Structural and Biochemical Analyses Reveal the Mechanism of Glutathione S-Transferase Pi 1 Inhibition by the Anti-cancer Compound Piperlongumine

Abstract

Glutathione S-transferase pi 1 (GSTP1) is frequently overexpressed in cancerous tumors and is a putative target of the plant compound piperlongumine (PL), which contains two reactive olefins and inhibits proliferation in cancer cells but not normal cells. PL exposure of cancer cells results in increased reactive oxygen species and decreased GSH. These data in tandem with other information led to the conclusion that PL inhibits GSTP1, which forms covalent bonds between GSH and various electrophilic compounds, through covalent adduct formation at the C7-C8 olefin of PL, whereas the C2-C3 olefin of PL was postulated to react with GSH. However, direct evidence for this mechanism has been lacking. To investigate, we solved the X-ray crystal structure of GSTP1 bound to PL and GSH at 1.1 Å resolution to rationalize previously reported structure activity relationship studies. Surprisingly, the structure showed that a hydrolysis product of PL (hPL) was conjugated to glutathione at the C7-C8 olefin, and this complex was bound to the active site of GSTP1; no covalent bond formation between hPL and GSTP1 was observed. Mass spectrometry (MS) analysis of the reactions between PL and GSTP1 confirmed that PL does not label GSTP1. Moreover, MS data also indicated that nucleophilic attack on PL at the C2-C3 olefin led to PL hydrolysis. Although hPL inhibits GSTP1 enzymatic activity in vitro, treatment of cells susceptible to PL with hPL did not have significant anti-proliferative effects, suggesting that hPL is not membrane-permeable. Altogether, our data suggest a model wherein PL is a prodrug whose intracellular hydrolysis initiates the formation of the hPL-GSH conjugate, which blocks the active site of and inhibits GSTP1 and thereby cancer cell proliferation.

Piperlongumine rapidly induces the death of human pancreatic cancer cells mainly through the induction of ferroptosis

“…The PANC-1 cells were pre-treated with the ROS scavenger, NAC, the ferroptosis inhibitors (Ferr-1 and Liprox), or the iron chelator, DFO, for 2 h, and were then further cultured in the presence or absence of PL for 16 h. PL dose-dependently decreased PANC-1 cell viability: 14 µM PL decreased cell viability to approximately 10% of the control. Consistent with findings from previous studies indicating that increased ROS levels are critical for cell death induced by PL (21,22,25), NAC (3 mM) almost completely abrogated the PL-induced decrease in the viability of the PANC-1 cells (Fig. 1B).…”

“…PL selectively induces the death of numerous cancer cell lines, as well as cancerous tumors in animal models, including pancreatic cancer, breast cancer and leukemia, but does not exhibit anti-proliferative behavior in non-transformed cells, thus rendering it a good candidate for cancer treatment (21)(22)(23)(24). PL directly binds to and inhibits the antioxidant enzyme, glutathione S-transferase Pi 1, resulting in elevated intracellular ROS generation and subsequent apoptotic cell death in cancers with no apparent toxicity to normal cells (21,25). Zou et al recently reported that PL interacted with thioredoxin reductase 1 to induce the ROS-mediated apoptosis of human gastric cancer cells (35).…”

“…PL selectively induces the death of numerous cancer cell lines in vitro and in vivo, including pancreatic cancer, breast cancer, and leukemia, but does not exhibit anti-proliferative behavior in non-transformed cells (21)(22)(23)(24). Therefore, PL is an attractive molecule for use in the development of novel treatment regimens for these types of cancer (25). PL directly binds to and inhibits the antioxidant enzyme, glutathione S-transferase Pi 1, resulting in elevated intracellular ROS levels and subsequent apoptotic cell death in cancers with no apparent toxicity to normal cells (21,25).…”

“…Therefore, PL is an attractive molecule for use in the development of novel treatment regimens for these types of cancer (25). PL directly binds to and inhibits the antioxidant enzyme, glutathione S-transferase Pi 1, resulting in elevated intracellular ROS levels and subsequent apoptotic cell death in cancers with no apparent toxicity to normal cells (21,25). Apart from the induction of apoptosis, PL has been shown to cause cell cycle arrest (26), induce autophagy (27), and to inhibit migration and invasion (28,29).…”

Two Natural Alkaloids Synergistically Induce Apoptosis in Breast Cancer Cells by Inhibiting STAT3 Activation

“…The killing effects of PL involve inhibiting proliferation [15], inducing apoptosis [16], promoting ROS production [17], inhibiting migration and invasion [18], as well as sensitizing other chemotherapy agents [19,20], which occurs regardless of p53 status [21]. In addition, multiple signaling pathways are activated or inactivated by PL, including MAPK [22], PI3K/Akt/mTOR [16], nuclear factor kappa B (NF-κB) [23], GSTP1 [24], and TrxR1 [25]. Besides, PL has been confirmed to be a natural inhibitor of STAT3.…”