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Research Paper
Tunicamycin induced endoplasmic reticulum stress promotes
apoptosis of prostate cancer cells by activating mTORC1
Prasun Guha1,4, Engin Kaptan1,5, Padmaja Gade2, Dhananjaya V. Kalvakolanu2,3
and Hafiz Ahmed1,3,6
1
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine and Institute of Marine and
Environmental Technology, Baltimore, Maryland, USA
2
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
3
University of Maryland Greenebaum Cancer Center, Baltimore, Maryland, USA
4
Current address: The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine,
Baltimore, Maryland, USA
5
Current address: Department of Biology, Istanbul University, Vezneciler, Istanbul, Turkey
6
Current address: GlycoMantra Inc., Baltimore, Maryland, USA
Correspondence to: Hafiz Ahmed, email: [email protected]
Keywords: apoptosis, tunicamycin, ER stress, oxidative burst, metastatic cancer
Abbreviations: ER, endoplasmic reticulum; ROS, reactive oxygen species; Tun, tunicamycin; CQ, chloroquine; PBS, phosphate-
buffered saline (10 mM phosphate, 140 mM NaCl, pH 7.5)
Received: May 04, 2017 Accepted: June 10, 2017 Published: July 15, 2017
Copyright: Guha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0
(CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source
are credited.
ABSTRACT
Figure 1: Tunicamycin-treated PC-3 cells show decreased N-glycosylation with an induction of ER stress. (A) Cell
viability of PC-3 cells. 1x 104 Cells were treated with 1-20 μg/ml of tunicamycin (Tun) for 24-96 h and cell viability was assessed with
WST-1 staining. (B) Glycan differentiation analysis of (I) PC-3 cell extract and (II) standard glycoproteins. PC-3 cells were treated with
10 μg/ml Tun for 72 h and the presence of N- and O-glycosylations in the cell extract and in the standard glycoprotein (as controls) were
analyzed by binding with various lectins as described in Materials and Methods. 70 kDa protein band (marked by red rectangle) was
subjected to mass-spectrometric analysis. (C) Mass spectrum of GRP78. 70 kDa protein (in Figure B-I) was identified as GRP78. (D)
Representative Western blot showing expression of calnexin. PC-3 cells were treated with 10 μg/ml Tun for 24-72 h and expression of
calnexin was determined on W. blot using anti-calnexin antibody. Actin was used as a loading control. The bar diagram at right shows
quantification of the calnexin expression from three experiments as measured by Image J software.
Figure 3: Tunicamycin-induced cell death of PC-3 cells was ROS-dependent. (A) Effect of Tun on ROS generation. PC-3 cells
were treated with Tun (10 μg/ml, 72 h) in the presence or absence of 2.5 mM N-acetyl cysteine (NAC) and ROS was measured with CM-
H2DCFDA. (B) Effect of ROS in mitochondrial membrane potential. PC-3 cells were treated with Tun (10 μg/ml, 72 h) in the presence
or absence of 2.5 mM NAC and membrane potential was measured. (C, D) Effect of ROS on cell death. PC-3 cells were treated with Tun
(10 μg/ml, 72 h) in the presence or absence of 2.5 mM NAC and cell death was measured by either cleaved caspase-3 staining on a flow
cytometer (C) or WST-1 staining (D).
Role of mTORC1 and RagC on p62 accumulation eNOS plays a role in DNA fragmentation or late
was determined in the presence of rapamycin (inhibitor of stage apoptosis
mTORC1) or after knocking down RagC. Rapamycin or
si-RagC reduced p62 accumulation by ~2 fold compared Role of eNOS in late stage apoptosis was confirmed
to Tun treatment alone (Figure 6F). Taken all data together, by TUNEL assay of eNOS knockout PC-3 cells in the
eNOS activates mTORC1 through eNOS-RagC pathway presence or Tun. To prepare a stable eNOS-/-PC-3 cells,
and inhibits autophagic degradation. cells were transfected with lentiviral shRNA targeting
eNOS. Lack of eNOS expression in eNOS-/-PC-3 cells
p62 accumulation facilitates aggregation of was first confirmed (Figure 8A). Negative shRNA
ubiquitinylated protein and ROS induced cell control transfected cells showed eNOS expression (data
death not shown). On TUNEL assay, Tun-treated eNOS-/-
cells showed almost 58% reduction (after subtracting
To ascertain the effect of p62 on cell death we background) (p<0.001) of apoptosis compared to Tun-
knocked down p62 and also over expressed p62 in treated wild type PC-3 cells (Figure 8B). Negative shRNA
PC-3 cells along with treatment with Tun (10 μg/ml, control transfected PC-3 cells showed similar results as the
72 h). Interestingly, we found that knockdown of p62 wild type PC-3 cells (data not shown).
significantly (p<0.01) reduced cell death, but over Taken all data (described above) together, sustained
expression of p62 significantly (p<0.001) increased Tun Tun-induced ER stress up-regulates eNOS, which in turn,
dependent cell death (Figure 7A). We next investigated activates mTORC1 (as measured by phosphorylation of S6
how accumulation of p62 stimulated cell death. Our kinase) through RagC. This results into an accumulation
data showed an accumulation of ubiquitinylated of p62 which facilitates aggregation of ubiquitinated
proteins in Tun treated cells which markedly increased protein thus compromising clearance of misfolded toxic
between 72 and 96 h (Figure 7B). To investigate if protein aggregates. Lastly, association of p62 proteins and
p62 could associate with ubiquitinylated proteins, misfolded proteins promote ROS mediated mitochondrial
co-immunoprecipitation and confocal imaging were apoptosis (Figure 8C).
performed. As shown in Figure 7C, p62 antibody
precipitated ubiquitinylated proteins indicating an DISCUSSION
association with p62 and ubiquitinylated proteins.
Further, confocal imaging showed a direct association Cell’s decision to live or die under stress conditions
of p62 with ubiquitinylated proteins (Figure 7D, merge is determined by the crosstalk between signaling pathways
picture). As shown in Figure 3A-3D, intracellular that regulate these processes. Cells make mutually exclusive
aggregation of misfolded proteins promoted ROS decisions (‘live’ or ‘die’) by employing pleiotropic
generation leading to cell death [25]. Taken together, data pathways or proteins. It is largely unknown how cells
suggest that p62 accumulation resulted in the cellular succeed to switch on or off robustly specific stress
deposition of misfolded of toxic protein aggregates response using an integrated network. From whole genome
leading to the ROS generation. Sustained accumulation expression microarray analysis of Tun-treated PC-3 cells at
of ROS destabilized mitochondrial membrane potential two time points, we show that under sustained ER-stress,
and triggered mitochondrion-dependent apoptosis. up-regulation of eNOS triggers death switch.
Figure 7: p62 accumulation facilitates aggregation of ubiquitinylated protein and ROS induced cell death. (A) Effect of
p62 down-regulation and up-regulation on PC-3 cell death. The cells were transfected with siRNA specific to p62 (for knockdown) or p62
construct (for ectopic expression) or scrambled siRNA or empty vector for the corresponding negative control followed by treatment with
10 μg/ml of Tun for 72 h and cell viability was measured by WST-1 staining. (B) Western blot showing accumulation of ubiquitinylated
proteins. PC-3 cells were treated with 10 μg/ml of Tun for 72 h and the cell extracts were subjected to W. blot using anti-ubiquitin antibody.
(C) Immunoprecipitation of ubiquitinylated proteins with anti-p62 antibody as described in Materials and Methods. Lower panel shows
the antibody (p62) load. (D) Confocal image showing co-localization of p62 and ubiquitinylated protein in Tun (72 h) treated PC-3 cells
(indicated by arrow).