Glutathione S-Transferase pi-1 Knockdown Reduces Pancreatic Ductal Adenocarcinoma Growth by Activating Oxidative Stress Response Pathways

Abstract

Glutathione S-transferase pi-1 (GSTP1) plays an important role in regulating oxidative stress by conjugating glutathione to electrophiles. GSTP1 is overexpressed in breast, colon, lung, and prostate tumors, where it contributes to tumor progression and drug resistance; however, the role of GSTP1 in pancreatic ductal adenocarcinoma (PDAC) is not well understood. Using shRNA, we knocked down GSTP1 expression in three different PDAC cell lines and determined the effect on cell proliferation, cell cycle progression, and reactive oxygen species (ROS) levels. Our results show GSTP1 knockdown reduces PDAC cell growth, prolongs the G0/G1 phase, and elevates ROS in PDAC cells. Furthermore, GSTP1 knockdown results in the increased phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun and the decreased phosphorylation of extracellular signal-regulated kinase (ERK), p65, the reduced expression of specificity protein 1 (Sp1), and the increased expression of apoptosis-promoting genes. The addition of the antioxidant glutathione restored cell viability and returned protein expression levels to those found in control cells. Collectively, these data support the working hypothesis that the loss of GSTP1 elevates oxidative stress, which alters mitogen-activated protein (MAP) kinases and NF-κB signaling, and induces apoptosis. In support of these in vitro data, nude mice bearing orthotopically implanted GSTP1-knockdown PDAC cells showed an impressive reduction in the size and weight of tumors compared to the controls. Additionally, we observed reduced levels of Ki-67 and increased expression of cleaved caspase-3 in GSTP1-knockdown tumors, suggesting GSTP1 knockdown impedes proliferation and upregulates apoptosis in PDAC cells. Together, these results indicate that GSTP1 plays a significant role in PDAC cell growth and provides support for the pursuit of GSTP1 inhibitors as therapeutic agents for PDAC.

Glutathione S-Transferases in Cancer

“…Similar findings have been reported about the interaction of GSTM3 and TRAF6 in cervical cancer cells [ 13 ]. Further, GSTP1 knockdown in pancreatic [ 59 ] and GSTM knockdown in cervical cancer cells [ 13 ] showed reduced phosphorylation of extracellular signal-regulated kinase (ERK1/2),which plays a pivotal role in promoting cell growth and proliferation in many mammalian cell types. We have previously reported that GSTP1 knockdown pancreatic ductal adenocarcinoma (PDAC) cells have impaired growth compared to the control [ 59 ].…”

“…Given that GSTP1 is a direct inhibitor of JNK activity, it is speculated that GSTP1 overexpression is associated with many drug-resistant tumors [ 90 , 91 , 92 ]. Elevated levels of GSTP1 are shown in pancreatic [ 59 ] and triple-negative breast cancer cells [ 12 ], where it interferes with the cellular signaling processes that influence cell survival, proliferation, and apoptosis. These non-enzymatic functions of GSTP1 provide an explanation for drug-resistance in GSTP1-overexpressing tumors to chemotherapeutic agents that are poor substrates for this enzyme [ 108 ].…”

“…Drug discovery and development have identified GST inhibitors as promising therapeutic targets to counter drug resistance in cancer patients. Convincing data suggest inhibiting GSTP1 protein levels and activity can increase oxidative stress, impair cancer-cell survival, reduce chemoresistance, and improve overall survival in patients [ 12 , 13 , 59 ]. However, one of the significant roadblocks that GST inhibitors encounter in clinical trials is their insufficient specificity.…”

“…Further, identical correlations were found for GSTM1 and GSTP1 for glioma ( n = 153) ( Figure 8 G), urothelial cancer ( n = 406) ( Figure 8 H), ovarian cancer ( n = 373) ( Figure 8 I), breast cancer ( n = 1075) ( Figure 8 J) [ 210 ], lung cancer ( n = 994) ( Figure 8 K), and pancreatic cancer ( n = 176) ( Figure 8 L) [ 59 ] patients. However, poor patient survival with the overexpression of GST proteins is not uniformly corroborated.…”

“…The contrasting reports can be attributed to the patient population, polymeric forms of GST proteins [ 213 ], and treatment regime variations among the studies. Despite the lack of clarity on the impact of overactive GST proteins on the overall survival of cancer patients, there is a popular consensus that higher expression of GST proteins drives tumor pathogenicity and results in poor outcomes [ 12 , 13 , 59 , 214 ]. The conflicting data currently makes GST proteins an unreliable prognostic marker for cancer-patient survival.…”