SET protein accumulates in HNSCC and contributes to cell survival: antioxidant defense, Akt phosphorylation and AVOs acidification.
OBJECTIVE
Determination of the SET protein levels in head and neck squamous cell carcinoma (HNSCC) tissue samples and the SET role in cell survival and response to oxidative stress in HNSCC cell lineages.


METHODS
SET protein was analyzed in 372 HNSCC tissue samples by immunohistochemistry using tissue microarray and HNSCC cell lineages. Oxidative stress was induced with the pro-oxidant tert-butylhydroperoxide (50 and 250μM) in the HNSCC HN13 cell lineage either with (siSET) or without (siNC) SET knockdown. Cell viability was evaluated by trypan blue exclusion and annexin V/propidium iodide assays. It was assessed caspase-3 and -9, PARP-1, DNA fragmentation, NM23-H1, SET, Akt and phosphorylated Akt (p-Akt) status. Acidic vesicular organelles (AVOs) were assessed by the acridine orange assay. Glutathione levels and transcripts of antioxidant genes were assayed by fluorometry and real time PCR, respectively.


RESULTS
SET levels were up-regulated in 97% tumor tissue samples and in HNSCC cell lineages. SiSET in HN13 cells (i) promoted cell death but did not induced caspases, PARP-1 cleavage or DNA fragmentation, and (ii) decreased resistance to death induced by oxidative stress, indicating SET involvement through caspase-independent mechanism. The red fluorescence induced by siSET in HN13 cells in the acridine orange assay suggests SET-dependent prevention of AVOs acidification. NM23-H1 protein was restricted to the cytoplasm of siSET/siNC HN13 cells under oxidative stress, in association with decrease of cleaved SET levels. In the presence of oxidative stress, siNC HN13 cells showed lower GSH antioxidant defense (GSH/GSSG ratio) but higher expression of the antioxidant genes PRDX6, SOD2 and TXN compared to siSET HN13 cells. Still under oxidative stress, p-Akt levels were increased in siNC HN13 cells but not in siSET HN13, indicating its involvement in HN13 cell survival. Similar results for the main SET effects were observed in HN12 and CAL 27 cell lineages, except that HN13 cells were more resistant to death.


CONCLUSIONS
SET is potential (i) marker for HNSCC associated with cancer cell resistance and (ii) new target in cancer therapy.
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café, s/n, 14040-930 Ribeirão Preto, SP, Brazil. andreiaml@usp.br
Andréia M
AM
Leopoldino
Cristiane H
CH
Squarize
Cristiana B
CB
Garcia
Luciana O
LO
Almeida
Cezar R
CR
Pestana
Lays M
LM
Sobral
Sérgio A
SA
Uyemura
Eloiza H
EH
Tajara
J
J
Silvio Gutkind
Carlos
C
Curti
D000975:Antioxidants / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002470:Cell Survival; D053938:DNA Fragmentation; D004268:DNA-Binding Proteins; D005470:Fluorometry; D005978:Glutathione / Q000378:metabolism; D006258:Head and Neck Neoplasms / Q000378:metabolism*; D056488:Histone Chaperones / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay; D054778:NM23 Nucleoside Diphosphate Kinases / Q000378:metabolism; D015388:Organelles / Q000378:metabolism; D018384:Oxidative Stress; D010766:Phosphorylation; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism*
D000975:Antioxidants; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D054778:NM23 Nucleoside Diphosphate Kinases; C513473:SET protein, human; D014157:Transcription Factors; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D051057:Proto-Oncogene Proteins c-akt; C517938:NME1 protein, human; D053148:Caspase 3; D053453:Caspase 9; D005978:Glutathione