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Effect of Snake Venom Toxin on Inhibition of Colorectal Cancer HT29 Cells Growth via Death Receptors Mediated Apoptosis

Objectives : We investigated whether snake venom toxin(SVT) from Vipera lebetina turanica sensitizes HT29 human epithelial colorectal cancer cells to tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL) induced apoptosis in cancer cells. Methods : Cell viability assay was used to assess the inhibitory effect of TRAIL on cell growth of HT29 human colorectal cancer cells. And 6-diamidino-2-phenylindole(DAPI), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay(TUNEL) staining assay were used to evaluate cell-apoptosis. Western blot analysis were conducted to observe apoptosis related proteins and death receptor. To assess whether the synergized inhibitory effect of SVT and TRAIL on reactive oxygen species(ROS) generation was reversed by strong anti-oxidative agent. Results : SVT with TRAIL inhibited HT29 cell growth different from TRAIL alone. Consistent with cell growth inhibition, the expression of TRAIL receptors; Expression of death receptor(DR)4 and DR5 was significantly increased and intrinsic pro-apoptotic cleaved caspase-3, -9 was subsequently increased together with increase of Bax/Bcl-2 ratio and extrinsic pro-apototic caspase-8 was also activated. In addition, the expression of anti-apoptotic survival proteins, a marker of TRAIL resistance(eg, cFLIP, survivin, X-linked inhibitor of apoptosis protein(XIAP) and Bcl-2) was suppressed by the combination treatment of SVT and TRAIL. Pretreatment with the ROS scavenger N-acetylcysteine abolished the SVT and TRAIL-induced upregulation of DR4 and DR5 expression and expression of the intrinsic pro-apoptotic caspase-3 and-9. Conclusion : The collective results suggest that SVT facilitates TRAIL-induced apoptosis in HT29 human epithelial colorectal cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 and consecutive induction of bilateral apoptosis via regulating apoptosis related proteins.


Ⅰ. Introduction
Colorectal carcinoma is mostly common and fatal, even holding a third rank in males and a second in females in the causes of cancer-related death worldwide 1,2) , due to current medical limitations such as incomplete visualization of its cells 3) , relatively rare, highly chemo-resistance 4,5) and its quiescent or slow proliferation 6) .
A single layer of epithelial cells on colorectum play a major role in maintaining a protective barrier to various immune cells between the lumen and the lamina propria 7,8) and preserving homeostasis between cell proliferation and apoptosis. Frequent exposure to imbalance of their dynamic equilibrium leads to inflammatory condition 9) and subsequently results in fatal colorectal cancer 10) .
Therefore, a novel therapeutic strategy and agent is urgently needed to cope with this cancer overcoming the tough challenge on the basis of better understanding the molecular mechanisms of apoptosis. Apoptosis, a programmed cell death can be triggered by two major pathways including the mitochondria-dependent intrinsic pathway and the death receptor (DR)-related extrinsic pathway 11,12) .
Recently, more and more attention is paid to the latter for a promising therapeutic approach of various cancers including colorectal cancer because of the well recognized attributes that DR signaling apoptosis is so selective and specific as to do little harm to normal cells and to effectively induce intrinsic apoptosis as well as extrinsic one 13,14) .
Of the above DR associated apoptotic pathway, DR4 and DR5 draws researcher s attraction as representatives, which exists on the cell surface of Tumors and binds to its ligand Tumor necrosis factor(TNF)α-related apoptosis-inducing ligand(TRAIL), a member of the TNF ligand super family, and subsequently induces apoptosis through up-regulation of pro-apoptotic caspases via activation of caspase-8 15,16) and concomitant initiation of mitochondria regulated apoptosis [17][18][19][20][21] . According to previous studies [22][23][24][25][26][27] , agents such as curcumin, baicalein, ursolic acid, gossypol and 15-deoxy-deltaprostaglandin J2 actually up-regulated the DR4 and DR5 TRAIL receptors in various cancer cells. However, another challenge, resistance of DR4 and DR5 to TRAIL makes clinicians or researchers not eagerly use TRAIL alone but decide to use it with other chemotherapeutic agents together, for they synergistically sensitize cancer cells through enhancement of DR4 and DR5 expressions via overcoming the TRAIL resistance [28][29][30][31] .
Natural snake venom toxin(SVT) from Vipera lebetina turanica was previously demonstrated as a promising chemotherapeutic agent against the growth of human prostate cancer cell and neuroblastoma cell through induction of apoptotic cell death that is mediated by the modulated expression of apoptosis regulatory proteins [32][33][34] .
In this study, I evaluated whether SVT from VVipera lebetina turanica combined with TRAIL represents anticancer effects in HT29 epithelial human colorectal cancer cells, and how it overcomes TRAIL resistance and restore the sensitivity in them.

B. Cell culture
The human colorectal cancer HT29 cell lines were purchased from the American Type Culture Collection (Manassas, VA). They were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS) and penicillin/streptomycin(100 U/㎖). Cell cultures were then maintained at 37 ℃ in a humidified atmosphere with 5 % CO 2 .

C. Cell viability assay
To determine the cell number, human colorectal cancer HT29 cells were plated in 24 well plates(5×10 4 cells/well) with or without TRAIL, and subconfluent cells were subsequently treated with SVT(0.1, 0.5, 1, 2 and 5 ㎍/㎖) for 24 hr. After treatment, cells were trypsinized and pelleted by centrifugation for 5 min at 1,500 rpm, resuspended in 5 ㎖ of phosphate-buffered saline(PBS), and 0.1 ㎖ of 0.2 % trypan blue was added to the cancer cell suspension in each of the solutions (0.9 ㎖ each). Subsequently, a drop of suspension was placed into a Neubauer chamber and the living cancer cells were counted. Cells that showed signs of staining were considered to be dead, whereas those that excluded trypan blue were considered viable. Each assay was carried out in triplicate.

D. Measurement of ROS
Generation of ROS was assessed by 2,7dichlorofluorescein diacetate(DCFH-DA, Sigma Aldrich, StLouis, MO, USA), anoxidation-sensitive fluorescent probe. Intracellular H2O2 or low-molecular-weight peroxides can oxidize 2,7-dichlorofluorescein diacetate to the highly fluorescent compound dichlorofluorescein (DCF). Briefly, human colorectal cancer HT29 cells were plated in 6 well plates(5×10 4 cells/well), and subconfluent cells were subsequently treated with SVT and TRAIL for 30 min. After the cells were trypsinized, the 1×10 4 cells were plated in black 96 well plate and incubated with 10 µM DCFH-DA at 37 ℃ for 4 hr. The fluorescence intensity of DCF was measured in a microplate-reader at an excitation wave length of 485 ㎚ and an emission wave length of 538 ㎚. were separated on SDS/12 %-polyacrylamide gels and then transferred to a nitrocellulose membrane(Hybond ECL; Amersham Pharmacia Biotech). Blots were blocked for 2 hr at room temperature with 5 %(w/v) non-fat dried milk in tris buffered saline(10 mM Tris, pH 8.0, 150 mM NaCl) containing 0.05 % Tween-20. The membrane was incubated for 5 hr at room temperature with following specific antibodies: mouse monoclonal antibodies directed against cleaved caspase-8(1:1,000 dilutions; Cell Signaling Technology, Beverly, MA), against Bax(1:500 dilutions; Santa Cruz Biotechnology), and against XIAP, survivin, bcl-2, cleaved caspase-3, -9 and c-FLIP(1:1,000 dilutions; Cell Signaling Technology, Beverly, MA). The blot was then incubated with the corresponding anti-rabbit/goat immunoglobulin G-horseradish peroxidase-conjugated secondary antibody(Santa Cruz Biotechnology Inc). Immunoreactive proteins were detected with the Enhanced Chemiluminescence Western blotting detection system(Amersham Pharmacia Biotech Inc, Buckingham shire, United Kingdom).

F. Apoptosis evaluation
Human colorectal HT29 cells(2.5×10 5 cells/well) were cultured on 8-chamber slides. The cells were treated with SVT(0.5 ㎍/㎖). The cells were washed twice with PBS and fixed by incubation in 4 % paraformaldehyde in PBS for 1 hr at room temperature. Membrane was permeabilized by exposure to 0.1 % Triton X-100 in phosphate-buffered saline for 5 min at room temperature. TdT-mediated dUTP nick and labeling (TUNEL) assays were performed by using the in situ Cell Death Detection Kit(Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. For 4'-6-Diamidino-2-phenyl indole (DAPI) staining, slides were incubated for 15 min at room temperature in the dark with mounting medium for fluorescence containing DAPI(Vector Laboratories Inc, Burlingame, CA). The cells were then observed through a fluorescence microscope(Leica Microsystems AG, Wetzlar, Germany).

G. Statistical analysis
The data were analyzed using the GraphPad Prism 4 ver. 4.03 software(GraphPad Software, La Jolla, CA). Data are presented as mean± SD. The differences in all data were assessed by one-way analysis of variance (ANOVA). When the p value in the ANOVA test indicated statistical significance, the differences were assessed by the Dunnett s test. A value of p<0.05 was considered to be statistically significant.

Ⅲ. Results
A. Effect of snake venom toxin on cell growth in HT29 human colorectal cancer cells       (Fig. 4).

E. Reverse effect of NAC on snake venom toxin-combined TRAIL induced ROS generation in HT29 human colorectal cancer cells
To assess whether the synergized inhibitory effect of SVT and TRAIL on ROS generation was reversed by strong anti-oxidative agent, NAC(10 mM) on cell growth of HT29 human colorectal cancer cells, I analyzed ROS measurement as described in materials

F. Synergic effect of snake venom toxin and TRAIL on apoptosis in HT29 human colorectal cancer cells
To determine whether SVT enhances TRAIL-induced apoptotic cell death of TRAIL-insensitive HT29 human colorectal cancer cells. I found that SVT alone increased about 39 % of caspase active cells, compared to control, 2.6 %, inducing 41.6 % apoptosis in HT29 human colorectal cancer cells, where TRAIL, 2.8 % represented little increase of them in contrast. However, combination treatment with SVT and TRAIL synergistically enhanced apoptotic cell death to 68.2 % in HT29 human colorectal cancer cells, compared to SVT alone (Fig. 6).

G. Effect of snake venom toxin on the expression of death receptors 4 and 5 in HT29 human colorectal cancer cells
To ascertain the underlying mechanism that may be responsible for enhancement of TRAIL-induced apoptotic cell death by SVT, I examined the effect of SVT on the expression of death receptors. SVT increased the expressions of both DR4 and DR5 in TRAIL-insensitive HT29 cells (Fig. 7). Especially, it represented dose dependent enhancement in DR5 expression (Fig. 7). Contrary to SVT, TRAIL exerted little influence on the expression of DR4 and DR5 in TRAIL-insensitive HT29 human colorectal cancer cells (Fig. 7). However, expression of DR4 and DR5 was synergistically further increased by the combined treatment of SVT and TRAIL (Fig. 7).

H. Effect of snake venom toxin on
TRAIL-induced expression of apoptosis related proteins I selected HT29 human colorectal cancer cell line to confirm its sensitivity to TRAIL and to ascertain the effect of SVT and/or TRAIL on the activation of caspase-8, caspase-3, caspase-9 cleavage and the expression of Bax in it. Although TRAIL alone had little effect on the activation of caspases cleavage and Bax expression, SVT increased the their expression, compared to control. Moreover, combination treatment of SVT and TRAIL significantly increased expression of the pro-apoptotic proteins (Fig. 8). On the basis of the previous reports that various anti-apoptotic proteins including survivin, Bcl-2, XIAP and cFLIP have been shown to induce resistance to TRAIL-induced apoptosis 35,36) . I investigate whether SVT sensitized HT29 cells to TRAIL through down-regulation of the expression of these anti-apoptotic proteins irrespective of their TRAIL resistance. As shown in Fig. 8, SVT decreased expression of XIAP, survivin, bcl-2 and cFLIP concentration dependently. These results indicated that the SVT enhances TRAIL-induced I. Reversal of synergistic effect of snake venom toxin-combined TRAIL on pro-apoptotic proteins in HT29 human colorectal cancer cells HT29 cells were pretreated with SVT for 24 hr and washed out. After that the cells were treated with TRAIL for 24 hr, and whole cell extracts were analyzed by western blotting using antibodies against caspase-3, caspase-9, β-actin. β-actin protein was used an internal control. Each band is representative for three experiments. I found that pretreatment(1 hr) of ROS scavenger N-acetylcysteine(NAC, 10 mM) reversed the activation of cleavaged caspase-3 and -9 Fig. 9. Increased expressions of snake venom tToxin-combined TRAIL-induced apoptotic protein was reversed by anti-oxidative NAC Whole cell extracts were analyzed by western blotting using the antibodies against caspase-3, caspase-9 and β-actin. β-actin protein was used an internal control. Each band is representative for three experiments.
by the combination treatment of SVT and TRAIL, suggesting TRAIL should be a promising target of apoptosis in HT29 cells (Fig. 9).
J. Reversal of synergistic effect of snake venom toxin-combined TRAIL on expression of DR4 and DR5 in HT29 human colorectal cancer cells HT29 cells were pretreated with SVT for 24 hr and washed out. After that the cells were treated with TRAIL for 24 hr, and whole cell extracts were analyzed by western blotting using antibodies against DR4, DR5 and β-actin. β-actin protein was used an internal control. Each band is representative for three experiments. I found that pretreatment(1 hr) of ROS scavenger N-acetylcysteine(NAC, 10 mM) reversed the increased activation of DR4 and DR5 by the combination treatment of SVT and TRAIL (Fig. 10).

Ⅳ. Discussion
Apoptosis is a form of programmed cell death, which can be triggered by two major pathways including mitochondria-dependent intrinsic pathway and DRassociated extrinsic pathway 11,12,37,38) .
In the intrinsic apoptotic pathway, ROS stimuli exerts influence upon intrinsic mitochondrial regulated apoptotic pathway, translocating a truncated form of Bid(tBid) into mitochondria and consecutively making a change of the mitochondrial outer membrane potential due to increased Bax/Bcl-2 ratio, and subsequently resulting in mitochondrial intrinsic apoptosis through activation of caspase-9 and caspase-3 via cytochrome C release, whereas it consecutively let DR4 and DR5 binding to its ligand, subsequently causing apoptosis through caspase-8-mediated rapid activation of caspase cascades in the extrinsic apoptotic pathway [39][40][41][42][43] .
Therefore, this implicate that ROS generation could be involved in extrinsic apoptosis as well as intrinsic one, that is substantiated by previous reports 25,44) .
In this study, it was found that Vipera lebetina turanica snake venom toxin inhibited cell growth of HT29 human epithelial colorectal cancer cells through induction of both intrinsic, mitochondria-mediated, and extrinsic DR-mediated apoptosis via increased binding of DR4, DR5 with TRAIL, and that the combination of SVT and TRAIL increased apoptotic cell death synergistically in spite of TRAIL insensitivity to them, representing SVT overcame TRAIL resistance and let the apoptosis going through enhancement of expression of proapoptotic proteins and control of anti-apoptotic ones simultaneously.
That is, SVT significantly inhibited cell proliferation of HT29 human colorectal cancer cells (Fig. 1). However, inconsistent with a previous report 45) , TRAIL had little influence upon cell viability of them (Fig. 2). However, TRAIL(50 ng/㎖) plus SVT(0.5 ㎍/㎖) synergistically and more significantly hindered cell growth of them, compared to SVT(0.5 ㎍/㎖) alone (Fig. 3). After treatment of strong anti-oxidant, N-acetyl-cysteine, NAC(1 or 10 mM), antiproliferation of HT29 human colorectal cancer cells and increase of ROS generation by combined SVT and TRAIL was abolished dose-dependently, suggesting inhibition of the cell growth was related with bilateral apoptosis induced by SVT together with TRAIL. I reconfirmed the above in the TUNEL assay that combination treatment with SVT and TRAIL synergistically increased apoptotic cell death to 68.2 % in HT29 human colorectal cancer cells, compared to SVT alone or TRAIL alone (Fig. 6). TRAIL mediates apoptotic cell death through enhanced expression of two death receptors, DR4 and DR5 which are expressed on the surface of cancer cells 46,47) . The binding of TRAIL to DR4 and DR5 result in DR mediated extrinsic apoptosis 48,49) .
Expression of DR4 and DR5 was also synergistically further increased by the combined treatment of SVT and TRAIL than SVT or TRAIL alone (Fig. 7). Taken together, it was suggested that SVT should play a major role in inducing DR mediated extrinsic apoptosis through compensating TRAIL resistance in HT29 human epithelial colorectal cancer cells.
Caspases, a family of cysteine aspartyl-specific proteases, control the triggering and executing apoptosis in both pathways including mitochondria dependent intrinsic apoptotic and DR mediated extrinsic apoptotic pathway 50) . In the activation of caspase cascade, of three main pathways including DR-ligand complex-activated caspase-8 pathway, the mitochondria dependent pathway characterized by mitochondrial membrane permeability change according to pro-apototic Bax/anti-apoptotic Bcl-2 ratio, consecutive cytochrome c release and subsequent activation of apoptosome-associated caspases-9 and -3 and the endoplasmic reticulum(ER)-specific apoptotic pathway involving caspase-12 activation, former two pathways are more noteworthy regarding both apoptotic pathways in various cancer cells and considered a potential target of developing new anticancer drugs [51][52][53] where induction of apoptosis critically depends on upregulation of proapoptotic caspases known as executioners of apoptosis [54][55][56] .
In the expression of apoptosis related proteins, SVT with TRAIL in HT29 cells represented increased Bax/Bcl-2 ratio, subsequently upregulated intrinsic apoptotic pathway related cleaved caspase-9, -3 as well as activated extrinsic DR-mediated apoptotic pathway related caspase-8, suggesting SVT exerts substantial influence upon bilateral apoptosis in them nevertheless of TRAIL resistance (Fig. 8).
Several studies have reported that resistance of cancer cells to TRAIL-induced apoptosis is known to be related to intracellular levels of anti-apoptotic proteins and that DR up-regulation and cell survival protein down regulation may be a promising strategy for sensitizing TRAIL-resistant cancer cells to TRAIL-induced apoptotic cell death [57][58][59][60][61] .
Coincident to the above, SVT decreased expression of anti-apoptotic XIAP, survivin, bcl-2 and cFLIP in addition to DR4, 5 enhancement in the HT29 human epithelial colorectal cancer cells, indicating that the SVT strengthened TRAIL-induced apoptotic cell death through the over-expression of DR4 and DR5, as well as the down-regulation of anti-apoptotic protein expression.
These results also reconfirmed through experiment to ascertain whether pretreatment(1 hr) of ROS scavenger N-acetylcysteine(NAC, 10 mM) abolish the activation of DR4,5 and pro-apoptotic cleavaged caspase-3 and -9 induced by the combination treatment of SVT and TRAIL (Fig. 9, 10) In conclusion, this study shows that SVT strengthen sensitizing HT29 human epithelial colorectal cancer cells to the TRAIL induced apoptosis, and that the combination treatment of SVT and TRAIL could get over TRAIL resistance. The results suggest that SVT may be a potent agent for the treatment or prevention of colorectal cancer.

V. Conclusion
The collective results suggest that SVT facilitates TRAIL-induced apoptosis in HT29 human epithelial colorectal cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 and consecutive induction of bilateral apoptosis via regulating apoptosis related proteins.