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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 thapsigargin and cigarette smoke on expression of BiP. Panel A: A549 cells were treated with 1 mM thapsigargin for the time periods specified. U = untreated cell control; V = vehicle (DMSO) control; T = thapsigargin. Panel B: A549 cells were cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke (CS = smoke treatment) for 20 minutes (35 cc puffs were diluted in 250 cc air), 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 BiP and GAPDH. TC = thapsigargin treated cells as a positive internal control for Panel B.
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Figure 6: Effect of thapsigargin and cigarette smoke on expression of BiP. Panel A: A549 cells were treated with 1 mM thapsigargin for the time periods specified. U = untreated cell control; V = vehicle (DMSO) control; T = thapsigargin. Panel B: A549 cells were cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke (CS = smoke treatment) for 20 minutes (35 cc puffs were diluted in 250 cc air), 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 BiP and GAPDH. TC = thapsigargin treated cells as a positive internal control for Panel B.

Mentions: The transient accumulation of misfolded proteins, a key indicator of ER stress, results in the dissociation of the ER-resident master chaperone regulator BiP from all three ER resident sensors (i.e., PERK, IRE1 and ATF6) triggering their activation and subsequent induction of various functions of the UPR that provide the cell with a mosaic of options to counter ER stress and reestablish homeostasis [61,62]. For example, when ER stress becomes chronic, one pro-survival mechanism is to upregulate the master regulator, BiP, which allows the cell to endure an increased level of unfolded proteins and forestall apoptosis [63,64]. In one recent publication, Hengstermann and Muller [31] saw a modest increase in BiP RNA levels in mouse cells after 4 h continuous treatment with PBS into which CS was previously bubbled [31]. A second report by Kelsen et al, [30] observed increased BiP protein levels in virally transformed human bronchial epithelial cells after 24 h continuous treatment with 15% cigarette smoke extract. In contrast to these studies, our results show that when A549 cells (Figure 6, Panel A) or NHBE cells (data not shown) were briefly exposed to freshly generated whole CS for 20 min, Western blot analysis showed no increase of BiP protein levels up to 72 h post-exposure. Control cells treated with thapsigargin show strong induction of BiP by 24 h. We speculated that this apparent discrepancy in BiP expression could be due to the fact that we exposed short-term cultures of untransformed NHBE cells (as opposed to rodent cells or virally-transformed bronchial cells). Alternatively, since there are significant differences between our exposure parameters and those detailed in these two recent reports, a more likely explanation may be that the brief CS treatment protocol (20 min followed by washout and addition of fresh medium) used in our experiments resulted in an abbreviated UPR response that fails to trigger BiP upregulation. Since BiP induction is a relatively late event in the UPR signaling cascade, differing stress-inducing exposure conditions can lead to short term perturbations without durable long-term changes [12]. To test this idea, we prepared CS-bubbled media (CSE) according to the procedure used by Hengstermann and Muller [31] and treated NHBE cells for 24 h in 15% or 30% CSE. Moreover, instead of our usual procedure, we also exposed NHBE cells to CS for 20 min, except that after this treatment, the cells were not placed in fresh media but were incubated in the CS-containing medium for another 24 hrs. We observed that both of these much longer exposure procedures did result in the upregulation of BiP, although not to the degree seen with the more potent ER-stress inducer thapsigargin (Figure 7). We note that when either A549 or NHBE cells are treated with 1 μM thapsigargin continuously for 72 h, BiP expression is still only seen after 24 hours post-treatment, indicating that BiP induction is a much later consequence of ER stress than is eIF2α phosphorylation in these cells (see Figure 6, Panel B). Consequently, these data, while agreeing with previous reports showing that BiP expression is induced by CS [30,31], clearly show that BiP induction can be modulated depending on level and duration of stress-inducing conditions. It has been shown that different cell types and different stress conditions can selectively activate one or more of the ER sensors [65]. Therefore, the induction of each UPR component (e.g., BiP) is likely to be dependent on its intrinsic set-point for biological activation, specific cell context, and the defined ability of an agent to induce ER stress over time.


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 thapsigargin and cigarette smoke on expression of BiP. Panel A: A549 cells were treated with 1 mM thapsigargin for the time periods specified. U = untreated cell control; V = vehicle (DMSO) control; T = thapsigargin. Panel B: A549 cells were cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke (CS = smoke treatment) for 20 minutes (35 cc puffs were diluted in 250 cc air), 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 BiP and GAPDH. TC = thapsigargin treated cells as a positive internal control for Panel B.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Effect of thapsigargin and cigarette smoke on expression of BiP. Panel A: A549 cells were treated with 1 mM thapsigargin for the time periods specified. U = untreated cell control; V = vehicle (DMSO) control; T = thapsigargin. Panel B: A549 cells were cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke (CS = smoke treatment) for 20 minutes (35 cc puffs were diluted in 250 cc air), 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 BiP and GAPDH. TC = thapsigargin treated cells as a positive internal control for Panel B.
Mentions: The transient accumulation of misfolded proteins, a key indicator of ER stress, results in the dissociation of the ER-resident master chaperone regulator BiP from all three ER resident sensors (i.e., PERK, IRE1 and ATF6) triggering their activation and subsequent induction of various functions of the UPR that provide the cell with a mosaic of options to counter ER stress and reestablish homeostasis [61,62]. For example, when ER stress becomes chronic, one pro-survival mechanism is to upregulate the master regulator, BiP, which allows the cell to endure an increased level of unfolded proteins and forestall apoptosis [63,64]. In one recent publication, Hengstermann and Muller [31] saw a modest increase in BiP RNA levels in mouse cells after 4 h continuous treatment with PBS into which CS was previously bubbled [31]. A second report by Kelsen et al, [30] observed increased BiP protein levels in virally transformed human bronchial epithelial cells after 24 h continuous treatment with 15% cigarette smoke extract. In contrast to these studies, our results show that when A549 cells (Figure 6, Panel A) or NHBE cells (data not shown) were briefly exposed to freshly generated whole CS for 20 min, Western blot analysis showed no increase of BiP protein levels up to 72 h post-exposure. Control cells treated with thapsigargin show strong induction of BiP by 24 h. We speculated that this apparent discrepancy in BiP expression could be due to the fact that we exposed short-term cultures of untransformed NHBE cells (as opposed to rodent cells or virally-transformed bronchial cells). Alternatively, since there are significant differences between our exposure parameters and those detailed in these two recent reports, a more likely explanation may be that the brief CS treatment protocol (20 min followed by washout and addition of fresh medium) used in our experiments resulted in an abbreviated UPR response that fails to trigger BiP upregulation. Since BiP induction is a relatively late event in the UPR signaling cascade, differing stress-inducing exposure conditions can lead to short term perturbations without durable long-term changes [12]. To test this idea, we prepared CS-bubbled media (CSE) according to the procedure used by Hengstermann and Muller [31] and treated NHBE cells for 24 h in 15% or 30% CSE. Moreover, instead of our usual procedure, we also exposed NHBE cells to CS for 20 min, except that after this treatment, the cells were not placed in fresh media but were incubated in the CS-containing medium for another 24 hrs. We observed that both of these much longer exposure procedures did result in the upregulation of BiP, although not to the degree seen with the more potent ER-stress inducer thapsigargin (Figure 7). We note that when either A549 or NHBE cells are treated with 1 μM thapsigargin continuously for 72 h, BiP expression is still only seen after 24 hours post-treatment, indicating that BiP induction is a much later consequence of ER stress than is eIF2α phosphorylation in these cells (see Figure 6, Panel B). Consequently, these data, while agreeing with previous reports showing that BiP expression is induced by CS [30,31], clearly show that BiP induction can be modulated depending on level and duration of stress-inducing conditions. It has been shown that different cell types and different stress conditions can selectively activate one or more of the ER sensors [65]. Therefore, the induction of each UPR component (e.g., BiP) is likely to be dependent on its intrinsic set-point for biological activation, specific cell context, and the defined ability of an agent to induce ER stress over time.

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