This month we’re excited to feature an article from the lab of Hafiz Ahmed and others at the University of Maryland, School of Medicine involving the treatment of PC-3 prostate cancer cells with tunicamycin. The article was published 15 July 2017 in the journal Oncotarget and includes gene expression data generated by the Phalanx Biotech service lab using our Human Whole Genome OneArray Microarray platform.
This study focuses on treatment of PC-3 prostate cancer cells with tunicamycin, a natural antibiotic known to induce ER stress. ER stress occurs when there is an imbalance between the demand for protein synthesis and the ability of the ER to carry out proper protein maturation and transport. The unfolded protein response is the cell’s natural response to alleviating ER stress. ER stress is also linked to cell death, as it can initiate both autophagy and apoptosis. This study ultimately reveals a signaling mechanism by which tunicamycin causes ER stress and reactive oxygen species (ROS)-induced apoptosis.
Tunicamycin Blocks N-Glycosylation and Induces ER Stress
After finding an effective dose of tunicamycin, the authors note that a 72-hour treatment blocked N-glycosylation. This is the known mechanism by which tunicamycin induces ER stress. N-glycosylation is the process by which sugars are added to proteins. If this system breaks down, then many proteins fail to mature in the ER (the hallmark of ER stress). The authors note that PC-3 cells begin to undergo apoptosis after 72 hours, and interestingly, they find that the cells undergo autophagy after 24 and 48 hours of tunicamycin treatment. The idea here is that the induction of autophagy allows the cell to survive, but prolonged ER stress eventually causes apoptosis at the 72-hour mark. The authors further prove this model by treating the cells with both tunicamycin and a chloroquine, a drug that prevents autophagy. In this co-treatment scenario, the cells die faster compared to being treated with tunicamycin alone.
Tunicamycin-Induced Cell Death is Related to Oxidative Stress
The authors find that ROS are markedly increased in tunicamycin-treated PC-3 cells. In the presence of an antioxidant, the ROS accumulation is attenuated. The authors also measure mitochondrial membrane potential in tunicamycin-treated PC-3 cells, with and without co-treatment with an antioxidant. Indeed, tunicamycin leads to the loss of mitochondrial membrane potential, whereas the presence of an antioxidant helps to reduce the loss of membrane potential. These results indicate that cell death in PC-3 cells (resulting from prolonged ER stress) is mediated by oxidative stress.
Microarray Analysis Reveals Genes and Pathways Involved in Tunicamycin Responses
Using Human OneArray Whole Genome Microarrays, the authors look at tunicamycin-treated PC-3 cells at 24 and 72 hours of treatment. This is a nice comparison because the PC-3 cells are undergoing autophagy at 24 hours and apoptosis at 72 hours. There are hundreds of differentially expressed genes between these two time points, including many pro-apoptotic markers up-regulated at 72 hours. Interestingly, the highest up-regulated gene at 72 hours is endothelial nitric oxide synthase or eNOS. They validate this finding with qPCR, showing an immense 250-fold up-regulation of eNOS. Furthermore, Western blot analysis of eNOS protein confirms up-regulation at 72 hours of tunicamycin treatment.
The Role of eNOS in Cell Death of Tunicamycin-Treated PC-3 Cells
The authors next look at how eNOS modulates autophagy. They find that knockdown of eNOS increases autophagy, whereas overexpression of eNOS prevents autophagy. This fits in nicely with their working model where 24 hour treated PC-3 cells have low levels of eNOS and high levels of autophagy, and 72 hour treated cells have high levels of eNOS and low levels of autophagy. The next piece of the puzzle is how the cellular response to tunicamycin shifts from autophagy to apoptosis after 72 hours of treatment. The authors find that upregulation of eNOS activates mTORC1 and promotes accumulation of p62, and that this response is mediated via the RagC pathway. Lastly, p62 accumulation results in the cellular deposition of misfolded toxic protein aggregates, thereby leading to ROS generation.
In summary, the authors reveal a potent cell signaling axis underlying how tunicamycin mediates cell death in a prostate cancer cellular model. They take a genomic approach by utilizing Human OneArray Whole Genome Microarrays, and find that sustained ER stress can trigger both autophagy and apoptosis. At the tipping point between autophagy and apoptosis, upregulation of eNOS negatively regulates autophagy via activation of mTORC1. Inhibition of autophagy causes p62 and ROS accumulation in the cells, which then triggers apoptosis. Ultimately, these experiments point to co-treatment with tunicamycin and an autophagy-blocker, such as chloroquine, possibly functioning as a potent cancer treatment.
At Phalanx Biotech, we promise to fast-track your research. Please contact us if you need a quality genomics service provider. We offer services on either Agilent or our own OneArray microarray platforms. We are also expert providers of NGS services, qPCR services, and miRNA expression microarray services. No matter what genomics approach you need, contact us, and we’ll be sure to advance your research efforts.
Guha P et al. Tunicamycin induced endoplasmic reticulum stress promotes apoptosis of prostate cancer cells by activating mTORC1 (2017). Oncotarget, Advance Publication.