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Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells.

Jorgensen E, Stinson A, Shan L, Yang J, Gietl D, Albino AP - BMC Cancer (2008)

Bottom Line: We show that: 1) CS induces ER stress and activates components of the UPR; 2) reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3) CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4) several major UPR regulators are increased either in expression (i.e., BiP and eIF2 alpha) or phosphorylation (i.e., phospho-eIF2 alpha) in a majority of human lung cancers.These data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2 alpha and BiP may have diagnostic and/or therapeutic potential.Furthermore, we speculate that upregulation of UPR regulators (in particular BiP) may provide a pro-survival advantage by increasing resistance to cytotoxic stresses such as hypoxia and chemotherapeutic drugs, and that UPR induction is a potential mechanism that could be attenuated or reversed resulting in a more efficacious treatment strategy for lung cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Public Health Division, Vector Research LLC, New York, NY, USA. ejorgensen@vectorgroupltd.com

ABSTRACT

Background: Although lung cancer is among the few malignancies for which we know the primary etiological agent (i.e., cigarette smoke), a precise understanding of the temporal sequence of events that drive tumor progression remains elusive. In addition to finding that cigarette smoke (CS) impacts the functioning of key pathways with significant roles in redox homeostasis, xenobiotic detoxification, cell cycle control, and endoplasmic reticulum (ER) functioning, our data highlighted a defensive role for the unfolded protein response (UPR) program. The UPR promotes cell survival by reducing the accumulation of aberrantly folded proteins through translation arrest, production of chaperone proteins, and increased degradation. Importance of the UPR in maintaining tissue health is evidenced by the fact that a chronic increase in defective protein structures plays a pathogenic role in diabetes, cardiovascular disease, Alzheimer's and Parkinson's syndromes, and cancer.

Methods: Gene and protein expression changes in CS exposed human cell cultures were monitored by high-density microarrays and Western blot analysis. Tissue arrays containing samples from 110 lung cancers were probed with antibodies to proteins of interest using immunohistochemistry.

Results: We show that: 1) CS induces ER stress and activates components of the UPR; 2) reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3) CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4) several major UPR regulators are increased either in expression (i.e., BiP and eIF2 alpha) or phosphorylation (i.e., phospho-eIF2 alpha) in a majority of human lung cancers.

Conclusion: These data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2 alpha and BiP may have diagnostic and/or therapeutic potential. Furthermore, we speculate that upregulation of UPR regulators (in particular BiP) may provide a pro-survival advantage by increasing resistance to cytotoxic stresses such as hypoxia and chemotherapeutic drugs, and that UPR induction is a potential mechanism that could be attenuated or reversed resulting in a more efficacious treatment strategy for lung cancer.

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Effect of antioxidant treatment on eIF2α phosphorylation following cigarette smoke exposure. A549 cells were exposed to air (mock treatment) or 2R4F cigarette smoke for 20 minutes with 35 cc puffs diluted in 250 cc air, with or without concurrent treatment with NAC or GSH (at 5 and 25 mM concentrations), after which the cells were placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α and GAPDH.
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Figure 10: Effect of antioxidant treatment on eIF2α phosphorylation following cigarette smoke exposure. A549 cells were exposed to air (mock treatment) or 2R4F cigarette smoke for 20 minutes with 35 cc puffs diluted in 250 cc air, with or without concurrent treatment with NAC or GSH (at 5 and 25 mM concentrations), after which the cells were placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α and GAPDH.

Mentions: The results shown in Figure 9 indicated that the vapor phase is as efficient as whole CS in inducing ER stress and the phosphorylation of eIF2α. Since a primary constituent of the vapor phase of CS is a large amount of highly reactive organic and inorganic substances that either are free radicals or reactive species (such as •O2-, •NO, •OH, etc), and more stable organic reactive species/oxidants (such as phenols, hydroquinone, epoxides, etc.) that can cause a marked imbalance in an individual's redox state and an overall increase in oxidative stress in the respiratory tract [72-75], we were interested in determining if free radical scavengers could effectively suppress the induction of eIF2α phosphorylation and the activation of the UPR program. Figure 10 shows that the thiol N-acetyl-L-cysteine (NAC), a potent free radical scavenging compound with antioxidant activity, can completely prevent CS-induced phosphorylation of eIF2α at 1 h or 4 h post-exposure in A549 cells. NAC is freely taken up by cells, and can impact a broad range of compounds, many of which cause some form of oxidative stress. For example, NAC can scavenge several reactive oxygen species (ROS) generated in CS such as •O2-, •OH, and H2O2, as well as directly bind and attenuate various reactive CS compounds that can generate free radicals intracellularly (e.g. aldehydes, epoxides, quinones, etc.). Figure 10 also shows that treatment with reduced glutathione (GSH), a free radical scavenger that does not cross the cell membrane, also suppresses the ability of CS to induce eIF2α phosphorylation.


Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells.

Jorgensen E, Stinson A, Shan L, Yang J, Gietl D, Albino AP - BMC Cancer (2008)

Effect of antioxidant treatment on eIF2α phosphorylation following cigarette smoke exposure. A549 cells were exposed to air (mock treatment) or 2R4F cigarette smoke for 20 minutes with 35 cc puffs diluted in 250 cc air, with or without concurrent treatment with NAC or GSH (at 5 and 25 mM concentrations), after which the cells were placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α and GAPDH.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2527015&req=5

Figure 10: Effect of antioxidant treatment on eIF2α phosphorylation following cigarette smoke exposure. A549 cells were exposed to air (mock treatment) or 2R4F cigarette smoke for 20 minutes with 35 cc puffs diluted in 250 cc air, with or without concurrent treatment with NAC or GSH (at 5 and 25 mM concentrations), after which the cells were placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α and GAPDH.
Mentions: The results shown in Figure 9 indicated that the vapor phase is as efficient as whole CS in inducing ER stress and the phosphorylation of eIF2α. Since a primary constituent of the vapor phase of CS is a large amount of highly reactive organic and inorganic substances that either are free radicals or reactive species (such as •O2-, •NO, •OH, etc), and more stable organic reactive species/oxidants (such as phenols, hydroquinone, epoxides, etc.) that can cause a marked imbalance in an individual's redox state and an overall increase in oxidative stress in the respiratory tract [72-75], we were interested in determining if free radical scavengers could effectively suppress the induction of eIF2α phosphorylation and the activation of the UPR program. Figure 10 shows that the thiol N-acetyl-L-cysteine (NAC), a potent free radical scavenging compound with antioxidant activity, can completely prevent CS-induced phosphorylation of eIF2α at 1 h or 4 h post-exposure in A549 cells. NAC is freely taken up by cells, and can impact a broad range of compounds, many of which cause some form of oxidative stress. For example, NAC can scavenge several reactive oxygen species (ROS) generated in CS such as •O2-, •OH, and H2O2, as well as directly bind and attenuate various reactive CS compounds that can generate free radicals intracellularly (e.g. aldehydes, epoxides, quinones, etc.). Figure 10 also shows that treatment with reduced glutathione (GSH), a free radical scavenger that does not cross the cell membrane, also suppresses the ability of CS to induce eIF2α phosphorylation.

Bottom Line: We show that: 1) CS induces ER stress and activates components of the UPR; 2) reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3) CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4) several major UPR regulators are increased either in expression (i.e., BiP and eIF2 alpha) or phosphorylation (i.e., phospho-eIF2 alpha) in a majority of human lung cancers.These data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2 alpha and BiP may have diagnostic and/or therapeutic potential.Furthermore, we speculate that upregulation of UPR regulators (in particular BiP) may provide a pro-survival advantage by increasing resistance to cytotoxic stresses such as hypoxia and chemotherapeutic drugs, and that UPR induction is a potential mechanism that could be attenuated or reversed resulting in a more efficacious treatment strategy for lung cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Public Health Division, Vector Research LLC, New York, NY, USA. ejorgensen@vectorgroupltd.com

ABSTRACT

Background: Although lung cancer is among the few malignancies for which we know the primary etiological agent (i.e., cigarette smoke), a precise understanding of the temporal sequence of events that drive tumor progression remains elusive. In addition to finding that cigarette smoke (CS) impacts the functioning of key pathways with significant roles in redox homeostasis, xenobiotic detoxification, cell cycle control, and endoplasmic reticulum (ER) functioning, our data highlighted a defensive role for the unfolded protein response (UPR) program. The UPR promotes cell survival by reducing the accumulation of aberrantly folded proteins through translation arrest, production of chaperone proteins, and increased degradation. Importance of the UPR in maintaining tissue health is evidenced by the fact that a chronic increase in defective protein structures plays a pathogenic role in diabetes, cardiovascular disease, Alzheimer's and Parkinson's syndromes, and cancer.

Methods: Gene and protein expression changes in CS exposed human cell cultures were monitored by high-density microarrays and Western blot analysis. Tissue arrays containing samples from 110 lung cancers were probed with antibodies to proteins of interest using immunohistochemistry.

Results: We show that: 1) CS induces ER stress and activates components of the UPR; 2) reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3) CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4) several major UPR regulators are increased either in expression (i.e., BiP and eIF2 alpha) or phosphorylation (i.e., phospho-eIF2 alpha) in a majority of human lung cancers.

Conclusion: These data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2 alpha and BiP may have diagnostic and/or therapeutic potential. Furthermore, we speculate that upregulation of UPR regulators (in particular BiP) may provide a pro-survival advantage by increasing resistance to cytotoxic stresses such as hypoxia and chemotherapeutic drugs, and that UPR induction is a potential mechanism that could be attenuated or reversed resulting in a more efficacious treatment strategy for lung cancer.

Show MeSH
Related in: MedlinePlus