Yonsei Med J.  2013 Nov;54(6):1407-1415. 10.3349/ymj.2013.54.6.1407.

Human Leptin Protein Induces Proliferation of A549 Cells via Inhibition of PKR-Like ER Kinase and Activating Transcription Factor-6 Mediated Apoptosis

• Lai Q
• Sun Y

• KMID: 1798137
• DOI: http://doi.org/10.3349/ymj.2013.54.6.1407

Abstract

PURPOSE
To investigate the anti-apoptotic mechanism of leptin in non-small cell lung cancer.
MATERIALS AND METHODS
The influences of leptin on apoptosis were investigated, analyzing the mechanism that triggers growth of A549 cells. The effects of leptin on cell proliferation were examined by XTT analysis. Leptin, C/EBP homologous protein (CHOP), phosphorylated-PKR-like ER kinase (p-Perk), inositol requiring proteins-1, spliced X-box transcription factor-1 (XBP1), cleaved activating transcription factor-6 (ATF6), eukaryotic translation initiation factor-2alpha, caspase-12 and CHOP protein were detected in four groups by western blot, and endoplasmic reticulum (ER) stress related mRNA were detected by reverse transcription PCR.
RESULTS
The expression of leptin in A549 and leptin transfected cells inhibited cisplatin activated ER stress-associated mRNA transcription and protein activation. Two ER stress unfolded protein response pathways, PERK and ATF6, were involved, and XBP1 and tumor necrosis factor receptor-associated factor 2 (TRAF2) were increased significantly when treated with cisplatin in A549-siRNA against leptin cells. Furthermore, CHOP expression was inhibited upon leptin expression in A549, LPT-PeP and LPT-EX cells.
CONCLUSION
Leptin serves as an important factor that promotes the growth of A549 cells through blocking ER stress-mediated pathways. This blocking is triggered by p-Perk and ATF6 via inhibition of CHOP expression.

Keyword

Apoptosis; ER stress; cell growth; leptin; TRAF2; XPB1

MeSH Terms

Activating Transcription Factor 6/genetics/*metabolism
Apoptosis/*drug effects/genetics
Blotting, Western
Cell Line, Tumor
Cell Proliferation/*drug effects
Humans
Leptin/*pharmacology
Reverse Transcriptase Polymerase Chain Reaction
eIF-2 Kinase/*metabolism
Activating Transcription Factor 6
Leptin
eIF-2 Kinase

Figure

• 

  Fig. 1 Detection of intrinsic leptin in A549, LPT-PeP, LPT-EX, and A549-siLPT cells with western blot assay. (A) Intrinsic or expressed leptin detected with leptin-specific monoclonal antibody. Various leptin proteins are indicated above the western blot bands. Protein molecular weights are shown on the right. (B) Statistical analysis. In the figure, LPT-PeP represents the leptin in peptide incubated BEAS2B cells, LPT-EX represents the expressed leptin in BEAS2B cells, and A549-siLPT represents the leptin in the interfered A549 cells. The relative value of each preparation is calculated by each gray numerical value of the specific product vs. that of β-actin. The average data of each preparation are evaluated based on three independent reactions and represented as mean±SD. Statistical differences of the data of cisplatin treatment compared with that of non-treatment are illustrated as *p<0.01, †p<0.001, respectively. siLPT, siRNA against leptin.

• 

  Fig. 2 Cell proliferation effects of leptin in the four groups. (A) A549, LPT-PeP, LPT-EX and A549-siLPT cells were incubated with cisplatin, respectively. The cell proliferations were measured by the XTT method. (B) TUNEL analysis of leptin expressing cells treated with or without cisplatin. The arrows show TUNEL positive cells. The average data of each preparation are evaluated based on three independent reactions and represented as mean±SD. Statistical differences of the data of cisplatin treatment compared with that of non-treatment are illustrated as *p<0.001. siLPT, siRNA against leptin.

• 

  Fig. 3 Early and late apoptosis phenomena were observed in cisplatin treatment and non-treatment groups. (A) Annexin V/PI double staining assays of cells incubated with or without cisplatin. (B) Statistical analysis. Y axis indicates the numbers of PI stained cells. X axis indicates the numbers of Annexin V-FITC strained cells. Results for three independent experiments are shown. The mean data of each preparation were the results of three independent western blots scan and calculation, and indicated as mean±SD. Statistical differences of the data of cisplatin treatment compared with that of non-treatment are illustrated as *p<0.01, †p<0.001, respectively. PI, propidium iodide; siLPT, siRNA against leptin.

• 

  Fig. 4 Detection of three UPR pathway proteins. (A) Detection of IRE1, p-Perk and cleaved ATF6. Statistical analysis of p-Perk (B), IRE1 (C) and Cleaved ATF6 (D). The average gray value of each preparation was calculated by the gray numerical value of each blot vs. that of β-actin. The mean data of each preparation were the results of three independent western blots scan and calculation, and indicated as mean±SD. Statistical differences of the data of cisplatin treatment compared with that of non-treatment are illustrated as *p<0.001. p-Perk, phosphorylated-PKR-like ER kinase; ER, endoplasmic reticulum; ATF6, activating transcription factor-6; UPR, unfolded protein response; IRE1, inositol requiring proteins-1; siLPT, siRNA against leptin.

• 

  Fig. 5 Hemi-quantification PCR detects the mRNA levels of ER stress (UPR) associated genes. (A) Detection of spliced XBP1 mRNA. (B) Detection of TRAF2 mRNA. (C) Detection of the eIF-2α mRNA. The products were separated in 1.5% agarose gels. The relative values of spliced XBP1, TRAF2 and eIF-2α were evaluated by the calculation of gray scan value of each preparation vs. that of β-actin protein. The mean data of each preparation were the results of three independent western blots scan and calculation, and indicated as mean±SD. Statistical differences of the data of cisplatin treatment compared with that of non-treatment are illustrated as *p<0.001, respectively. siLPT, siRNA against leptin; ER, endoplasmic reticulum; UPR, unfolded protein response; XBP1, X-box transcription factor-1; eIF-2α, eukaryotic translation initiation factor-2α; TRAF2, tumor necrosis factor receptor-associated factor 2.

• 

  Fig. 6 Detection of ER stress (UPR) associated genes. (A) Detection of spliced XBP1 protein, TRAF2 protein, e-IF-2α protein. (B) Detection of spliced XBP1 protein. (C) Detection of TRAF2 protein. (D) Detection of eIF-2α protein. The relative values of spliced XBP1, TRAF2 and eIF-2α proteins were evaluated by the calculation of gray scan value of each preparation vs. that of β-actin protein. The mean data of each preparation were the results of three independent western blots scan and calculation, and indicated as mean±SD. Statistical differences of the data of cisplatin treatment compared with that of non-treatment are illustrated as *p<0.001 respectively. ER, endoplasmic reticulum; UPR, unfolded protein response; XBP1, X-box transcription factor-1; eIF-2α, eukaryotic translation initiation factor-2α; siLPT, siRNA against leptin; TRAF2, tumor necrosis factor receptor-associated factor 2.

• 

  Fig. 7 Changes of ER stress-associated events in the cells expressing leptin. (A) The levels of CHOP and caspase-12 in A549, A549-siLPT, LPT-PeP, and LPT-EX cells were evaluated by individual western blots. (B) Statistical analysis of Cleaved caspase-12. (C) Statistical analysis of CHOP. The average gray value of each preparation was evaluated by the calculation of gray scan value of each preparation vs. that of β-actin protein. The mean data of each preparation were the results of three independent western blots scan and calculation, and indicated as mean±SD. Statistical differences of the data of cisplatin treatment compared with that of non-treatment are illustrated as *p<0.001. ER, endoplasmic reticulum; siLPT, siRNA against leptin.

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