siRNA-mediated downregulation of TC21 sensitizes esophageal cancer cells to cisplatin.
OBJECTIVE
To determine the functional significance of TC21 in esophageal squamous cell carcinoma (ESCC).


METHODS
TC21 siRNA transfection was carried out using Hyperfectamine to knock down TC21, and transcripts were analyzed by reverse transcription-polymerase chain reaction and protein by Western blotting. We demonstrated the effect of TC21 downregulation of cell signaling in esophageal cancer cells by assessing the phosphorylation status of its downstream targets, phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog (PTEN), protein kinase B (pAkt), nuclear factor-κB (NF-κB) and cyclinD1 using specific antibodies. Cell survival analysis after cisplatin treatment was carried out by cell viability assay and cell cycle analysis using flow cytometry.


RESULTS
TC21 knockdown in human ESCC cell line TE13 cells, showed only a marginal increase (14.2%) in cell death compared with control cells. The expressions of the signaling proteins PI3K and pAkt, transcription factor NF-κB, and cell cycle protein cyclin D1 were markedly decreased in response to TC21 downregulation, whereas the level of pPTEN, an antagonist of PI3K, was increased. In addition, we evaluated the potential of TC21 as a putative target for sensitizing ESCC cells to the chemotherapeutic agent cisplatin. Increased cell death (38.4%) was observed in cells treated with cisplatin after TC21 knockdown compared with cells which were treated with cisplatin alone (20% cell death).


CONCLUSIONS
Results suggest that TC21 mediates its effects via the PI3K-Akt pathway, NF-κB and cyclin D1, and enhances chemoresistance in esophageal cancer cells.
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi 110029, India.
Raghibul
R
Hasan
Shyam Singh
SS
Chauhan
Rinu
R
Sharma
Ranju
R
Ralhan
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D019938:Cyclin D1 / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects* / Q000235:genetics; D004938:Esophageal Neoplasms / Q000378:metabolism / Q000473:pathology*; D055785:Gene Knockdown Techniques; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
D000970:Antineoplastic Agents; D008565:Membrane Proteins; D016328:NF-kappa B; D034741:RNA, Small Interfering; D019938:Cyclin D1; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; C087360:RRAS2 protein, human; D020559:Monomeric GTP-Binding Proteins; D002945:Cisplatin