Bax and APPL1 are involved in DCC-ADD induced colorectal ...
Bax and APPL1 are involved in DCC-ADD induced colorectal ...
Bax and APPL1 are involved in DCC-ADD induced colorectal ...
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I. Introduction<br />
Deleted <strong>in</strong> <strong>colorectal</strong> carc<strong>in</strong>oma (<strong>DCC</strong>) is a<br />
tumor suppressor, which was discovered <strong>in</strong> rectal<br />
carc<strong>in</strong>oma tissues <strong>in</strong> the early 1990s. <strong>DCC</strong> gene<br />
locates on chromosome 18q21.3 <strong>and</strong> spans 1.4 Mb<br />
encod<strong>in</strong>g a transmembrane prote<strong>in</strong> composed of 1447<br />
am<strong>in</strong>o acids (Fearon ER, et al., 1990). The<br />
extracellular region of <strong>DCC</strong> is a receptor for netr<strong>in</strong>-1<br />
conta<strong>in</strong><strong>in</strong>g four immunoglobul<strong>in</strong> doma<strong>in</strong>s <strong>and</strong> six<br />
fibronect<strong>in</strong> type III doma<strong>in</strong>s. And the transmembrane<br />
<strong>and</strong> cytoplasmic region of <strong>DCC</strong> conta<strong>in</strong>s an addiction<br />
dependence doma<strong>in</strong> (<strong>ADD</strong>), which <strong>in</strong>troduces signals<br />
<strong>in</strong>to cytoplasm (Bernet A, et al., 2007). Expression of<br />
<strong>DCC</strong> is markedly reduced <strong>in</strong> more than 50% of<br />
<strong>colorectal</strong> tumors, as well as <strong>in</strong> many other neoplasms<br />
(Horstmann MA, et al., 1997; Mehlen, P, et al.,<br />
2004). And loss of <strong>DCC</strong> is associated with poor<br />
prognosis <strong>and</strong> potentially decreased response to<br />
adjuvant chemotherapy <strong>in</strong> <strong>colorectal</strong> cancer patients<br />
(Gal R, et al., 2004; Shibata D, et al., 1996; Saito M,<br />
et al.,1999; Banerjee AK, 1997). The Rat-1 cell l<strong>in</strong>e<br />
with high levels of <strong>DCC</strong> antisense RNA expression<br />
showed a faster growth rate, <strong>and</strong> had tumorigenicity<br />
<strong>in</strong> nude mice (Narayanan R, et al., 1992). While<br />
restoration of <strong>DCC</strong> expression can suppress<br />
tumorigenic growth properties <strong>in</strong> vitro <strong>and</strong> <strong>in</strong> nude<br />
mice (Goyette MC, et al., 1992; Tanaka K, et al.,<br />
1991; Mehlen, P, et al., 2004).<br />
<strong>DCC</strong> functions as a tumor suppressor via its<br />
ability to trigger tumor cell apoptosis (Castets M, et<br />
al., 2011). By function<strong>in</strong>g as a dependence receptor,<br />
<strong>DCC</strong> <strong>in</strong>duces apoptosis unless engaged by its lig<strong>and</strong>,<br />
netr<strong>in</strong>-1. Over expression of the <strong>DCC</strong> lig<strong>and</strong> netr<strong>in</strong>-1<br />
<strong>in</strong> the digestive tract has been shown to result <strong>in</strong> the<br />
<strong>in</strong>hibition of epithelial cell death <strong>and</strong> the promotion of<br />
tumor progression. The pro-apoptotic signal<strong>in</strong>g<br />
<strong>in</strong>duced by <strong>DCC</strong> requires its <strong>in</strong>tracellular doma<strong>in</strong><br />
cleavage by caspase3 after Asp 1290 (Mazel<strong>in</strong> L, et<br />
al., 2004; Mehlen P, et al., 1998). The mutation of<br />
Asp 1,290 to Asn <strong>in</strong>hibits the cleavage <strong>and</strong> suppresses<br />
the pro-apoptotic activity of <strong>DCC</strong>. The residues 1243-<br />
1264 of <strong>DCC</strong> exposed by cleavage were ascerta<strong>in</strong>ed<br />
to be the very doma<strong>in</strong> required to <strong>in</strong>duce apoptosis.<br />
This doma<strong>in</strong>, named <strong>ADD</strong>, <strong>in</strong>teracts with caspase9<br />
<strong>and</strong> activates downstream caspases, by which the<br />
apoptotic signal is amplified <strong>and</strong> <strong>in</strong>duces cell<br />
apoptosis (Mehlen P, et al., 1998; Forcet C, et al.,<br />
2001). Although some studies had shown that the<br />
<strong>in</strong>teraction with caspases might be the mechanism of<br />
<strong>DCC</strong>-<strong>in</strong>duced apoptosis, whether other apoptosis<br />
signal pathway is <strong><strong>in</strong>volved</strong> has not been affirmed. In<br />
the present study, we found that the expression of <strong>Bax</strong><br />
<strong>and</strong> <strong>APPL1</strong> <strong>in</strong> <strong>colorectal</strong> cancer cells SW1116 is<br />
evidently <strong>in</strong>creased after the transfection of <strong>DCC</strong>-<br />
<strong>ADD</strong> gene, <strong>in</strong>dicat<strong>in</strong>g that both <strong>Bax</strong> <strong>and</strong> <strong>APPL1</strong> <strong>are</strong><br />
closely related to the pro-apoptotic activity of <strong>DCC</strong>.<br />
Gene Therapy <strong>and</strong> Molecular Biology Vol 14, page 59<br />
57<br />
II. Materials <strong>and</strong> Methods<br />
2.1 Vector construction<br />
The cod<strong>in</strong>g sequence of <strong>DCC</strong>-<strong>ADD</strong> (NM_005215.3,<br />
3,727-3,792 bp) was obta<strong>in</strong>ed by anneal<strong>in</strong>g of the follow<strong>in</strong>g<br />
two oligodeoxynucleotides (synthesized by Sangon Biotech<br />
Co., Ltd, Ch<strong>in</strong>a) at 55 o C for 1 m<strong>in</strong> follow<strong>in</strong>g denaturat<strong>in</strong>g<br />
at 94 °C for 30 s. The sense str<strong>and</strong> was 5'-<br />
CTAGCGCCACCATGAACAATCCTGCTGTCGTGAGC<br />
GCCATCCCGGTGCCAACGCTAGAAAGTGCCCAGTA<br />
CCCAGGAATCTGAC-3', <strong>and</strong> the anti-sense str<strong>and</strong> was 5'-<br />
TCGAGTCAGATTCCTGGGTACTGGGCACTTTCTAG<br />
CGTTGGCACCGGGATGGCGCTCACGACAGCAGGA<br />
TTGTTCATGGTGGCG-3'. (The underl<strong>in</strong>ed letters were<br />
cohesive end sites of restriction enzyme XhoI <strong>and</strong> NdeI, <strong>and</strong><br />
the italic letters were Kozak sequence). The <strong>ADD</strong> sequence<br />
was then ligated with XhoI <strong>and</strong> NdeI digested plasmid<br />
vector pIRES2-EGFP. The recomb<strong>in</strong>ed plasmid was<br />
transformed <strong>in</strong>to E.coli JM109 competent cells. After<br />
screen<strong>in</strong>g, amplify<strong>in</strong>g, extract<strong>in</strong>g <strong>and</strong> purify<strong>in</strong>g, the<br />
recomb<strong>in</strong>ant plasmid pIRES2-EGFP-<strong>ADD</strong> was used for<br />
transfection.<br />
2.2 Cell culture <strong>and</strong> transfection<br />
Human <strong>colorectal</strong> carc<strong>in</strong>oma cells SW1116 were at<br />
37 o C <strong>in</strong> a 5% CO 2 humidified <strong>in</strong>cubator <strong>and</strong> ma<strong>in</strong>ta<strong>in</strong>ed <strong>in</strong><br />
Dulbecco’s modified Eagle’s medium (DMEM)<br />
supplemented with 10% heat-<strong>in</strong>activated fetal bov<strong>in</strong>e serum<br />
(FBS, GIBCO) <strong>and</strong> antibiotics (100 IU/mL of penicill<strong>in</strong> <strong>and</strong><br />
100 IU/mL of streptomyc<strong>in</strong>).<br />
The SW1116 cells were seeded <strong>in</strong>to 96, 24 or 6-well<br />
plates (5×10 3 , 1×10 4 or 2×10 5 cells/well) <strong>and</strong> cultured for<br />
12 hours to confluence. The plasmid pIRES2-EGFP-<strong>ADD</strong><br />
or pIRES2-EGFP was then transfected <strong>in</strong>to SW1116 cells<br />
with Lipofectam<strong>in</strong>e 2000 (Invitrogen, USA) accord<strong>in</strong>g to<br />
the manufacturer’s <strong>in</strong>struction, <strong>and</strong> the ratio of<br />
Lipofectam<strong>in</strong>e 2000 (μL) to DNA (μg) was 5:2.<br />
2.3 Cell count kit-8 (CCK-8) cell<br />
proliferation assay<br />
The SW1116 cells were seeded <strong>in</strong>to a 96-well plate<br />
<strong>and</strong> cultured for 12 hours to confluence followed by<br />
trasfection. Each group comprised 10 wells. 36 h post<br />
transfection, 10 μL of CCK-8 solution (Doj<strong>in</strong>do, Japanese)<br />
was added <strong>in</strong>to each well, which conta<strong>in</strong>ed 100 μL of<br />
medium. After <strong>in</strong>cubation for 3 h at 37 o C, the amount of<br />
generated formazan was measured for absorbance at a<br />
wavelength of 450 nm. The optical density (OD) values<br />
recorded represented the proliferation of cells, <strong>and</strong> were<br />
used to calculate the growth <strong>in</strong>hibition rate by the formula:<br />
Inhibition rate = (1 - OD value of transfection group/OD<br />
value of medium group) × 100%. All the above experiments<br />
were performed <strong>in</strong> triplicate.<br />
2.4 Acrid<strong>in</strong>e orange/ethidium bromide<br />
(AO/EB) fluorescence sta<strong>in</strong><strong>in</strong>g assay<br />
Little coverslips were putted <strong>in</strong>to each well of a<br />
24-well plate before seed<strong>in</strong>g SW1116 cells <strong>in</strong>to it. The<br />
cells were cultured for 12 hours to confluence followed<br />
by transfection. 24 h post transfection, the coverslips<br />
were took out for AO/EB fluorescence sta<strong>in</strong><strong>in</strong>g.