistance (Osborne and Schiff, 2011). Endocrine resistance may be induced by the loss of ERα, increased nonnuclear ERα or growth factor receptor signaling, deranged signal transducers (such as ERK1/ERK1, JNK and STAT3), cell cycle regulators (such as cyclin D1 and the cyclin-dependent kinase inhibitors p21^WAF1 and p27^kip1) or apoptosis regulators (such as the anti-apoptotic Bcl-2) (Musgrove and Sutherland, 2009, Osborne and Schiff, 2011).

Pawpaw (Asimina triloba) and soursop (graviola, Annona muricata) are annonaceous plants used as anticancer folk therapies in (North, Central and South) America and Southeast Asia and have been studied in a few observational clinical studies (Cassileth, 2008, Coothankandaswamy et al., 2010, Liaw et al., 2010, McLaughlin, 2008). Annonaceous acetogenins are cytotoxic to multidrug-resistant MCF-7 cells (Oberlies et al., 1997). Annonacin (C₃₅H₆₄O₇), an annonaceous acetogenin containing a mono-tetrahydrofuran ring with two flanking hydroxyls, also inhibits growth in MCF-7 cells (Yuan et al., 2003). However, the molecular mechanisms are not understood.

Thus, we studied the growth-inhibitory mechanisms of annonacin in terms of ERα-related pathways (p-ERK1/2, p-JNK, p-STAT3, cyclin D1, Bcl-2, p21^WAF1 and p27^kip1) in MCF-7 cells. Moreover, the effects of annonacin on MCF-7 xenografts in nude mice were also investigated.

Section snippets

Cell culture and reagents

ERα-positive MCF-7 cells and ERα-negative MDA-MB-231 cells were purchased on February 18, 2009 from Bioresource Collection and Research Center (Hsinchu, Taiwan), where cells were authenticated by DNA fingerprints of short tandem repeat profiling. Cells were cultured in DMEM/F-12 (1:1) medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) in a humidified 5% CO₂ incubator at 37 °C. Cells were fasted for 24 h before adding fresh medium

Effects of annonacin on cell cycle distribution and cell death in MCF-7 cells

Annonacin (0.1 μM) time-dependently (6–48 h) arrested cells in the G₀/G₁ phase of the cell cycle (Fig. 1A) while increasing apoptosis at 48 h (Fig. 1B). Additionally, annonacin dose-dependently (0.5–1 μM) increased cell death at 48 h (Fig. 1C). Annonacin also dose-dependently (0.05–1 μM) decreased cell survival at 48 h (supplementary Fig. 2A). Moreover, annonacin (0.1 μM) time-dependently (24–48 h) decreased cell survival (supplementary Fig. 2B). However, annonacin (0.1 μM) did not affect cell survival

Discussion and conclusions

This study adds mechanistic insights to our previous finding that annonacin inhibits growth in MCF-7 cells (Yuan et al., 2003).We found that annonacin induced cell death only at high doses (≥0.5 μM). In contrast, low-dose (0.1 μM) annonacin, like tamoxifen and fulvestrant (Cariou et al., 2000, Musgrove and Sutherland, 2009), induced cell-cycle-dependent (G₀/G₁) growth arrest concomitantly with the induction of p21^WAF1 and p27^kip and the inhibition of cyclin D1 protein expression.

Cell cycle (G₀/G₁

Acknowledgement

This work was supported by the National Science Council of Taiwan (NSC-94-2321-B-037-006) to Lea-Yea Chuang.

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