RateofSOXQ6Secretionineightcellslineslungcancer

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Rate of SOX/Q6 Secretion in eight cells lines lung cancer
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Université Tahri Mohammed Béchar
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PhytoChem &BioSub Journal Vol. 14(3) 2020
ISSN 2170-1768
CAS-CODEN:PBJHB3
267
Rate of SOX/Q6 Secretion in eight cells lines lung cancer
Lamia Seddiki
Faculty of Nature and life science. University Tahri Mohamed. Bechar 08000 Algeria
Received: June24, 2020; Accepted: September 28, 2020
Corresponding author Email: [e-mail]
Copyright © 2020-POSL
DOI:10.163.pcbsj/2020.14.‐3‐267
Abstract. Bronchopulmonary cancer is a serious public health problem dueto its prognosis and its
frequency, which is why tobacco and pollution are the main causes. Our work focused on the
functional study of sulfhydryl oxidase/ quiescineQ6 (SOx / Q6). Sulfhydryls oxidases / quiescine
Q6 are enzymes that catalyze the formation of disulfide bridges. Their secretion has been studied
depending on cell proliferation. Using DTT as asubstrate, weassessed theoxidaseactivity secreted
in pulmonary fibroblast supernatants (WI38) and non-small cell epithelial tumor cell lines. Western
blot analyzesshow twotypesof SOx / Q6, onelongand theother short in WI38. However, western-
blot analyzesof themedium with 10% FCSshow thepresenceof bovineSOx / Q6, thisis confirmed
by theoxidaseactivity. On theother hand, theresults show that thesecretion of SOx / Q6 varies
according tothetumor cell lineseither a secretion increasesat thefirst stageof proliferation, second
stageof confluence, or thirdstageafter confluence.
Key Words: Sulfyhydryl oxydas; Human quiescin Q6; Oxidativeactivity; Secretion; Lung cancer.
1. INTRODUCTION
Lung cancer is the leading cause of cancer death worldwide. The root cause is smoking,
including 15,000 in Algeria and 25,000 new patients are diagnosed every year and 22,000 of
them have a poor prognosis in France. The severity of the disease is due to late diagnosis in
metastatic stage. The majority (80%) cases of lung cancer tumors are non-small cell
(NSCLC). We distinguish in particular adenocarcinoma and squamous cell carcinoma is the
most represented forms. The gene expression of these two major types of cancer profiles
reveal molecular changes particularly with over expression of genes for detoxification and
anti-oxidants (Coppock et al.; 1998).
At the cellular or tissue level, the level balance reducing and oxidizing is in equilibrium.
Deficiency or overproduction of molecular players will influence the behavior of the cell
(Schafer et al., 2001). The two systems are better known antioxidants, glutathione system
reduced / oxidized glutathione (2GSH/GSSG) and reduced thioredoxin / thioredoxin system
oxidized (TRX (SH) 2/TRXSS). A deficiency of one of them or a significant increase in pro-
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PhytoChem &BioSub Journal Vol. 14(3) 2020
ISSN 2170-1768
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oxidants and free radicals (ROS, NO) induced DNA damage, including mutations in genes
anti-and pro-oncogene and thereby causes cell proliferation (Ghosh et al., 1999). The super-
family of thioredoxin includes thioredoxin (TRX1) Protein disulfide isomerase (PDI), which
are associated with functionally related proteins such as thioredoxin reductase (Nordberget
al, 2001).
Thioredoxin is a small ubiquitous protein (Mr 11,500) containing a highly conserved
cysteine-glycine-proline-cysteine (CPMF), which is the active site motif. She is involved in
the activity of transcription factors (NF B, AP-1, ASK1) which will modulate the
expression of target genes and cellular functions associated (proliferation, apoptosis,
resistance to antitumor agents). Over expression of thioredoxin in cancer cells promotes cell
proliferation and increases the resistance of a variety of cells to chemotherapy (Soini et al.
2001).
Protein disulfide isomerase (PDI) is very abundant in the endoplasmic reticulum where it
catalyzes the oxidation, reduction of disulfide bonds and their isomerization using the active
site cysteine-glycine-histidine-cysteine (CHCH) (Freedman et al ., 1994). It is characterized
by the presence of two functional domains thioredoxin and a similar sequence in the
endoplasmic reticulum retention Lysine-Glutamate-Aspartate-leucine (KDEL).
Recently, a new family of FAD-dependent sulfhydryl oxidase has been described. The first
members of this family have been appointed sulfhydryl oxidase / Quiescine Q6 (SOx/Q6)
(Coppock et al., 1998, Benayoun et al., 2001).
Figure 1. Structures of thioredoxin protein, the protein disulfide isomerase of sulphydryl oxidase /
Quiescine Q6 (SOx/Q6) and sulphydryl oxidase neuroblastoma (SOxN). The thioredoxin domains are
shown in orange and green areas ERV1, transmembrane sequences in black, the C-terminal
extension in purple. N: potential glycosylation site.
Indeed, the expression of human SOx/Q6 gene is specifically induced in WI38 fibroblasts
upon entry into quiescence (Coppock et al., 1993). This leaves suggest a potential
involvement of this gene in cell proliferation. A second member of this family was recently
SOx/Q6
’courte ’
SOx/Q6
’longue ’
PDI
Thiorédoxine
SOxN
CGPC
CGHC CGHC
N N
C73GHC75 CRDC CSAC
512455 515452
C73GHC75 CRDC CSAC
NN
452 455 515
512
CGHC CRDC CSAC
N
SOx/Q6
’courte ’
SOx/Q6
’longue ’
PDI
Thiorédoxine
SOxN
CGPC
CGHC CGHCCGHC CGHC
N N
C73GHC75 CRDC CSAC
512455 515452
N N
C73GHC75 CRDC CSACC73GHC75 CRDC CSACC73GHC75 CRDC CSAC
512455 515452
C73GHC75 CRDC CSAC
NN
452 455 515
512
C73GHC75 CRDC CSAC
NN
C73GHC75 CRDC CSACC73GHC75 CRDC CSACC73GHC75 CRDC CSAC
NN
452 455 515
512
CGHC CRDC CSAC
N
CGHC CRDC CSACCGHC CRDC CSACCGHC CRDC CSAC
N
Short
Long

PhytoChem &BioSub Journal Vol. 14(3) 2020
ISSN 2170-1768
CAS-CODEN:PBJHB3
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described from neuroblastoma cells and called sulfhydryl oxidase derived from
neuroblastoma (SOxN), this protein plays a role in sensitizing cells to interferon gamma
(IFN-) inducer apoptosis. The protein is localized to the plasma membrane and in the
nuclear membrane (Wittke et al. 2003). These sulfhydryl oxidase (SOX) are flavoenzymes
which oxidize thiol groups in proteins and small molecules to form disulfide bridges
according to the reaction
2R-SH + O2R-S-S-R+ H2O2.
The gene for human quiescine Q6 produces two major transcripts, a 5600 nucleotides long
transcript encoding a long form of the protein (Mr 80000) and a short transcript of 4800
nucleotides encoding a short form (Mr 60000) (Figure 1 ). The protein structure deduced
from the cDNA of sulfhydryl oxidase / Quiescine Q6 has a thioredoxin domain of PDI type
in the N-terminal region and a catalytic domain VRE (essential for respiration and viability)
in the C-terminal region. SOx/Q6 has a long extension of 143 amino acids C-terminal
position which has a potential transmembrane sequence.
Figure 2. Diagram showing the activity of sulfhydryl oxydase/QuiescineQ6 (SOx/Q6). SOx/Q6 form
the disulfide bridge in the presence of molecular oxygen to produce the hydrogen peroxide.
The four proteins mentioned above, the TRX, PDI, and SOxN SOx/Q6 possess in structure
at least one thioredoxin domain but is functionally antagonistic actions vary between
oxidation and reduction, suggesting that there may be functional interactions between these
proteins.
Studies show the presence of SOx/Q6 in many human tissues. This protein can be secreted
by cells (Coppock et al., 1993). This suggests that SOx/Q6 may be involved in disulfide
bond formation of a wide variety of proteins within the cell (Thorpe et al., 2002) and that
this molecule may also participate in the deposit and the state of crosslinking of the
extracellular matrix by the catalysis of disulfide bridges (Thorpe et al., 2002) (Figure 2). In
addition the results obtained in the laboratory Inserm U 618 suggest that the SOx/Q6 is
involved in lung cancer non-small cell. In this context, it was important to assess the possible
disruption of the expression of SOx/Q6 in lung tumor cell lines models by analyzing the
level of secretion of human SOx/Q6 at different stages of growth by measuring the oxidase
activity.
2. MATERIAL AND METHODS
2-1) Biological Material
2-1-1) Cell lines
The work was carried out from a line WI38 human embryonic lung fibroblast and lung
tumor epithelial cells (Calu-1, H1838, H358, H460, H23, A549, H520). These lines come

PhytoChem &BioSub Journal Vol. 14(3) 2020
ISSN 2170-1768
CAS-CODEN:PBJHB3
270
from the ATCC (American Type Culture Collection). They are grown in their appropriate
culture media (MEM, RPMI 1640 (GIBCO)) with 10% fetal calf serum (FCS) (reference
10106-169, Biological Industries) at 37 ° C and 5% CO2.
2-1-2) The cell culture supernatants
The culture supernatants were collected at pre-confluence three days after sowing (T1), at
confluence five days after sowing (T2) and post-confluence seven days after sowing (T3).
The samples are stored at -20 ° C and then concentrated 20 times with 2 mL Vivaspin
concentrators (Viva sciences) with a cutoff of 10,000 by repeated centrifugation at 5000 g
for 3 minutes at 4 ° C (Universal 16 R). A protease inhibitor cocktail (Sigma, P8340) was
added to the supernatants concentrated in the proportion of 1/50.
FCS control was performed in parallel. The culture medium RPMI with 10% FCS was
incubated at 37 ° C and 5% CO 2 for 3, 5 and 7 days before concentration.
2-2) Specific antibodies directed against the sulfhydryl oxidase
The antibodies used are polyclonal antibodies produced by chicken. Extraction and
purificationdes antibody was made from chicken eggs immunized, but the purified anticoprs
have undergone tests recognition SOX molecule Q6 rat and human.
2-3) Measurement of activity oxidize
2-3-1) Principle of the assay oxidation of DTT by DTNB
To perform this measurement, we used as substrate dithiothreitol (DTT) (lot No. 708984
85849121, Boehringer) which has two SH groups. Sulphydryl oxidase, put into the presence
of DTT reduced, catalyzes the formation of an internal disulfide.
The amount of free thiols present in the medium was measured using the thiol reagent 5,5 '-
dithiobis-2-nitrobenzoic acid (DTNB) (Ellman et al., 1956) acid. DTNB (reference D-8130,
lot 54H0479, Sigma) reacts with free thiols to form a mixed disulfide and releasing 5-thio-2-
nitrobenzoic acid (TNB) chromophore which has a maximum of absorption at 412 nm with a
molar extinction coefficient of 13,600 M-1.cm-1. This method allows us to measure the
disappearance of free thiols present in the reaction medium as a function of time and derive
the rate of oxidation.
2-3-2) Measurement of oxidase activity in culture supernatants.
Measures of oxidase activity in concentrated culture supernatants were performed under the
following conditions:
Culture supernatant
Concentrated 20 times sulfhydryl oxidase (rSOx) Control DTT Control
Mu.l buffer volume 34.6µl 37.6 µl 39.6 µl
volume rSOx 0 µl 2 µl 0 µl
sample volume 5 μL 0 0 µl
Volume of DTT 200
mM 0.4 µl 0.4 µl 0.4 µl
total volume 40 µl 40 µl 40 µl