TRAIL-Based High Throughput Screening Reveals a Link between TRAIL-Mediated Apoptosis and Glutathione Reductase, a Key Component of Oxidative Stress Response

Abstract

A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR), a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS) and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH) fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model.

Targeting mitochondria in cancer therapy could provide a basis for the selective anti-cancer activity

“…Starting compounds should [1] elicit a reproducible response in at least two assay types and have a dose-response over a hundred-fold concentration range; [2] be analytically validated in terms of integrity and purity; [3] demonstrate adequate potency; [4] possess a tractable starting point of chemical optimization with no obvious major chemical liabilities. Most of these requirements were addressed in our previous publication [8]. Thus, we demonstrated that our lead compound, NSC130362 (Fig 1, A) selectively induces reproducible responses in either oxidative stress or caspase 3/7 activity assays in cancer cells.…”

“…Elevated levels of reactive oxygen species (ROS) and subsequent oxidative stress are hallmarks of carcinogenesis and metastasis providing a potential therapeutic index [2–4]. Our data and recent studies by others demonstrated that elevated levels of ROS can be exploited in vitro and in vivo to preferentially target cancer cells while sparing normal cells [5–8]. The ROS-based approach to induce apoptosis in cancer cells is conceptionally different from conventional therapy targeting well known oncogenes and tumor suppressors - a therapy which is often ineffective due to multiple genetic and epigenetic alterations in cancer cells and the ability of cancer cells to upregulate compensatory mechanisms [9, 10].…”

“…We also showed that its combination with different oxidative stress inducers, such as arsenic trioxide (ATO), myricetin (Myr), and buthionine sulfoximine (BSO), caused cell death in a variety of breast, pancreatic, prostate, and lung carcinoma cell lines as well as in human melanoma MDA-MB-435 and AML cells from patients. Finally, NSC130362 demonstrated anti-tumor activity in vivo [8]. To complement these results, we performed dose-response studies in pancreatic carcinoma MIA PaCa-2 cells (Fig 1, B).…”

“…For comparison purposes, we also included FDA-approved ATO in this analysis. Our selection of ATO was based on our previous findings showing that ATO and NSC130362 exhibited synergistic responses against multiple cancer cells [8]. As we expected, NSC130362 efficiently potentiated the effect on cell viability of either ATO or MDV3100.…”

“…We recently reported the discovery of 1,4-naphthoquinine (1,4-NQ) derivative, NSC130362, which inhibits GSR and, as a consequence, induces oxidative stress and subsequent apoptosis in cancer cells but not in normal human primary hepatocytes. NSC130362 also showed anti-tumor activity in vivo [8]. In addition to inhibiting GSR, 1,4- NQs can be reduced by NADH/NADPH dehydrogenase followed by autoxidation, which results in the formation of ROS and potential oxidative stress.…”