Profiling of apoptosis- and autophagy-associated molecules in human lung cancer A549 cells in response to cisplatin treatment using stable isotope labeling with amino acids in cell culture
Authors: Guifeng Liu
Zongqiang Wang
Jinlan Jiang
Journal: International journal of oncology (2019)
Discipline: CLINICAL MEDICINE
Keywords: autophagy; chemotherapy; non-small cell lung cancer; stable isotope labeling by amino acids in cell culture.
Affiliations: Department of Medical Services, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China., Department of Radiology, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China., Science Research Center, Department of Orthopedics, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China.

Abstract:  Cis‑diammine‑dichloro‑platinum II‑based adjuvant chemotherapy provides an alternative therapy to improve the survival of patients with lung tumors, especially those with non‑small cell lung cancer (NSCLC). However, drug resistance is a large clinical problem and its underlying mechanism remains unclear. In the present study, NSCLC A549 cells were treated with a low concentration of cisplatin in order to observe and determine the development of chemoresistance, via growth curves, colony forming assays and apoptosis assays. Then the induction of autophagy was examined in the cisplatin‑treated A549 cells with a fluorescence reporter. Profiled proteins in the cisplatin‑treated A549 cells were also assessed using the stable isotope labeling by amino acids in cell culture (SILAC) method, and then the differentially expressed molecules were verified. The results demonstrated that A549 cells became less sensitive to cisplatin [resistant A549 cells (A549R)] following 20 passages in the medium containing a low concentration of cisplatin, with less apoptotic cells post‑cisplatin treatment. A549R cells grew more efficiently in the cisplatin medium, with more colony formation and more cells migrating across the baseline. In addition, NSCLC results demonstrated that more autophagy‑related proteins (ATGs) were expressed in the A549R cells. Furthermore, the western blotting results confirmed this upregulation of ATGs in A549R cells. In addition, two repeated SILAC screening experiments recognized 15 proteins [glucose‑regulated protein, 78 kDa (GRP78), heat shock protein 71, pre‑mRNA processing factor 19, polypyrimidine tract binding protein 1, translationally controlled tumor protein, Cathepsin D, Cytochrome c, thioredoxin domain containing 5, MutS homolog (MSH) 6, Annexin A2 (ANXA2), BRCA2 and Cyclin dependent kinase inhibitor 1A interacting protein, MSH2, protein phosphatase 2A 55 kDa regulatory subunit Bα, Rho glyceraldehyde‑3‑phosphate‑dissociation inhibitor 1 and ANXA4] that were upregulated by &gt;1.5‑fold in heavy (H)‑ and light (L)‑labeled A549R cells. In addition, 16 and 14 proteins were downregulated by &gt;1.5‑fold in the H‑ and L‑labeled A549R cells, respectively. The majority of the downregulated proteins were associated with apoptosis. In conclusion, the present study isolated a cisplatin‑resistant human lung cancer A549 cell clone, with reduced apoptosis and high levels of autophagy, in response to cisplatin treatment. In cisplatin‑resistant A549R cells, SILAC proteomics recognized the high expression of GRP78 and other proteins that are associated with anti‑apoptosis and/or autophagy promotion. 

Retracted: No

PubPeer comments:
 Figure 4C. These two bands are very similar. ![file] , 
 Dear Dr. Parashorea Tomentella, I am the first author of this article, Zongqiang Wang. For some reason, the e-mail registration audit failed, so I used anonymous registration. We are really appreciating your careful check and raising this concern. The two bands are very similar at first glance, however different. We would like to show you how different they are in the published paper. 1.Downloaded the pdf file and then cut Figure 4 from the file. We just took a screenshot of the figure, as following: ![file] 2.Adjust the brightness contrast of Figure 4, magnify the adjusted Figure. The following figure showed the magnified local parts of the two bands. Of course, we have labeled the significantly different parts between them. Software: Photoshop. Adjustment references: adding 65% brightness and reducing 30% contrast, magnified 6 times of the figure (Of course, not exact, just approximate references are ok.). Note: Adjust the figure as a whole Figure, not just any local part of it. ![file] Detailed parts with difference, 1) to 5): 1) A sharp protruding angle at the top-right of left band, not at the right one; 2) An insufficient angle at the bottom-right of left band, not at the right one; 3) A wider protruding angle at the left-top of left band, not at the right one; 4) A wider protruding angle at the left of left band, not at the right one; 5) The grayscale edge at the bottom of left band is heavy at left and light at right, however same for both sides at the right one. In addition, the adjusted and magnified bands also show their natural and fluent continuity with their backgrounds. However, this response is just a method to magnify the difference between the two bands. You are welcome to check them with such method at your convenient moment; and we are confident that you get same conclusion about them. Thank you very much. Sincerely yours, Dr Zongqiang Wang ,