CDT toxicity is associated with cell cycle arrest and eventually cause cell death through the activation of apoptotic cascade (35,36). Our preliminary data showed that cell cycle was arrested at G2/M phase after treatment of cells with NP-CdtB by using flow cytomerty analysis (Figure 7). We next detected the cell viability after cell was treatment with NP-CdtB for 24 hours by using MTT assay. As shown in Figure 8, cell viability was decrease with a dose-dependent manner when AGS cells were treated with variety concentrations (20nM, 200nM, 500nM and 1000nM) of NP-CdtB for 24 hours. We also detected sub-G1 population using flow cytometry to confirm the our
20
observation. When AGS cells were treated with NP-CdtB for different incubation times (24, 48, 72 hours), the population of the sub-G1 shown increased in a time-dependent manner (Figure 9A). Our results showed that NP-CdtB-induced cell apoptosis at 24-72 h with a significant increase when compare to control groups (Figure 9B).
It has been reported that several molecules were involved in the apoptosis pathway, including the Bcl-2 families, caspase families, p53 (67-69). The Bcl-2 families function as an anti-apoptosis which included Bcl-2 and Bcl-xl. On the other hand, the Bax, Bak, Bad, and Bid were molecules that involved in pro-apoptotic. We next assessed whether the cell apoptosis induced by NP-CdtB in the AGS cells was associated with an anti-apoptosis family. As show in Figure 10, the levels of the Bax and Bak were increased after treatment of cells with CDT holotoxin or NP-CdtB when compared with control groups (only AGS cell and only nanoparticle). Additonally, the expression of anti-apoptosis molecular, Bcl-2, was decreased. This result suggested that NP-CdtB has the ability to delivery CdtB subunit enter the AGS cell nucleus and then activate the apoptotic cascade. The results from this study suggested that NP-CdtB has ability to delivery into the host cell nucleus and thus induced cell cycle arrest at G2/M phase and cell apoptosis (Figure 11).
21
Discussion
Campylobacter jejuni is one of the major cause of infection diarrhea world-wide
as well as the most commonly reported bacterial cause of food-borne illness in the United States (3), C. jejuni was found can cause disease in different hosts including human, beast animal, and pets. This bacterium can also induce several types of diseases in human including gastritis, gastroenteritis, septicemia and serious neurological disease like Guillian-Barré syndrome. Recently, the infection of C. jejuni was became a seriously problem around the word.
The cytolethal distending toxin (CDT) is one of the important virulence factors secreted by C. jejuni. CDT also contributes to survival and establishment of disease in the host cells. One of the most important function of CDT is to induce cell distending and lead to a severe genotoxin effects on host (19). The earlier study indicated that CDT is a family of heat-labile protein cytotoxin found in various gram-negative bacteria pathogens of human (70,71). CDT is typically compose by three subunits, including CdtA, CdtB and CdtC (72). CDT holotoxin function as an AB2 toxin in which CdtA and CdtC serve as a binding subunits, and CdtB plays an important role in destruction of the host DNA. Once CDT holotoxin get into the host cell from the plasma membrane, CdtB using an endoplasmic pathway through Golgi and ER, after which CdtB translocated into the nucleus (73). The catalytic subunit CdtB has DNase-I like activity, whereas CdtA and CdtC serve as the binding subunit which associate with the cell surface and delivering of CdtB into the target cell lead to cell apoptosis (74). The research in 2001 by Lara-Tejero et al. indicated that Cdt activity requires the function of three subunits, when applied individually, purified CdtA, CdtB or CdtC doesn’t exhibit toxic activity in the host cells (75). Moreover, several
22
groups have shown that CdtA-CdtC complex can inhibit subsequent intoxication by holotoxin (76,77). The results from these studies suggested that the binding subunits CdtA and CdtC play a crucial role in the association with the host cell membrane.
Thus we attempt to utilize other substances to substitute as the binding subunits and achieve the same results.
Nanoparticle is a microscopic particle which contains biodegradable and safety used in vivo. Nanoparticle has a widely potential application in several fields. Recent years, nanoparticle was commonly used in cancer therapy due to its ability to conjugate and inject some bacterial toxins as well as delivery of drugs into target cells which may induce the toxin effects (51). In 2007 Townsend et al. who used nanoparticle to directly deliver therapies to neurons in the central nervous system, which is protected by blood-brain barrier (BBB). However, nanoparticle has ability to bypass the blood-brain barrier (BBB) because of its small volume, (50). Due to the widely utilized of nanoparticle, the major object of this study is to develop a novel nanoparticle encapsulated bacterial toxin for gastric cancer therapy.
The acidic environment in gastric probability cause drug decomposition or loss of their function. Thus, this is one of the problems we consider in this study: the nanoparticle used in this study was consisted of chitosan and heparin. Chitosan has structural characteristics similar to glycosaminoglycans which is non-toxin and biodegradable (78). Heparin is a well-know anticoagulant and has low-moelcular-weight about 15kDa. Heparin harbors a polyanionic mucopolysaccharide which has ability to bind to cell receptors and associate with mucosal regeneration, angiogenesis, and proliferation (59,79), Recently, polymer nanoparticle was wide application in target therapy in which serve as a carrier for drug delivery. Those two components has a pH sensitivity characteristic that are stable at pH 1.2-2.5 but unstable and break apart at pH 7.0. A previous study also indicated
23
that the drug was release by nanoparticle at pH 1.2 (35%) and at pH6.8 (80%). This result acknowledges that the nanoparticle can protect drugs from destruction by gastric acids (54).
The current study created a system which has ability to encapsulate CdtB and deliver CdtB into the AGS cells. This system was substituted CDT holotoxin for nanoparticle to achieved the same results. The nanoparticle-encapsulated drug for delivery into the cells must be smaller than 200 nm to be taken up by epithelial cells (54,80). Figure 2 shows the size of NP-CdtB is between 200nm and 300nm. This is not in the range from previous study. Thus, we first determined whether the NP-CdtB have ability to enter the AGS cells. As show in Figure 4, the images from CLSM indicated that upon treatment of cells with NP-CdtB for 2 hours, NP-CdtB was located in intercellular spaces and cell cytoplasm. When changes the fresh medium and incubation for further 4 hours, NP-CdtB was found co-localized with the cell nucleus. This result suggests that nanoparticle play an important role in the delivery of CdtB subunit into the cell nucleus, In addition, the western blot results from Figure 5 also indicated the maximum of NP-CdtB get into the AGS cell is around 4 hours. This is better than 8 hours after CDT holotoxin treatment. Thus, the results from these studies suggest that NP-CdtB has better efficiency then CDT holotoxin in the delivery of CdtB into the AGS cells.
Several groups have been utilized nanoparticle as a delivery system which has been approved can translate in vivo for cancer therapy (50,51,81). In this study we used the bacterial toxin CDT to destroy the gastric cancer cells. CdtB function as a phosphatidylinositol-3,4,5-triphosphate phosphates which is analogous to the tumor suppressor phosphatases, PTEN and SHIP 1 (40,82). CdtB cause cell cycle arrest at G2/M phase on host cell was reported in 1997 (33). In our current study, as shown in Figure 6, the cell cycle analysis indicated that NP-CdtB not only has ability to deliver
24
into the host cells, but also cause cell cycle arrest at G2/M phase (83). The data from these studies also indicated that nanoparticle cannot cause cell cycle arrest in AGS cells.
Upon cells treated with CDT holotoxin and NP-CdtB for 24 hours, the cell viability was significantly decrease in a dose-dependent manner (Figure 8). In addition, CDT holotoxin cause cell cycle arrest at G2/M phase then induced cell death via the apoptosis pathway. We thus detected the cell apoptosis after treatment of cells with NP-CdtB. The population of apoptotic cells was graduated increased with a time-dependent manner.
CDT holotoxin can be produced by some Gram-negative bacteria, which may associate with double-stranded DNA breaks resulting in cell cycle arrest at G2/M phase and leading to cell apoptosis via the mitochondrial-dependent apoptosis pathway (84). Our study by western blot analysis suggests that the expression level of the Bax and Bak was increase after AGS cells treated with Cdt holotoxin or nona-CdtB. This result demonstrated that the NP-CdtB have the ability in the activation of the apoptotic cascade (Figure 10). Most important, our study also found that the caspase families are not involved in NP-CdtB-induced apoptosis pathway (data not show).
Because of the previously research indicated that cell exposure to CDT holotoxin can triggers activation of checkpoint responses that lead to cell DNA damage, cell cycle arrest as well as cell apoptosis. The key molecules involved in DNA double strand breaks was refered to Ataxia telangiectasia mutated (ATM) kinase. A previous study suggest that CDT holotoxin can trigger full activation of ATM and subsequent phosphorylation of its downstream effectors such as DNA repair complex (H2AX), checkpoint kinase (chk2) and the tumor suppressor gene (p53) (85). Thus, we suppose that NP-CdtB-induced cell apoptosis may through the ATM-dependent DNA damage
25
checkpoint responses. However whether those molecules involved in the NP-CdtB-induced apoptosis of AGS cells required further investigation.
In conclusion, we developed a novel nanoparticle which carried heparin and chitosan for encapsulation of bacterial toxin CdtB. We also proposed that nanoparticle can successfully replaced CdtA and CdtC to deliver CdtB subunit into the AGS cells.
In this study we can demonstrate that NP-CdtB can induced cell cycle arrest at G2/M phase and finally leaded to cell apoptosis in AGS cells. This system we created have a higher efficiently to delivery CdtB into the human gastric adenocarcinoma cells.. The mechanism of NP-CdtB delivery into host cell nucleus and induce cell apoptosis is described in Figure 11.
26
References
1. Smibert, R. M. (1975) In Bergy's Manual of Determinative Bacteriology, 8th ed., 207-211
6. Kuipers, E. J. (1999) Aliment Pharmacol Ther 13 Suppl 1, 3-11 7. Ketley, J. M. (1997) Microbiology 143 ( Pt 1), 5-21
8. Beuchat, L. R. (1985) Appl Environ Microbiol 50, 934-939
9. Rollins, D. M., and Colwell, R. R. (1986) Appl Environ Microbiol 52, 531-538 10. Corry, J. E., and Atabay, H. I. (2001) Symp Ser Soc Appl Microbiol, 96S-114S 11. Morooka, T., Umeda, A., and Amako, K. (1985) J Gen Microbiol 131,
1973-1980
12. Neal-McKinney, J. M., Christensen, J. E., and Konkel, M. E. Mol Microbiol 76, 918-931
13. Wassenaar, T. M., and Blaser, M. J. (1999) Microbes Infect 1, 1023-1033 14. Wassenaar, T. M. (1997) Clin Microbiol Rev 10, 466-476
15. Caroff, M., Karibian, D., Cavaillon, J. M., and Haeffner-Cavaillon, N. (2002) Microbes Infect 4, 915-926
16. Park, S. F., and Richardson, P. T. (1995) J Bacteriol 177, 2259-2264
17. Arimi, S. M., Park, R. W., and Fricker, C. R. (1990) J Appl Bacteriol 69, 384-389
18. Johnson, W. M., and Lior, H. (1988) Microb Pathog 4, 103-113 19. Johnson, W. M., and Lior, H. (1988) Microb Pathog 4, 115-126
20. Shenker, B. J., McKay, T., Datar, S., Miller, M., Chowhan, R., and Demuth, D.
(1999) J Immunol 162, 4773-4780
21. Ohguchi, M., Ishisaki, A., Okahashi, N., Koide, M., Koseki, T., Yamato, K., Noguchi, T., and Nishihara, T. (1998) Infect Immun 66, 5980-5987
22. Okuda, J., Kurazono, H., and Takeda, Y. (1995) Microb Pathog 18, 167-172 23. Gelfanova, V., Hansen, E. J., and Spinola, S. M. (1999) Infect Immun 67,
6394-6402
24. Chien, C. C., Taylor, N. S., Ge, Z., Schauer, D. B., Young, V. B., and Fox, J. G.
27 (2000) J Med Microbiol 49, 525-534
25. Young, V. B., Knox, K. A., and Schauer, D. B. (2000) Infect Immun 68, 184-191
26. Pickett, C. L., Pesci, E. C., Cottle, D. L., Russell, G., Erdem, A. N., and Zeytin, H. (1996) Infect Immun 64, 2070-2078
27. Pickett, C. L., and Whitehouse, C. A. (1999) Trends Microbiol 7, 292-297 28. Nesic, D., Hsu, Y., and Stebbins, C. E. (2004) Nature 429, 429-433
29. Shenker, B. J., Besack, D., McKay, T., Pankoski, L., Zekavat, A., and Demuth, D. R. (2005) J Immunol 174, 2228-2234
30. Lara-Tejero, M., and Galan, J. E. (2000) Science 290, 354-357
31. Hickey, T. E., McVeigh, A. L., Scott, D. A., Michielutti, R. E., Bixby, A., Carroll, S. A., Bourgeois, A. L., and Guerry, P. (2000) Infect Immun 68, 6535-6541
32. Watson, R. O., and Galan, J. E. (2005) Cell Microbiol 7, 655-665
33. Comayras, C., Tasca, C., Peres, S. Y., Ducommun, B., Oswald, E., and De Rycke, J. (1997) Infect Immun 65, 5088-5095
34. Sugai, M., Kawamoto, T., Peres, S. Y., Ueno, Y., Komatsuzawa, H., Fujiwara, T., Kurihara, H., Suginaka, H., and Oswald, E. (1998) Infect Immun 66, 5008-5019
35. Shenker, B. J., Demuth, D. R., and Zekavat, A. (2006) Infect Immun 74, 2080-2092
36. Bielaszewska, M., Sinha, B., Kuczius, T., and Karch, H. (2005) Infect Immun 73, 552-562
37. Boesze-Battaglia, K., Besack, D., McKay, T., Zekavat, A., Otis, L., Jordan-Sciutto, K., and Shenker, B. J. (2006) Cell Microbiol 8, 823-836
38. Nesic, D., and Stebbins, C. E. (2005) PLoS Pathog 1, e28
39. Mise, K., Akifusa, S., Watarai, S., Ansai, T., Nishihara, T., and Takehara, T.
(2005) Infect Immun 73, 4846-4852
40. Shenker, B. J., Dlakic, M., Walker, L. P., Besack, D., Jaffe, E., LaBelle, E., and Boesze-Battaglia, K. (2007) J Immunol 178, 5099-5108
41. Thelestam, M., and Frisan, T. (2004) Rev Physiol Biochem Pharmacol 152, 111-133
42. Whitehouse, C. A., Balbo, P. B., Pesci, E. C., Cottle, D. L., Mirabito, P. M., and Pickett, C. L. (1998) Infect Immun 66, 1934-1940
43. Akifusa, S., Poole, S., Lewthwaite, J., Henderson, B., and Nair, S. P. (2001) Infect Immun 69, 5925-5930
44. Lewis, D. A., Stevens, M. K., Latimer, J. L., Ward, C. K., Deng, K., Blick, R., Lumbley, S. R., Ison, C. A., and Hansen, E. J. (2001) Infect Immun 69,
28 5626-5634
45. Chithrani, B. D., Ghazani, A. A., and Chan, W. C. (2006) Nano Lett 6, 662-668 46. Hochella, M. F., Jr., Lower, S. K., Maurice, P. A., Penn, R. L., Sahai, N.,
Sparks, D. L., and Twining, B. S. (2008) Science 319, 1631-1635
47. Pridgen, E. M., Langer, R., and Farokhzad, O. C. (2007) Nanomedicine (Lond) 2, 669-680
48. Bisht, S., Feldmann, G., Soni, S., Ravi, R., Karikar, C., and Maitra, A. (2007) J Nanobiotechnology 5, 3
49. Brannon-Peppas, L., and Blanchette, J. O. (2004) Adv Drug Deliv Rev 56, 1649-1659
50. Townsend, S. A., Evrony, G. D., Gu, F. X., Schulz, M. P., Brown, R. H., Jr., and Langer, R. (2007) Biomaterials 28, 5176-5184
51. Showalter, S. L., Huang, Y. H., Witkiewicz, A., Costantino, C. L., Yeo, C. J., Green, J. J., Langer, R., Anderson, D. G., Sawicki, J. A., and Brody, J. R. (2008) Cancer Biol Ther 7, 1584-1590
52. Wu, Y., Wang, W., Chen, Y., Huang, K., Shuai, X., Chen, Q., Li, X., and Lian, G. Int J Nanomedicine 5, 129-136
53. He, X. W., Liu, T., Chen, Y. X., Cheng, D. J., Li, X. R., Xiao, Y., and Feng, Y.
L. (2008) Cancer Gene Ther 15, 193-202
54. Lin, Y. H., Chang, C. H., Wu, Y. S., Hsu, Y. M., Chiou, S. F., and Chen, Y. J.
(2009) Biomaterials 30, 3332-3342
55. Kas, H. S. (1997) J Microencapsul 14, 689-711
56. Mansouri, S., Cuie, Y., Winnik, F., Shi, Q., Lavigne, P., Benderdour, M., Beaumont, E., and Fernandes, J. C. (2006) Biomaterials 27, 2060-2065
57. Li, Y., Wang, H. Y., and Cho, C. H. (1999) J Pharmacol Exp Ther 290, 789-796
58. Andersson, L. O., Barrowcliffe, T. W., Holmer, E., Johnson, E. A., and Soderstrom, G. (1979) Thromb Res 15, 531-541
59. Hirsh, J., Warkentin, T. E., Shaughnessy, S. G., Anand, S. S., Halperin, J. L., Raschke, R., Granger, C., Ohman, E. M., and Dalen, J. E. (2001) Chest 119, 64S-94S
60. WHO. (1994) IARC monographs on the evaluation of carcinogenic risks to humans. In: Schistosomes, liver flukes and Helicobacter pylori. in International Agency for Research on Cancer (WHO ed., WHO, Lyon, France 61. Wu, T. L., Su, L. H., Chia, J. H., Kao, T. M., Chiu, C. H., Kuo, A. J., and Sun,
C. F. (2002) Epidemiology and infection 129, 227-231
62. Li, Y., Wang, W. P., Wang, H. Y., and Cho, C. H. (2000) Eur J Pharmacol 399, 205-214
29
63. Shu, X. Z., Zhu, K. J., and Song, W. (2001) Int J Pharm 212, 19-28
64. Lin, Y. H., Chung, C. K., Chen, C. T., Liang, H. F., Chen, S. C., and Sung, H.
W. (2005) Biomacromolecules 6, 1104-1112
65. Grenha, A., Seijo, B., and Remunan-Lopez, C. (2005) Eur J Pharm Sci 25, 427-437
66. Ma, Z., Yeoh, H. H., and Lim, L. Y. (2002) J Pharm Sci 91, 1396-1404
67. Alaoui-El-Azher, M., Mans, J. J., Baker, H. V., Chen, C., Progulske-Fox, A., Lamont, R. J., and Handfield, M. PLoS One 5, e11714
68. Ohara, M., Hayashi, T., Kusunoki, Y., Nakachi, K., Fujiwara, T., Komatsuzawa, H., and Sugai, M. (2008) Infect Immun 76, 4783-4791
69. Matangkasombut, O., Wattanawaraporn, R., Tsuruda, K., Ohara, M., Sugai, M., and Mongkolsuk, S. Infect Immun 78, 783-792
70. Frisan, T., Cortes-Bratti, X., and Thelestam, M. (2002) Int J Med Microbiol 291, 495-499
71. Lara-Tejero, M., and Galan, J. E. (2002) Trends Microbiol 10, 147-152
72. Pickett, C. L., Cottle, D. L., Pesci, E. C., and Bikah, G. (1994) Infect Immun 62, 1046-1051
73. Guerra, L., Teter, K., Lilley, B. N., Stenerlow, B., Holmes, R. K., Ploegh, H. L., Sandvig, K., Thelestam, M., and Frisan, T. (2005) Cell Microbiol 7, 921-934 74. Montecucco, C., Papini, E., and Schiavo, G. (1994) FEBS Lett 346, 92-98 75. Lara-Tejero, M., and Galan, J. E. (2001) Infect Immun 69, 4358-4365 76. McSweeney, L. A., and Dreyfus, L. A. (2005) Infect Immun 73, 2051-2060 77. Akifusa, S., Heywood, W., Nair, S. P., Stenbeck, G., and Henderson, B. (2005)
Microbiology 151, 1395-1402
78. Muzzarelli, R., Baldassarre, V., Conti, F., Ferrara, P., Biagini, G., Gazzanelli, G., and Vasi, V. (1988) Biomaterials 9, 247-252
79. Benoit, D. S., and Anseth, K. S. (2005) Acta Biomater 1, 461-470
80. Chang, C. H., Lin, Y. H., Yeh, C. L., Chen, Y. C., Chiou, S. F., Hsu, Y. M., Chen, Y. S., and Wang, C. C. Biomacromolecules 11, 133-142
81. Peng, W., Anderson, D. G., Bao, Y., Padera, R. F., Jr., Langer, R., and Sawicki, J. A. (2007) Prostate 67, 855-862
82. Dlakic, M. (2001) Science 291, 547
83. DiRienzo, J. M., Cao, L., Volgina, A., Bandelac, G., and Korostoff, J. (2009) R E S EARCH L E T T E R 291, 222–231
84. Liyanage, N. P., Manthey, K. C., Dassanayake, R. P., Kuszynski, C. A., Oakley, G. G., and Duhamel, G. E. Helicobacter 15, 98-107
85. Guerra, L., Albihn, A., Tronnersjo, S., Yan, Q., Guidi, R., Stenerlow, B., Sterzenbach, T., Josenhans, C., Fox, J. G., Schauer, D. B., Thelestam, M.,
30
Larsson, L. G., Henriksson, M., and Frisan, T. PLoS One 5, e8924
86. Konkel, M. E., Monteville, M. R., Rivera-Amill, V., and Joens, L. A. (2001) Curr Issues Intest Microbiol 2, 55-71
!
31
Tables
Table 1. The species of Campylobacter and the related diseases.
Species Reservoir Host Human Disease Frequency C. jejuni subsp. jejuni Poultry, pigs, bulls,
dogs, cats, rabbits,
C. jejuni subsp. doylei Humans Gastroenteritis, gastritis, septicemia
Uncommon
C. coli subsp. fetus Pigs, poultry, bulls, sheep, birds
C. upsaliensis Dogs, cats Gastroenteritis, septicemia, abscesses
Uncommon
C. fetus subsp. fetus Cattle, sheep Septicemia, gastroenteritis, spontaneous abortion, meningitis
Uncommon
C. fetus subsp. venereal Cattle Septicemia Uncommon
Adapted from On (5)
32
Table 2. Particle sizes and zeta potentials of nanoparticles prepared with different chitosan and heparin concentrations in deionized water (n = 5).
Chitosan Concentration (mg/mL)
Heparin Concentration (mg/mL)
Mean Particle Size (nm)
Zeta Potential (mV)
0.3 1.0 ● ●
0.6 1.0 208.4 r 10.6 30.4 r 1.5
0.9 1.0 248.5 r 8.7 33.6 r 0.8
1.2 1.0 273.1 r 10.6 37.4 r 1.9
1.5 1.0 298.1 r 9.6 39.4 r 21.4
● Precipitation of aggregates was observed.
33
Table 3. Particle sizes and zeta potentials of the prepared bacterial toxin-loaded nanoparticles in deionized water (n = 5).
Protein:Heparin Concentration (mg/mL)
Chitosan Concentration
(mg/mL)
Mean Particle Size (nm)
Zeta Potential (mV)
12.0:1.0 1.2 2788.1 r 300.9 14.8 r 5.9
6.0:1.0 1.2 983.5 r 163.9 20.8 r 3.1
3.0:1.0 1.2 414.3 r 8.3 29.9 r 1.7
1.5:1.0 1.2 312.4 r 15.7 32.3 r 0.7
34
Figures
Figure 1. The infection routes of Campylobacter jejuni. (Adapted from Konkel (86))
Food processing and delivering
Ingestion by an impressionable host
Passage through the stomach into the intestine
Colonization
Cell invasion and toxin production Diarrheal disease
Fecal runoff Livestock and
wildlife
Inflammatory response
Contamination of surface water
Transmission
Human illness Environment
35
Figure 2. The polydispersity index (left panel) and transmission electron microscopy examination (right panel) of CdtB-loaded chitosan/heparin nanoparticles.
36
Figure 3. Determination of CdtB by using SDS-PAGE and western bloting.
CdtB was first constructed from C. jejuni and ligated into pET21d. The molecular weight was determined by using SDS-PAGE (A) and western blot analysis against His-tag (B) and CdtB (C). The molecular weight marker was denoted at the left side.
37
Figure 4. Confocal microscopy analysis of Nanoparticle -CdtB delivery.
Cells were pre-treated with HBSS (containing 50mM glucose, pH6.5) for 30 mins.
After that, the cells were treated with HBSS containing NP-CdtB for 2 hours, and then remove the NP-CdtB. Cells were incubated with fresh culture medium for 4 hours, the CLSM was employed to observe the cellular uptake of NP-CdtB in cells. Fluorescent of NP-CdtB was used in the study and the distribution of Cy3-chitosan (red), NP-CdtB (green), and nuclei (blue, stained with DAPI) in the intracellular spaces was observed by a confocal microscope.
38
(A)
(B)
Figure 5. Detection the efficiency of CDT holotoxin or Nanoparticle -CdtB delivered into cells.
Cells were incubated with 200nM of (A) NP-CdtB or (B) CDT holotoxin for the indicated periods. The expression of CdtB was determined using western blot analysis against CdtB.
39
(A)
(B)
Figure 6. Nanoparticle -CdtB induced cell cycle arrest at G2/M phase in AGS cells.
AGS cells were treated with NP-CdtB (200nM) for the indicated time (1, 2, 4, 6, 7 and 24 hours). The population of cell cycle arrest was analyzesd by flow cytometry.
(B) The population of G2/M phase was shown in time-cause dependent manner, the G2/M phase population was increased after AGS cells incubated with NP-CdtB for 2 hours, compare with the negative control (only AGS and nanoparticle). IRCF193 and CDT holotoxin are represented as positive controls. Result are expressed as the Mean±S.E. īp <0.05 compared with control; **p < 0.01 compared with control.
40
Figure 7. Cells were detachment after Nanoparticle -CdtB treatment for 24 to 72 hours.
The cells were detached after incubation with CDT holotoxin or NP-CdtB for 24 hours. The detached cells was more obvious after treatment with NP-CdtB or CDT holotoxin for 72 hours. Representative image are show. Scale bar, 100 Pm.
41
Figure 8. The effects of Nanoparticle -CdtB on cell viability in AGS cells.
AGS cells were incubated with various concentrations (20, 200, 500, 1000, 2000 nM) for 24 hours. The percentage of cell viability was analysis by MTT assay (n=3).
Result are expressed as the Mean±S.E. īp <0.05 compared with control; **p < 0.01 compared with control.
42
(A)
(B)
Figure 9. Nanoparticle -CdtB induced apoptosis of human gastric adenocarcinoma cells.
(A) AGS cells were treated with CDT holotoxin, NP-CdtB, and nanoparticle for the indicated times (24, 48, 72 hours). The population of apoptotic cell was analyzed by flow cytometry of PI-stained cells (n=3). (B) The data represented percentage of sub-G1 cells was increase with a time-dependent manner compare with control groups (NP and control). Results are expressed as the Mean±S.E. īp <0.05 compared with control; **p < 0.01 compared with control.
43
Figure 10. Bax, Bak, Bcl-2 activation are involved in Nanoparticle -CdtB mediated cell apoptosis in human gastric adenocarcinoma cells.
AGS cells were incubated with CDT holotoxin, NP-CdtB and nanoparticle for 24 hours. Expression of Bax, Bak, Bcl-2 were examined by western blot analysis. The expression of Bax and Bak were increased after treatment of cells with CDT holotoxin and NP-CdtB.
44
Figure 11. Depiction of the mechanism of Nanoparticle-CdtB delivers into host cell nucleus and induces cell apoptosis.