| Thiazolidinediones (TZDs), including troglitazone, ciglitazone, rosiglitazone, and pioglitazone, are specific ligands of peroxisome proliferator-activator receptor gamma(PPARgamma). Although best known as drugs for the treatment of type-2 diabetes that work via a receptor-dependent mechanism, in recent years, the TZDs have been shown to possess antitumor activity as well. However, the role of PPARgamma-agonist activity in the antitumor mechanism of TZDs remains controversial. In this study, we provide evidence that the antitumor activity of TZDs is independent of PPARgamma activation. This evidence includes the nearly equivalent sensitivities of the prostate cancer cells lines LNCaP, PC3 and DU145 to the antiproliferative effects of TZDs, despite wide differences among these cells in PPARgamma expression (LNCaP, low expression PC3 and DU145, high expression). Also, the Delta2-TZD analogs, STG28 and OSU-CG12, which are devoid of PPARgamma-agonist activity, exhibit multifold greater anti-proliferative potency than the parental TZDs (troglitazone and ciglitazone, respectively).Our ongoing work to characterize the mechanism of the PPARgamma-independent antitumor effects of TZDs revealed that these agents act as energy restriction mimetics through the inhibition of glucose metabolism. Using multiple approaches to assess glucose uptake, glycolytic rate, NADH production, and lactate formation, we show that TZDs perturb intracellular glucose homeostasis in a manner similar to that of known energy restriction mimetic agents (ERMAs), such as 2-deoxyglucose and resveratrol, as well as glucose starvation. Moreover, TZDs induce rapid effects in cancer cells that are identical to the hallmark cellular responses associated with energy restriction. These include the rapid induction of silent information regulator 1 (Sirt1) expression and activating phosphorylation of AMP-associated protein kinase (AMPK) within the first 10 min of treatment, followed by induction of endoplasmic reticulum (ER) stress responses at 2h post-treatment. TZD-induced AMPK phosphorylation elevates tuberous sclerosis complex 2 (TSC2) complex activity, resulting in suppression of mTOR kinase and its downstream signaling component p70S6K. This deactivation of mTOR signaling leads to type II programmed cell death i.e. autophagy, which accounts for approximately 20% of TZD-mediated cell death. In contrast, while TZD-mediated induction of ER stress leads to the expression of many stress-responsive proteins, such as GRP78, GADD153, and IRE1alpha, the precise role of ER stress in the antitumor activity of TZDs remains unclear.The induction of Sirt1 by TZDs is transient and short-lived with a duration of approximately 1h, and leads to reduced expression of Cop9 signalosome subunit 5 (CSN5), the negative regulator of Skp1-Cul1-F-box protein (SCF) complex activity, at the transcriptional level at 1h post-treatment. The resultant activation of the SCF complex leads to the ubiquitin-dependent proteasomal degradation of the F-box protein S-phase kinase-associated protein 2 (Skp2), which causes the upregulation of a Skp2 substrate, the CDK inhibitor p27, and subsequent G1 arrest. Another F-box protein, beta-transducin repeat-containing protein (beta-TrCP), was first identified in our laboratory as a Skp2 substrate. Thus, the suppression of Skp2 expression by TZDs results in the accumulation of beta-TrCP protein, which is evident after 12h of treatment, which in turn results in a wave of proteolytic degradation of a number of beta-TrCP substrates that include important apoptotic- and cell cycle-regulatory proteins, such as beta-catenin, cyclin D1, Wee1, Cdc25A, NFkappaB/p105, IkappaBalpha, Emi1, and particularly the transcriptional factor, specificity protein 1 (Sp1). It is intriguing that one of the target genes of Sp1 is Skp2. Thus, reduction of Sp1 levels resulting in a negative feedback regulation on Skp2 repression leading to further accumulation of beta-TrCP protein.The proteolysis of Sp1 is important to the antitumor effects of TZDs in that it represses the transcriptional expression of a host of Sp1 target genes, such as Skp2, Dnmt1, ERalpha, EGFR, and androgen receptor (AR), all of which are pivotal to cancer cell proliferation and survival. The transcriptional repression of AR provides a mechanistic basis for one of our earliest observations in TZD-treated prostate cancer cells, which is the reduced expression/secretion of prostate specific antigen (PSA), an important biomarker for prostate cancer progression.In conclusion, our findings show that the antitumor mechanism of TZDs in prostate cancer cells is PPARgamma-independent and is involves mimicking glucose restriction to induce three major signaling pathways: the Sirt1-CSN5-Skp2-beta-TrCP-Sp1 cascade which leads to G1 arrest and apoptosis, the AMPK-TSC1/2-mTOR-p70S6K cascade which results in autophagic cell death, and the ER stress signaling of which the role in TZD-mediated cancer cell death remains vague. |
