Transcriptome Analysis of Piperlongumine-Treated Human Pancreatic Cancer Cells Reveals Involvement of Oxidative Stress and Endoplasmic Reticulum Stress Pathways

Abstract

Piperlongumine (PL), an alkaloid obtained from long peppers, displays antitumorigenic properties for a variety of human cell-and animal-based models. The aim of this study was to identify the underlying molecular mechanisms for PL anticancer effects on human pancreatic cancer cells. RNA sequencing (RNA-seq) was used to identify the effects of PL on the transcriptome of MIA PaCa-2 human pancreatic cancer cells. PL treatment of pancreatic cancer cells resulted in differential expression of 683 mRNA transcripts with known protein functions, 351 of which were upregulated and 332 of which were downregulated compared to control-treated cells. Transcripts associated with oxidative stress, endoplasmic reticulum (ER) stress, and unfolded protein response pathways were significantly overexpressed with PL treatment. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to validate the RNA-seq results, which included upregulation of HO-1, IRE1a, cytochrome c, and ASNS. The results provide key insight into the mechanisms by which PL alters cancer cell physiology and identify that activation of oxidative stress and ER stress pathways is a critical avenue for PL anticancer effects.

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). In addition, PL has been shown to promote cell death by activating several mechanisms, including apoptosis, autophagy and necrosis, affecting the PI3K/AKT/mTOR, p38/JNK, MEK/ERK and NF-κB pathways (22,(27)(28)(29)36). In the present study, we found that PL alone induced the death of pancreatic cancer cells, at least in part, by the induction of ferroptosis, the execution of which requires the accumulation of ROS in an iron-dependent manner (16).…”

“…Piperlongumine (PL) is a biologically active alkaloid that largely exists in the long pepper (Piper longum L.). 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).…”

Matching cell lines with cancer type and subtype of origin via mutational, epigenomic and transcriptomic patterns

“…Similarly, for pancreatic adenocarcinoma, benefits were observed by focusing on cell lines that resemble the corresponding cancer type better: using only the ‘golden set’ plus ‘silver set’ cell lines, 10 new significant associations were found (Fig 4b). For instance, we detected that SMAD4-mutant cell lines are more resistant to piperlongumine, a natural product claimed to have antitumor properties exerted via multiple pathways (42,43). Mutations of the tumor suppressor gene EP300 were associated with higher sensitivity to three drugs in pancreatic cancer cell lines (Fig 4d).…”