Diindolylmethane and its halogenated derivatives induce protective autophagy in human prostate cancer cells via induction of the oncogenic protein AEG-1 and activation of AMP-activated protein kinase (AMPK)
Introduction
Prostate cancer is a major health problem worldwide, ranking as the second most common cancer in males [1] and the third leading cause of cancer-related deaths among American and Canadian men [2]. Treatment with drugs targeting androgen receptor (AR) signaling is the main therapy for early-stage androgen-dependent (AD) prostate cancer [3]. Unfortunately, many patients with AD prostate cancer will progress to an androgen-independent (AI) phenotype, which is harder to treat and often fatal [4], [5]. Diindolylmethane (DIM) is a promising anticancer agent derived from the ingestion of Brassica plants (cabbage, broccoli, etc.) [6]. We have shown that several di-halogenated analogs of DIM (ring-DIMs) have anti-androgenic effects in human AD LNCaP prostate cancer cells [7] and induce apoptosis and necrosis in LNCaP and human AI PC-3 prostate cancer cells with greater potencies than DIM [8]. More recently, we have shown that DIM and ring-DIMs induce ER stress, mitochondrial dysfunction, and autophagy in prostate cancer cells [9]. Autophagy is a self-digestion process activated by cellular stress, in which dysfunctional organelles and protein aggregates are sequestered in double-membraned vesicles [10], and then transported to lysosomes for proteolytic degradation and recycling to maintain cellular homeostasis [11].
Autophagy plays an important role in cancer cell progression; its induction in response to stresses following chemotherapy may promote cancer cell survival. However, excessive autophagy could activate a cell death mechanism known as cytotoxic autophagy, which is different from programmed cell death (apoptosis) [12], [13]. Uncontrolled growth of cancer cells is facilitated by the inactivation of cell death pathways, such as apoptosis, and stimulation of cell survival pathways [14]. Thus, the dysregulation of autophagic machinery in cancer cells, leading to imbalances in the activation of cell death- or survival-related pathways, may have a critical influence on either tumor progression or regression. Various Atg (autophagy-related) proteins are involved in the initiation and regulation of autophagy [15]. The conversion of LC3B (Atg3) from its diffuse LC3BI form to the punctuated LC3BII, which associates with the autophagosome [16], is used as a classic marker of autophagy. AMP-activated protein kinase (AMPK) regulates autophagy via phosphorylation of the autophagy-initiating protein ULK1 (Atg1) [17]. The oncogenic protein, astrocyte elevated gene-1 (AEG-1), also known as metadherin (MTDH) or protein LYRIC, is overexpressed in various cancer including that of the prostate [18], [19], where it acts as a mediator of AMPK activity and autophagy in response to cellular metabolic stress [20], [21]. AEG-1 contributes to chemoresistance in hepatocellular carcinoma (HCC) cells [22] and promotes hepato-carcinogenisis through inhibition of senescence in transgenic mice that overexpress hepatocyte-specific AEG-1 [23].
Although DIM is known to be a mitochondrial ATP synthase inhibitor [24] that alters AMP/ATP ratios leading to activation of AMPK [25], the exact mechanism(s) of DIM-induced protective autophagy has not been elucidated. Previous studies have shown that AEG-1 induces protective autophagy via activation of AMPK [21]. Therefore, we wished to determine the possible involvement of AEG-1 in AMPK activation as well as DIM- and ring-DIM-mediated induction of autophagy.
Section snippets
Cell culture and treatment
LNCaP AR-positive and AD human prostate cancer cells were purchased from the American Type Culture Collection (Manassas, VA); LNCaP C4-2B (C42B) AR-positive and AI human prostate cancer cells were purchased from the MD Anderson Cancer Centre (Houston, TX). LNCaP and C42B prostate cancer cells were cultured in RPMI 1640 medium supplemented with 10% FBS, and 1% penicillin/streptomycin (Life Technologies, Gaithersburg, MD) at 37 °C and 5% CO2. Ring-DIMs were synthesized in our laboratories as
DIM and ring-DIMs induce the formation of autophagic vacuoles in prostate cancer cells
An 8-h treatment of androgen-sensitive LNCaP and androgen-insensitive C42B cells with DIM, 4,4′-Br2DIM and 7,7′-Cl2DIM significantly increased the formation of autophagic vacuoles (Fig. 1A–D). The concentrations used in these experiments were based on our previous studies on DIM- and ring-DIM-mediated induction of protective autophagy in the same prostate cancer cells [8], [9]. We also confirmed the formation of autophagosomes by transmission electron microscopy after exposure of C42B cells to
Discussion
Our current findings demonstrate that DIM and ring-DIMs activate the AMPK signaling in LNCaP and C42B cells by increasing AMPK-, ACC-(a substrate uniquely phosphorylated by AMPK) and ULK1 phosphorylation in a time-dependent manner (Fig. 2). Chen and coworkers previously reported that a formulated DIM (B-DIM), which has increased bioavailability in vivo, can activate AMPK signaling as early as 3 h after exposure [25]. DIM has also been shown to activate AMPK signaling in ovarian cancer cells, and
Conclusion
We have identified a novel mechanism of DIM- and ring-DIM-induced protective autophagy, which is due to induction of AEG-1 and subsequent activation of AMPK. Our results suggest that development of novel drug therapies against prostate cancer could include selective autophagy inhibitors as adjuvants. Moreover, targeting DIM- and ring-DIM-mediated induction of AEG-1 and subsequent induction of senescence may be an effective novel therapy for treating prostate cancer.
The following are the
Acknowledgments
This work was funded by an operating grant from the Canadian Institutes of Health Research (CIHR grant no. MOP-115019) to JTS, EG and SS. HD received a scholarship from the Fonds de Recherche du Québec - Santé (FRQS). All authors declare to have no conflicts of interest.
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2022, BiochimieCitation Excerpt :Activation of AMPK can inhibit the occurrence of cancer, but may also promote the occurrence and development of cancer, depending on the specific situation. In LNCaP and C42B human prostate cancer cells, indolemethane and its halogenated derivatives induce protective autophagy in cancer cells by inducing phosphorylation of AMPK, ULK-1 and acetyl-CoA carboxylase [142]. Downregulation of AEG-1 or AMPK can inhibit DIM and RING-DIM-induced autophagy.