J. Analysis of cytochrome c release
4. Aspirin makes TRAIL-resistant HeLa cells TRAIL-sensitive through inhibition of
When aspirin was pretreated at least for 24hr, TRAIL-induced apoptotic cell death of HeLa was considerably enhanced in company with caspase activation. In order to
explore a
major mechanism(s) that
makes HeLa cells TRAIL-sensitive, I examined the changed expressions of various apoptotic regulators by western blotting following aspirin treatment (Fig. 9). It is well known that TRAIL can bind with death receptors, DR4 and DR5, which trigger extrinsic apoptotic signal pathway (Pan et al., 1997). Specially, DR5 levels are up-regulated by chemotherapeutic agents (Jun et al., 2011). Thus, I checked the effect of aspirin on the expression levels of DR4 and DR5. However, the results from western blot analysis showed that aspirin treatment did not alter the total cellular levels of DR4 and DR5 even at 32hr treatment.In addition, changes in the level of anti-apoptotic protein Bcl-2 and
pro-apoptotic protein Bak were negligible. On the other hand, aspirin affected the
activation and expression of ERK1/2 and Bax. The activation of ERK1/2 was
inhibited and
the level of Bax expression was increased by aspirin depending on time of treatment (Fig. 9A). Next, I examined those molecules in the combined treatment of aspirin with TRAIL. Interestingly, I observed obvious decrease of ERK1/2 activation and Mcl-1protein expression, which was up-regulated by TRAIL alone (Fig. 9B). To see whether inactivation of ERK1/2 by aspirin is an important mechanism to make HeLa cells TRAIL-sensitive, we tested the effect of combination treatment of ERK inhibitor PD98059 (20uM) for 2hr and TRAIL (100ng/ml) for 8hr in HeLa cells. The combination of PD98059 and TRAIL enhanced cell death (Fig. 10A). As shown in analysis of annexin V staining, sub G1 population, and MMP change analysis (Fig. 10B, C, and D), apoptotic cell death was enhanced by the combination treatment of PD98059 and TRAIL. As shown in Fig.10E, inhibition of ERK1/2 activation by PD98059 induced caspase activities and down-regulated Mcl-1 level. The results from the treatment of TRAIL and an ERK inhibitor were
similar to those from the treatment of aspirin and TRAIL. It indicates that
ERK1/2 inactivation by aspirin plays a critical role in TRAIL-induced apoptotic cell death through promoted caspase activities.- 20 -
Fig.9. Effect of various time of aspirin treatment and combined treatment of aspirin with TRAIL. A. HeLa cells were treated with 5mM aspirin for 0, 6, 12, 18, 24 and 32hr. B.
The cells were pretreated with 5mM aspirin for 24hr and then treated with 100ng/ml TRAIL for 8hr. Total protein extract was obtained and indicated proteins were analyzed by western blotting with appropriate antibodies. α-tubulin was used as loading control.
- 21 - A.
- 22 - E.
Fig.10. Effect of the combined treatment of PD98059 with TRAIL. HeLa cells were treated with TRAIL (100ng/ml) for 8hr in the presence or absence of PD98059 (20uM) for 2hr. A. Cytotoxicity was analyzed by LDH assay. B. Annexin-V/PI analysis. C. Sub G1 anlaysis. D. MMP change anlaysis. E. Total protein extracts were obtained and analyzed by western blotting. The cells were seeded at 5х103 cells in 96 flate well (A), 1.5 х105 cells in 6-well plate (B,C, and D) or 1х106 cells in 60ф dish (E), respectively and incubated for 20hr.
- 23 -
Fig. 11. Effect of caspase about expression of p-ERK and Mcl-1. HeLa cells were pretreated with 50uM of z-VAD for 1hr and aspirin and TRAIL were treated as previously described. Concentrations of aspirin and TRAIL were 5mM and 100ng/ml, respectively.
Cells were harvested and equal amount of protein was processed for western blot analysis to detect target proteins.
- 24 -
Suppl.1. Cytotoxicity was analyzed by LDH assay. HeLa cells were seeded at 2 х 105 cells in 6-well plate and incubated for 20hr. In the combination treatment, aspirin was pretreated for 12hr, 24hr, or at the same time with TRAIL.
- 25 -
Suppl. 2. Morphologies of HeLa cells treated with aspirin, TRAIL or combination of them in the presence or absence of z-IETD. HeLa cells (2 х 105) were plated onto 6-well tissue culture plates and stabilized for 20hr. Cells were pretreated with aspirin (5mM) for 24hr and treated continuously with TRAIL (100ng/ml) for 8hr without changing media. For the treatment of z-IETD, the cells were pretreated with z-IETD (50uM) for 1hr and followed by treatment with aspirin, TRAIL or the combination
- 26 -
Ⅳ. DISCUSSION
TRAIL has been shown to be selective in the induction of apoptosis in cancer cells with minimal toxicity to normal tissues. However, not all cancers are sensitive to TRAIL-mediated apoptosis (Pitti et al., 1996). TRAIL-resistance can occurs through dysfunctions of the death receptors DR4 and DR5 (S. Prasad et al., 2011), overexpression of 2 or Bcl-X(L) (Fulda et al., 2002, Langenau et al., 2005), loss of Bax or Bak function (Wang et al., 2001, LeBlanc et al., 2002), high expression of c-FLIP or inhibitor of apoptosis proteins (Irmler et al., 1997, Kim et al., 2008) and reduced release of second mitochondria-derived activator of caspases (Smac/Diablo) from the mitochondria to the cytosol ((Luo et al., 1998).
Finally, activation of different subunits of mitogen-activated protein kinases or nuclear factor-kappa B can lead to development of either TRAIL resistance or apoptosis in certain types of cancer cells (Zhang and Fang, 2005). Thus, researchers have attempted to solve a problem of TRAIL-resistance through combimation treatment with other chemical agent. I have tried for the combined treatment of aspirin with TRAIL in TRAIL-resistant HeLa cells.
Aspirin has been used as chemopreventive agents of cancers to induce apoptosis or to reduce the incidence of tumor formations in a variety of organs, i.e. colon (Qiao et al., 1998), lung (Hosomi et al.,2000), stomach (Wong et al., 1999), and colorectum (Williams et al., 1997).
In this work, I was interested in by what mechanism HeLa cells are resistant to TRAIL.
Interestingly, TRAIL enhanced ERK1/2 activation and increased level of anti-apoptotic protein Mcl-1 in HeLa cells. ERK1/2 has been implicated in the regulation of a variety of cellular processes. However, the precise molecular mechanism of ERK1/2 has still remained controversial. For example, ERK1/2 activation is required in cisplatin-induced apoptosis (Wang. et al., 2000). In contrast, ERK 1/2 activation promotes cell survival in neuronal PC12 cells (She et al., 2000). I observed that TRAIL-mediated ERK activation protects HeLa cells from TRAIL-induced apoptosis through up-regulating Mcl-1, not Bcl-2 (Lee et al. 2006).
Different molecules have been reported to play a role in conferring TRAIL resistance in accompany with ERK1/2 activation. For example, the ERK1/2 signaling pathway regulates the expression of Bcl-2, Bcl-XL, and Mcl-1 and promotes the survival of human pancreatic
- 27 -
tumor cells (Boucher et al. 2000). When HeLa cells were treated with TRAIL after pretreatment of aspirin for 24h, more cell death was induced than when they were treated at the same time or pretreated for 12hr (Suppl.1). ERK1/2 inactivation by aspirin affects caspase activiation rather than down-regulation of Mcl-1 level because the level of Mcl-1 was not changed by aspirin. In addition, down-regulated Mcl-1 by the combination treatment was restored by z-VAD (Fig. 11). Although several groups reported down-regulation of Mcl-1 level by aspirin (Park et al.20Mcl-10, Daniel et al. 20Mcl-10), they used the higher concentration (8mM, 10mM) of aspirin, compared with that I used (5mM). Once ERK activation was almost inhibited by aspirin, caspase-3, -8 and -9
were activated and cytochrome c was released from mitochondria. We observed that a caspase-8 inhibitor, zIETD, blocked the cell death induced by combined treatment of aspirin with TRAIL (Suppl.2).
These results imply that ERK activation largely suppressed the processing of caspase-8. Consistently with my results,
there have been several studies to indicate that ERK activation suppresses TRAIL-mediated apoptosis and inhibits the processing of caspase-8 and Bid thereby turning off the mitochondrial amplification loop and that ERK pathway acts as an important modulator of various apoptosis-inducing signals and prevents cell death in different systems including Fas and TRAIL-mediated cell death (Soderstrom et al., 2002, Tran et al., 2001).I also showed that combined treatment of aspirin with TRAIL downregulated level of Mcl-1 protein. In this system, Mcl-1 level was regulated by caspase activation (Fig. 11) as also suggested by Herrant at al. (2004). Downregulated Mcl-1 triggers a mitochondrial pathway and amplifies caspase activation It has been known that the anti-apoptotic Bcl-2 family proteins, including Bcl-2 and Mcl-1, can bind Bid and inhibit Bid's ability to activate Bax or Bak. As a result, the anti-apoptotic Bcl-2 family proteins inhibit apoptosis by sequestering Bid, leading to reduced Bax or Bak activation. Therefore, down-regulation of Mcl-1 can trigger translocation of tBid by caspase-8 from cytosol to the outer mitochondrial membrane (Li at al., 1998). It leads to tBid interacts with pro-apoptotic protein, Bax or Bak leading to the insertion of Bax or Bak into organelle membranes, primarily the outer mitochondrial membrane (Eskes at al., 2000, Clohessy at al., 2006). Activated Bax and/or
- 28 -
Bak form an oligomeric pore in the outer membrane and induce loss of MMP and the release of cytochrome c from mitochondria to cytosol (Desagher at al., 1999). As shown in this study, the released cytochrome c from mitochondria could induce the sequential activation of caspase-9 and caspase-3, which enhance cleavage of Mcl-1 and finally promote apoptosis via mitochondrial pathway in HeLa cells.
In this study, it is suggested that ERK inactivation by aspirin promotes TRAIL-induced caspase activation and activates caspase-dependent cleavage of Mcl-1 which triggers mitochondrial pathway, resulting in enhanced cell death. Taken together, inhibiton of mediated ERK1/2 activation is an important mechanism to overcome TRAIL-resistance in HeLa cells and aspirin plays a role as a ERK 1/2 inhibitor. In conclusion, TRAIL-resistant HeLa cells could be sensitized by pretreatment of aspirin.
- 29 -
REFERENCES
1. Ashkenazi A :Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat. Rev. Cancer 2: 420-430, 2002
2. Bertagnolli MM: The potential of non-steroidal anti-inflammatory drugs (NSAIDs) for colorectal cancer prevention. J. Surg. Oncol 84: 113–119, 2003
3. Bouralexis S, Findlay DM, Atkins GJ, Labrinidis A, Hay S, and Evdokiou A: Progressive resistance of BTK-143 osteosarcoma cells to Apo2L/TRAIL-induced apoptosis is mediated by acquisition of DcR2/TRAIL-R4 expression: resensitisation with chemotherapy. Br. J. Cancer 7: 206–214, 2003
4. Boucher MJ, Morisset J, Vachon PH, Reed JC, Laine J, Rivard N.: MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-X (L), and Mcl-1 and promotes survival of human pancreatic cancer cells. J Cell Biochem 79:355-69, 2000
5. Broaddus VC, Dansen T B, Abayasiriwardana KS, Wilson SM, Finch AJ, Swigart LB, Hunt, AE, and Evan GI: Bid mediates apoptotic synergy between tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and DNA damage. J. Biol. Chem 280: 12486–
12493, 2005
6. Büneker CK, Yu R, Deedigan L, Mohr A, Zwacka RM. IFN-γ combined with targeting of XIAP leads to increased apoptosis-sensitisation of TRAIL resistant pancreatic carcinoma cells.Cancer Lett. Nov 2, 2011
7. Chan AT: Aspirin, non-steroidal anti-inflammatory drugs and colorectal neoplasia: future challenges in chemoprevention. Cancer Causes Control 14: 413–418, 2003
8. Clohessy JG, Zhuang J, de Boer J, Gil-Gomez G, Brady HJM: Mcl-1 interacts with truncated Bid and inhibits its induction of cytochrome c release and its role in receptor-mediated apoptosis. J Biol Chem 281:5750–5759, 2006
- 30 -
9. Daniel IS, M. Pique, M. Barragan et al., “Aspirin induces apoptosis in human leukemia cell independently of NF-κB and MAPKs through alteration of the Mcl-1/Noxa balance,”
Apoptosis, vol. 15, pp. 219–229, 2010
10. Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B, and Martinou JC: Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol 144:
891–901, 1999
11. Degli-Esposti MA, Dougall WC, Smolak PJ, Waugh JY, Smith CA &Goodwin RG. The novel receptor TRAILR4 induces NF-kappaB and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity, 7(6), 813–820,1997
12. Eskes R, Desagher S, Antonsson B, and Martinou JC: Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol. Cell. Biol 20:929–935, 2000
13. Fulda S, Jeremias I, and Debatin KM: Cooperation of betulinic acid and TRAIL to induce apoptosis in tumor cells. Oncogene 23: 7611–7620, 2004
14. Green DR: Apoptotic pathways: the roads to ruin. Cell 94: 695-698, 1998
15. Green DR. and Reed, JC :Mitochondria and apoptosis. Science 281: 1309-1311, 1998 16. Gross A., McDonnell, J.M., and Korsmeyer, S.J.: Bcl-2 family members and the
mitochondria in apoptosis. Genes & Dev. 13: 1899-1911, 1999
17. Herrant M, Jacquel A, Marchetti S, Belhacene N, Colosetti P, et al.: Cleavage of Mcl-1 by caspases impaired its ability to counteract Bim-induced apoptosis. Oncogene.
23:7863–7873, 2004
- 31 -
18. Han J, Goldstein LA, Gastman BR, Rabinowich H: Interrelated roles for Mcl-1 and BIM in regulation of TRAIL-mediated mitochondrial apoptosis. J Biol Chem 281:10153–
10163, 2006
19. Hosomi Y., Yokose T., Hirose Y., Nakajima R., Nagai K., Nishiwaki Y., and Ochiai A : Increased cyclooxygenase 2 (COX-2) expression occurs frequently in precursor lesions of human adenocarcinoma of the lung. Lung Cancer 30, 73–81, 2000
20. Jane EP, Premkumar DR and Pollack IF. Bortezomib sensitizes malignant human glioma cells to TRAIL, mediated by inhibition of the NF-{kappa}B signaling pathway. Mol Cancer Ther 10: 198-208, 2011
21. Jun Yang, Yun Yang, Li Tian, Xi-Feng Sheng, Fei Liu, Jian-Guo Cao. Casticin-induced apoptosis involves death receptor 5 upregulation in hepatocellular carcinoma cells. World J Gastroenterol October 14; 17(38): 4298-4307, 2011
22. Kang MH and Reynolds CP. Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways incancer therapy. Clin Cancer Res. 15: 1126-1132, 2009
23. Kim S, Lee TJ, Park JW, Kwon TK .Overexpression of cFLIPs inhibits oxaliplatin-mediated apoptosis through enhanced XIAP stability and Akt activation in human renal cancer cells. J Cell Biochem 105:971–979, 2008
24. Langenau DM, Jette C, Berghmans S, Palomero T, Kanki JP, et al. Suppression of apoptosis by bcl-2 overexpression in lymphoid cells of transgenic zebrafish. Blood 105:
3278–3285,2005
25. LeBlanc H, Lawrence D, Varfolomeev E, Totpal K, Morlan J, Schow P, Fong S, Schwall R, Sinicropi D, and Ashkenazi A: Tumor-cell resistance to death receptor--induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax. Nat.
Med 8: 274–281, 2002
- 32 -
26. LeBlanc HN, and Ashkenazi A: Apo2L/TRAIL and its death and decoy receptors. Cell Death Differ 10: 66–75, 2003
27. Lee DY, Lee MW, Lee HJ, Noh YH, Park SC, Lee MY, Kim KY, Lee WB, Kim SS:
ERK1/2 activation attenuates TRAIL induced apoptosis through the regulation of mitochondria dependent pathway. Toxicol In Vitro 20: 816-23, 2006
28. Lee Y J, Lee KH, Kim HR., Jessup J M, Seol DW, Kim TH, Billiar TR, and Song, YK:
Sodium nitroprusside enhances TRAIL-induced apoptosis via a mitochondria-dependent pathway in human colorectal carcinoma CX-1 cells. Oncogene 20: 1476–1485, 2001 29. Li H, Zhu H, Xu CJ, and Yuan J: Cleavage of BID by caspase 8 mediates the
mitochondrial damage in the Fas pathway of apoptosis. Cell 94: 491–501, 1998
30. Luo X, Budihardjo I, Zou H, Slaughter C, and Wang X: Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94: 481–490, 1998
31. MacFarlane, M: TRAIL-induced signalling and apoptosis. Toxicol. Lett., 139(2–3), 89–
97, 2003
32. M.Gloria Luciani, Christoph Campregher and Christoph Gasche: Aspirin blocks proliferation in colon cells by inducing a G1 arrest and apoptosis through activation of the checkpoint kinase ATM. Carcinogenesis 10: 2207–2217, 2007
33. Marshall CJ: Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80: 179–185, 1995
34. Mezosi E, Wang SH, Utsugi S, Bajnok L, Bretz JD, Gauger PG, Thompson NW, Baker Jr JR Interleukin-1β and tumor necrosis factor (TNF)-α sensitize human thyroid epithelial cells to TNF-related apoptosis-inducing ligand-induced apoptosis through
- 33 -
increases in procaspase-7 and bid, and the down-regulation of p44/42 mitogen-activated protein kinase activity. J Clin Endocrinol Metab 89:250–257, 2004
35. Nyormoi O, Mills L, and Bar-Eli M: An MMP-2/MMP-9 inhibitor, 5a, enhances apoptosis induced by ligands of the TNF receptor superfamily in cancer cells. Cell Death Differ 10: 558–569, 2003
36. Pan G, Ni J, Wei YF, Yu G, Gentz R, and Dixit VM: An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 277: 815–818, 1997
37. Park SY, Billiar TR, and Seol DW: IFN-gamma inhibition of TRAIL-induced IAP-2 upregulation, a possible mechanism of IFN-gamma-enhanced TRAIL-induced apoptosis.
Biochem. Biophys. Res. Commun 291: 233–236, 2002
38. Park IS, Jo JR, Hong H, and Jang BC et al: Aspirin induces apoptosis in YD-8 human oral squamous carcinoma cells through activation of caspases, down-regulation of Mcl-1, and inactivation of ERK-1/2 and AKT. Toxicology in Vitro 24 713–720, 2010
39. Pitti RM, Marsters SA, Ruppert S, et al.: Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 271: 12687–12690, 1996
40. Qiao L, Hanif R., Sphicas E., Shiff SJ, and Rigas B.: Effect of aspirin on induction of apoptosis in HT-29 human colon adenocarcinoma cells. Biochem. Pharmacol. 55,53–64, 1998
41. S. Prasad, V. R. Yadav, R. Kannappan, and B. B. Aggarwal, Ursolic acid, a pentacyclin triterpene, potentiates TRAILinduced apoptosis through p53-independent up-regulation of death receptors: evidence for the role of reactive oxygen species and JNK, The Journal of Biological Chemistry, vol. 286, no. 7, pp. 5546–5557, 2011
- 34 -
42. Sale EM, Atkinson PG, Sale GJ: Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis. EMBO J 14: 674–684, 1995
43. Seger R and Krebs EG: The MAPK signaling cascade. Faseb J 9: 726–735, 1995
44. She, Q.B., Chen, N., Dong, Z. : ERKs and p38 kinase phosphorylate p53 protein at serine 15 in response to UV radiation. J. Biol. Chem. 275, 20444–20449 , 2000
45. Shuanglin Xiang, Zhenhua Sun, Qiongzhi He, Feng Yan, Yijun Wang and Jian Zhang:
Aspirin inhibits ErbB2 to induce apoptosis in cervical cancer cells. Med Oncol 379–387, 2010
46. Soderstrom TS, Poukkula M., Holmstrom TH., Heiskanen KM, & Eriksson JE :Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in :Mitogen-activated Tcells abrogates TRAIL-induced apoptosis upstream of the mitochondrial amplification loop and caspase-8. J. Immunol., 169(6),2851–2860, 2002
47. Strasser A., O'Connor L., and Dixit VM : Apoptosis signaling. Annu. Rev. Biochem 69:217-245, 2000
48. Thiyam Ramsing Singh, Sharmila Shankar, Xufen Chen, Mohammed Asim, and Rakesh K. Srivastava: Synergistic Interactions of Chemotherapeutic Drugs and Tumor Necrosis Factorrelated Apoptosis-inducing Ligand/Apo-2 Ligand on Apoptosis and on Regression of Breast Carcinoma in Vivo. Cancer Res 63: 5390-5400, 2003
49. Tillman DM, Izeradjene K, Szucs KS, Douglas L, and Houghton JA: Rottlerin sensitizes colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis via uncoupling of the mitochondria independent of protein kinase C. Cancer Res 63: 5118–5125, 2003
- 35 -
50. Tran SE, Holmstrom TH, Ahonen M, Kahari VM, & Eriksso JE :MAPK/ERK overrides the apoptotic signaling from Fas, TNF, and TRAIL receptors. J. Biol. Chem., 276(19), 16484–16490, 2001
51. Wang X, Martindale JL, Holbrook NJ: Requirement for ERK activation in cisplatin-induced apoptosis. J Biol Chem 275:39435-39443, 2000
52. Wang X : The expanding role of mitochondria in apoptosis. Genes & Dev. 15: 2922-2933, 2001
53. Williams CS, Smalley W, and DuBois RN.: Aspirin use and potential mechanisms for colorectal cancer prevention. J. Clin. Investig. 100, 1325–1329, 1997
54. Wong BC., Zhu GH., and Lam SK.: Aspirin induced apoptosis in gastric cancer cells.
Biomed. Pharmacother. 53, 315–318, 1999
55. Zhang L, Fang B: Mechanisms of resistance to TRAIL-induced apoptosis in cancer.
Cancer Gene Ther, 12(3):228-237, 2005
- 36 - - 국문요약 -
Aspririn 과 TRAIL 병합 처리시 자궁 경부암인 HeLa cell 에서 일어나는 효과에 관한 분석
아주대학교 대학원 의생명학과
임 세 란
(지도교수 : 장 영 주)
TRAIL(TNF-related apoptosis inducing ligand)는 세포 표면의 death receptor 들과 결합하여 세포질내 death domain 을 통해 caspase 을 활성화 시키는 방법으로 세포사멸을 유도한다. 이런 TRAIL 은 암세포에만 선별적으로 세포사멸을 유도하기 때문에 각광받는 물질이기도 하다. 그러나 TRAIL 에 저항성을 보이는 암세포들이 있어 이들 치료를 위해 저농도의 항암물질과 병합처리하는 방법이 사용되어지고 있다. 여기서 우리는 TRAIL 의 다른측면에 관점을 두고 실험에 임했다. 즉 TRAIL 은 암세포에 죽음의 신호뿐 아니라 생존의 신호로도 작용한다는 것이다. 그로인해 암세포가 TRAIL 에 저항성을
- 37 -
보이는 것이라 생각했고 이런 생존신호를 억제시킴으로써 TRAIL 에 민감해질 것이라 생각했다. 우리는 TRAIL 의 생존신호를 억제시키는 물질로 aspirin 을 사용했으며, 이것은 TRAIL 에 저항성을 보이는 자궁경부암세포인 HeLa cell 의 세포사멸을 향상시켰다.
Aspirin 은 HeLa cell 의 증식을 억제시키지만 세포독성이 나타나지는 않았고, TRAIL 은 약간의 세포사멸을 유도했으나, 그 농도가 증가한다고 해서 더 많은 사멸을 유도하지는 않음을 LDH 와 MTT 방법을 통해 알 수 있었다. 하지만
Aspirin 은 HeLa cell 의 증식을 억제시키지만 세포독성이 나타나지는 않았고, TRAIL 은 약간의 세포사멸을 유도했으나, 그 농도가 증가한다고 해서 더 많은 사멸을 유도하지는 않음을 LDH 와 MTT 방법을 통해 알 수 있었다. 하지만