Inhibition of autophagy stimulate molecular iodine-induced apoptosis in hormone independent breast tumors

doi:10.1016/j.bbrc.2011.10.054

Biochemical and Biophysical Research Communications

Volume 415, Issue 1, 11 November 2011, Pages 181-186

https://doi.org/10.1016/j.bbrc.2011.10.054 Get rights and content

Abstract

Estrogen receptor negative (ER^−ve) and p53 mutant breast tumors are highly aggressive and have fewer treatment options. Previously, we showed that molecular Iodine (I₂) induces apoptosis in hormone responsive MCF-7 breast cancer cells, and non-apoptotic cell death in ER^−ve–p53 mutant MDA-MB231 cells (Shrivastava, 2006). Here we show that I₂ (3 μM) treatment enhanced the features of autophagy in MDA-MB231 cells. Since autophagy is a cell survival response to most anti-cancer therapies, we used both in vitro and in vivo systems to determine whether ER^−ve mammary tumors could be sensitized to I₂-induced apoptosis by inhibiting autophagy. Autophagy inhibition with chloroquine (CQ) and inhibitors for PI3K (3MA, LY294002) and H+/ATPase (baflomycin) resulted in enhanced cell death in I₂ treated MDA-MB231 cells. Further, CQ (20 μM) in combination with I₂, showed apoptotic features such as increased sub-G1 fraction (∼5-fold), expression of cleaved caspase-9 and -3 compared to I₂ treatment alone. Flowcytometry of I₂ and CQ co-treated cells revealed increase in mitochondrial membrane permeability (p < 0.01) and translocation of cathepsin D activity to cytosol relative to I₂ treatment. For in vivo studies ICRC mice were transplanted subcutaneously with MMTV-induced mammary tumors. A significant reduction in tumor volumes, as measured by MRI, was found in I₂ and CQ co-treated mice relative to I₂ or vehicle treated mice. These data indicate that inhibition of autophagy renders ER^−ve breast tumor cells more sensitive to I₂ induced apoptosis. Thus, I₂ together with autophagy inhibitor could have a potential tumorostatic role in ER^−ve aggressive breast tumors that may be evaluated in future studies.

Highlights

► Molecular iodine (I₂) causes non-apoptotic cell death in MDA-MB231 breast tumor cells. ► Autophagy is activated as a survival mechanism in response to I₂ in MDA-MB231. ► Autophagy inhibition sensitizes tumor cells to I₂-induced apoptotic cell death. ► Autophagy inhibitor potentiates apoptosis and tumor regressive effects of I₂ in mice.

Introduction

Breast cancer, a leading cause of cancer related deaths in women, is one of the most frequently diagnosed cancers. Breast tumors are considered estrogen dependent and consequently, anti-estrogens and aromatase inhibitors are used as hormonal treatment for hormone responsive tumors, i.e. those with estrogen receptor (ER) and progesterone receptor (PR) expression in the tumor tissue hormone treatment is not effective in patients with ER/PR negative tumors. Furthermore, in many cases, ER positive (ER^+ve) tumors initially respond to hormonal treatment but subsequently become resistant to endocrine therapy [1].

About 50% of breast cancers have p53 deficient/mutant tumor suppressor gene, a critical mediator of cell death. Aggressive nature of ER negative p53 mutant/deficient breast tumors require better therapeutic choices. It has been recently shown that deletion, depletion or inhibition of p53 induces autophagy [2], whether autophagy represents a mechanism for cell death or survival is unclear and could lead to therapeutic resistance [3]. In this regard, inhibition of autophagy, by chloroquine (CQ), has been found to enhance therapeutic efficacy in cMyc lymphoma [4]. However, the concurrent use of CQ has not been evaluated with breast cancer therapies.

Iodine has long been known to maintain the normal physiology of thyroid and breast tissue. Molecular iodine (I₂) has been found effective in diminishing mammary dysplasia and atypia resulting from iodine deficiency, symptoms of mammary fibrosis in women and occurrence of chemically induced mammary cancer in rats (50–70%) [5]. Earlier we demonstrated an estrogen receptor and p53 status independent cytotoxic effect of I₂ in breast cancer cells [6]. Furthermore, chronic I₂ supplementation in rats had no demonstrable harmful secondary effects on either thyroid or general physiology [7].

In the present study we attempt to demonstrate that I₂ induces cell death as well as autophagic response. In addition, we evaluated if inhibition of autophagy by sub-therapeutic antimalarial dose of chloroquine (CQ) enhances I₂ mediated cell death by p53 independent and caspase mediated apoptosis, and whether CQ potentiates apoptotic and tumor regressive effects of I₂ treatment in vivo.

Section snippets

Cytotoxicity assay

Human breast cancer cell line MDA-MB231 was obtained and maintained as described previously [6]. Cells were treated with either vehicle, 3 μM I₂ alone or combination of 3 μM I₂ and 20 μM chloroquine (Sigma). The stock solution of 75 μM iodine was prepared as previously described [6]. In parallel experiments following inhibitors were used; baflomycin (Sigma, 20 nM); 3 methyladenine (3MA) (Sigma, 1 mM) and LY294002 (Sigma, 10 μM). Cell viability was assessed by trypan blue (0.01%) dye-exclusion assay.

Live cell staining

Molecular iodine induces cell death and autophagy in MDA-MB231 cells

Previously, we found that I₂ induces apoptosis in hormone responsive (ER positive) and p53 positive MCF-7 cells, however, in MDA-MB231 breast cancer cells I₂ induces non-apoptotic cell death [6]. In this study we confirmed that MDA-MB231 breast cancer cells are resistant to apoptotic effects of iodine. High levels of mutant p53 gene in these cells, stabilized by elevated phospholipase D (PLD) activity, may contribute to the suppression of apoptosis [11]. However, for the first time we now

Grant support

This work was supported by the Department of Science and Technology, and Department of Biotechnology New Delhi (Grant SR/SO/HS/17/2003 and BT/PR9822/MED/30/42/2007 to Godbole M.M.) and research fellowship from the Council of Scientific and Industrial Research, New Delhi (9/590(0065)/2006/EMR-I to Singh P.).

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Biochemical and Biophysical Research Communications

Abstract

Highlights

Introduction

Section snippets

Cytotoxicity assay

Live cell staining

Molecular iodine induces cell death and autophagy in MDA-MB231 cells

Grant support

References (20)

Molecular iodine induces caspase-independent apoptosis in human breast carcinoma cells involving the mitochondria-mediated pathway

J. Biol. Chem.

Method and timing of tumor volume measurement for outcome prediction in cervical cancer using magnetic resonance imaging

Int. J. Radiat. Oncol. Biol. Phys.

Bcl-2 antiapoptotic proteins inhibit beclin-1 dependent autophagy

Cell

Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells

Bioorg. Med. Chem.

Antiestrogen therapy for breast cancer

Cancer Therapy

Regulation of autophagy by cytoplasmic p53

Nat. Cell Biol.

The role of autophagy in cancer development and response to therapy

Nat. Rev. Cancer

Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma

J. Clin. Invest.

Is iodine a gatekeeper of the integrity of the mammary gland?

J. Mammary Gland Biol. Neoplasia

Uptake and gene expression with antitumoral doses of iodine in thyroid and mammary gland: evidences that chronic administration has no harmful effects

Thyroid

Cited by (29)

Excessive apoptosis and disordered autophagy flux contribute to the neurotoxicity induced by high iodine in Sprague-Dawley rat

LINC00470 Coordinates the Epigenetic Regulation of ELFN2 to Distract GBM Cell Autophagy

Autophagy regulates high concentrations of iodide-induced apoptosis in SH-SY5Y cells

Iodine and Thyroid Hormone Synthesis, Metabolism, and Action

Synergistic killing effect of chloroquine and androgen deprivation in LNCaP cells

Hsp90 inhibitors and drug resistance in cancer: The potential benefits of combination therapies of Hsp90 inhibitors and other anti-cancer drugs