Inhibition of JNK-Mediated Autophagy Promotes Proscillaridin A- Induced Apoptosis via ROS Generation, Intracellular Ca+2 Oscillation and Inhibiting STAT3 Signaling in Breast Cancer Cells.

Muhammad Khan,Muhammad Azhar Nisar,Muhammad Zubair Saleem,Gulzar Alam,Tonghui Ma,Mohammed Alshwmi,Lingling Jin,Shahid Alam,He Zhang,Syed Riaz Ud Din

doi:10.3389/fphar.2020.01055

Abstract

Breast cancer is the most heterogenous cancer type among women across the world. Despite concerted efforts, breast cancer management is still unsatisfactory. Interplay between apoptosis and autophagy is an imperative factor in categorizing therapeutics for cancer treatment. Proscillaridin A (PSD-A), a well-known cardiac glycoside used for cardiac arrest and arrythmias, has been unveiled in many cancer types but the underlying mechanism for apoptosis and autophagy in breast cancer is not fully understood. In our study, PSD-A restricted cell growth, inhibited STAT3 activation and induced apoptosis and autophagy in breast cancer cells via ROS generation and Ca+2 oscillation. Pretreatment of NAC and BAPTA-AM restored PSD-A induced cellular events in breast cancer cells. PSD-A induced apoptosis via DNA fragmentation, caspase-cascade activation, PARP cleavage, mitochondrial dysfunction, Bax/Bcl-2 proteins modulation and ER chaperone GRP78 inhibition along with decreased phosphorylation of ERK1/2. Inhibition of STAT3 activation was found to be associated with decreased phosphorylation of SRC. Moreover, PSD-A induced events of autophagy i.e. conversion of LC3-I to LC3-II, and Atg3 expression via JNK activation and decreased mTOR and AKT phosphorylation. In this study, pretreatment of SP600125, a JNK inhibitor, reduced autophagy and enhanced STAT3 inhibition and apoptosis. Additionally, SB203580, a commercial p38 inhibitor, stimulated STAT3 activation and improved autophagic events rate in breast cancer cells, displaying the role of the MAPK signaling pathway in interplay between apoptosis and autophagy. Our data suggest that the rate of apoptotic cell death is improved by blocking JNK-induced autophagy in PSD-A treated MCF-7 and MDA-MB-231 breast cancer cells.

Highlights

Breast cancer is the most prevalent heterogeneous type of disease with a high incidence and mortality rate
Collective data of Cell counting kit-8 (CCK-8) assay, morphological examination and clonogenic assay reveal that Proscillaridin A (PSD-A) inhibits proliferation and induces cytotoxic effect in MCF-7 and MDA-MB-231 breast cancer cell lines
We found that pretreatment of NAC decreased PSD-A induced poly (ADP ribose) polymerase (PARP) cleavage up to a low significant level while that of BAPTA-AM significantly decreased PARP cleavage in both cell types indicating the dynamic role of intracellular Ca+2 in apoptosis (Figure 3F)

Summary

Introduction

Breast cancer is the most prevalent heterogeneous type of disease with a high incidence and mortality rate. It is the second leading cause of cancer-related death in women after lung cancer (Yuan et al, 2017). Breast cancer has been classified into different molecular subtypes, based on presence or absence of hormone receptors (HR+/HR-) for estrogen or progesterone and excessive level of human epidermal growth factor receptor 2 (HER2+/HER2-). Luminal A (HR+/HER2-) and Luminal B (HR+/HER2+) are hormone receptor positive breast cancer subtypes, accounting for 70-80% of all breast cancer types (Haque et al, 2012). TNBC is identified by the absence of estrogen receptor, progesterone receptor, and overexpression of human epidermal growth factor receptor-2 (HR-/HER2-). Comparative to Luminal A and Luminal B, TNBC is highly recurrent and distantly metastatic breast cancer and irresponsive to commonly used hormone therapies to treat breast cancer due to lack of specific target sites (Qian et al, 2013)

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