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TNF-alpha mediates eosinophil cationic protein-induced apoptosis in BEAS-2B cells.

Chang KC, Lo CW, Fan TC, Chang MD, Shu CW, Chang CH, Chung CT, Fang SL, Chao CC, Tsai JJ, Lai YK - BMC Cell Biol. (2010)

Bottom Line: Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha).Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody.In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan.

ABSTRACT

Background: Eosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis.

Results: To address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody.

Conclusion: In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.

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Effect of rECP-induced apoptosis on ER response. BEAS-2B cells were treated with rECP or thapsigargin (TG; an ER toxin as a positive control) for the indicated times. The cells used to determine the ER response were incubated with 20 μM rECP or 1 μM TG for 0, 4, 8, 12, 16, 20 and 24 h. (A) Total cell lysates were resolved by 10% SDS-PAGE, and the relative accumulated GRP78 was investigated by western blotting with anti-GRP78 and anti-actin. (B) For de novo proteins synthesis, cells were labeled with [35S]methionine for 2 h before harvesting. Equal amounts of labeled cell lysates were resolved by 10% SDS-PAGE followed by quantitative analysis. All data represent the arithmetic mean ± SEM. *P < 0.05.
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Figure 4: Effect of rECP-induced apoptosis on ER response. BEAS-2B cells were treated with rECP or thapsigargin (TG; an ER toxin as a positive control) for the indicated times. The cells used to determine the ER response were incubated with 20 μM rECP or 1 μM TG for 0, 4, 8, 12, 16, 20 and 24 h. (A) Total cell lysates were resolved by 10% SDS-PAGE, and the relative accumulated GRP78 was investigated by western blotting with anti-GRP78 and anti-actin. (B) For de novo proteins synthesis, cells were labeled with [35S]methionine for 2 h before harvesting. Equal amounts of labeled cell lysates were resolved by 10% SDS-PAGE followed by quantitative analysis. All data represent the arithmetic mean ± SEM. *P < 0.05.

Mentions: The ER response is generally triggered by environmental stress and sometimes leads to apoptosis. Because GRP78 plays a crucial role in the ER response [50], the level of GRP78 expression in BEAS-2B cells treated with rECP was assessed by Western blotting and a de novo synthesis assay. Accumulated and newly synthesized GRP78 were detected using anti-GRP78 (Figure 4A) and metabolic labeling with [35S]methionine (Figure 4B), respectively. The ratio of both accumulated and nascent GRP78 to actin did not change during rECP treatment. As for positive control, when the cells were treated with 1 μM TG, an ER toxin, a 2- to 4-fold increase in accumulated GRP78 after 4 to 24 h treatment was observed; moreover, newly synthesized GRP78 revealed a 4- to 6-fold increase after 4 to 24 h under the same condition. Taken together, these results implied that rECP-induced apoptosis was ER-independent, in consistence with the results of caspase-12 inhibitor treatment (Figure 3C).


TNF-alpha mediates eosinophil cationic protein-induced apoptosis in BEAS-2B cells.

Chang KC, Lo CW, Fan TC, Chang MD, Shu CW, Chang CH, Chung CT, Fang SL, Chao CC, Tsai JJ, Lai YK - BMC Cell Biol. (2010)

Effect of rECP-induced apoptosis on ER response. BEAS-2B cells were treated with rECP or thapsigargin (TG; an ER toxin as a positive control) for the indicated times. The cells used to determine the ER response were incubated with 20 μM rECP or 1 μM TG for 0, 4, 8, 12, 16, 20 and 24 h. (A) Total cell lysates were resolved by 10% SDS-PAGE, and the relative accumulated GRP78 was investigated by western blotting with anti-GRP78 and anti-actin. (B) For de novo proteins synthesis, cells were labeled with [35S]methionine for 2 h before harvesting. Equal amounts of labeled cell lysates were resolved by 10% SDS-PAGE followed by quantitative analysis. All data represent the arithmetic mean ± SEM. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2819994&req=5

Figure 4: Effect of rECP-induced apoptosis on ER response. BEAS-2B cells were treated with rECP or thapsigargin (TG; an ER toxin as a positive control) for the indicated times. The cells used to determine the ER response were incubated with 20 μM rECP or 1 μM TG for 0, 4, 8, 12, 16, 20 and 24 h. (A) Total cell lysates were resolved by 10% SDS-PAGE, and the relative accumulated GRP78 was investigated by western blotting with anti-GRP78 and anti-actin. (B) For de novo proteins synthesis, cells were labeled with [35S]methionine for 2 h before harvesting. Equal amounts of labeled cell lysates were resolved by 10% SDS-PAGE followed by quantitative analysis. All data represent the arithmetic mean ± SEM. *P < 0.05.
Mentions: The ER response is generally triggered by environmental stress and sometimes leads to apoptosis. Because GRP78 plays a crucial role in the ER response [50], the level of GRP78 expression in BEAS-2B cells treated with rECP was assessed by Western blotting and a de novo synthesis assay. Accumulated and newly synthesized GRP78 were detected using anti-GRP78 (Figure 4A) and metabolic labeling with [35S]methionine (Figure 4B), respectively. The ratio of both accumulated and nascent GRP78 to actin did not change during rECP treatment. As for positive control, when the cells were treated with 1 μM TG, an ER toxin, a 2- to 4-fold increase in accumulated GRP78 after 4 to 24 h treatment was observed; moreover, newly synthesized GRP78 revealed a 4- to 6-fold increase after 4 to 24 h under the same condition. Taken together, these results implied that rECP-induced apoptosis was ER-independent, in consistence with the results of caspase-12 inhibitor treatment (Figure 3C).

Bottom Line: Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha).Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody.In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan.

ABSTRACT

Background: Eosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis.

Results: To address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody.

Conclusion: In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.

Show MeSH
Related in: MedlinePlus