[Frontiers in Bioscience E4, 2389-2395, June 1, 2012]
Adenovirus KH901 promotes 5-FU antitumor efficacy and S phase in LoVo cells
Wei Peng1, Jin Li1, XG Yin2, JF Xu1, LZ Cheng1
1
TABLE OF CONTENTS
1. ABSTRACT
A combination of oncolytic and chemotherapeutic agents has been used to kill cancer cells. However, the effect of oncolytic adenoviruses on the cell cycle remains to be determined. Cytotoxicity assays were performed to determine cell death in cells treated with 5-fluorouracil (5-FU) alone or in combination with the oncolytic adenovirus KH901. Dynamic changes in the cell cycle, cell proliferation, and apoptosis-related proteins including p-AKT, Bcl-2, Bax, and caspase 3 were investigated after treatment with 5-FU with or without KH901. A higher proportion of S-phase cells were observed after treatment with KH901 and 5-FU than with 5-FU alone. p-AKT, Bcl-2, and Bax expression was increased upon treatment with KH901, whereas the expression of caspase-3 was not induced upon treatment with KH901 with or without 5-FU. KH901 exhibited significant potential as an oncolytic adenovirus and increased cell death in combination with 5-FU in LoVo cells, as compared to 5-FU alone. In conclusion, KH901 stimulates LoVo cells to enter the S-phase by activation of p-AKT, which could partly explain its synergistic effect with 5-FU on LoVo cell cytotoxicity.
2. INTRODUCTION
The use of oncolytic adenoviruses for the treatment of tumors has gained popularity in recent years, and several phase I and II clinical trials to study their efficacy have been performed. Several recombinant adenoviruses have been designed with innovative modifications, in order to improve their specificity and efficacy of replicating in tumor cells and increasing cell killing. The E1A gene promoter of the virus has been replaced with either the TERT (telomerase reverse-transcriptase) gene (1), E2F (2, 3), or survivin (4), in order to induce conditional virus replication in targeted tumors. One of the most universal tumor-specific genes identified thus far is the TERT gene. TERT is a telomerase catalytic subunit that is responsible for the maintenance of the normal length of telomeres. It is believed that TERT is active and highly expressed in >90% of malignant tumors (5) but inactive in the overwhelming majority of somatic human cells (6). KH901 is a conditionally replicating oncolytic serotype 5 adenovirus (Ad5) in which the E1A gene is driven by a modified human TERT promoter. Shen reported that the replication ability of KH901 was more rapid in LNcap, Hep3B, A549, SW620, and HeLa cancer cells and lower in normal somatic cell lines such as MRC25 and WI38 than that of wild-type Ad5 (7).
In addition to the specific tumor-lysis function, KH901 also induces local and systemic antitumor activity due to the cDNA of human granulocyte macrophage colony-stimulating factor (GM-CSF) that is inserted into the E3 region under the control of the endogenous viral E3 promoter, and which is transactivated by E1A. Thus, the modified telomerase promoter possibly controls GM-CSF expression. Many studies have validated the therapeutic potential of manipulating the cytokine balance in the tumor microenvironment to promote immune-mediated tumor destruction. A comparative analysis of the relative abilities of multiple immunostimulatory molecules to enhance host responses by gene transfer into tumor cells has shown that GM-CSF is the most potent of 30 gene products tested. GM-CSF engenders protective immunity primarily by stimulating recruitment, maturation, and function of dendritic cells. The elevated level of systemic GM-CSF and its benefit in the treatment of tumors have been demonstrated in preclinical and clinical studies of KH901 (9).
5-Fluorouracil (5-FU) is a pyrimidine analog that requires cellular uptake and metabolic activation to exert its cytotoxic effects (10). It is widely used in the treatment of a range of cancers, particularly colorectal cancer, but also those of the breast, head and neck, and digestive tract (11).
Oncolytic adenoviruses have been shown to exert a synergistic effect on chemotherapeutic drugs (12-15), but the exact mechanism is not clear. In the present study, we aimed to evaluate the mechanism of action of the combination treatment of KH901 and 5-FU in colon cancer. We examined the effects of KH901 alone and in combination with 5-FU on the dynamic changes in the cell cycle as well as the expression of apoptosis-related proteins.
3. MATERIALS AND METHODS
3.1. Cell lines, cell culture, and drugs
Human colorectal carcinoma cell lines were purchased from the Shanghai Cell Collection (Shanghai, China). HEK293 and LoVo cells were maintained in RMPI-1640 supplemented with 2% or 10% heat-inactivated fetal bovine serum (Life Technologies Incorporation, Carlsbad, CA, USA), respectively, at 37�C in a 95% air/5% CO2 humidified incubator (Thermo Fisher Scientific, Massachusetts, USA). 5-FU was commercially purchased (Sigma High-Tech Co. Ltd., Shanghai, China).
3.2. CPE assay of KH901
The conditionally replicating oncolytic adenovirus KH901 and the wild-type adenovirus were obtained from Kanghong Pharmaceuticals (Chengdu, China). To determine the infectivity of KH901, MOI (multiplicity of infection) was used as a parameter in the cytopathic-effect assay (CPE). HEK293 cells were cultured in RPMI-1640 containing 2% fetal bovine serum, and 105 cells per well were plated in 10-well plates, incubated for 24 hours, and then infected with either KH901 or wild-type adenovirus at an increasing MOI of 0, 0.001, 0.01, 0.1, and 1 pfu/cell for an additional 48 hours. The culture medium was then removed, and the cells were stained with crystal violet (0.5% in 20% methanol; Sigma).
3.3. Cell-viability assay
For crystal-violet staining, 2 � 105 LoVo cells were incubated for 24 hours in 6-well plates, treated with 0.5 or 1 mg/mL 5-FU, alone or in combination with KH901 at an MOI of 10 pfu, and incubated for an additional 48 hours. The culture medium was then removed, and the cells were stained with crystal violet (0.5% in 20% methanol; Sigma).
For the quantitative cell-viability assay, 4000 LoVo cells were incubated in 96-well tissue culture plates for 24 hours and treated with various concentrations of 5-FU, alone or in combination with KH901, in PBS for an additional 72 hours. The medium was removed and 100 μL fresh medium containing cholecystokinin-8 (Dojindo, Kyushu Island, Japan) was added to each well. Cells were then incubated at 37�C for 3 hours, and the absorbance of each well was read on a Bio-Rad microplate reader (Hercules, CA) at 450 nm. The cell-inhibition rate is assessed by the equation: inhibition rate = 100% - absorption of the treated group/control.
3.4. Flow cytometry assay
LoVo cells were seeded in 6-well plates at 2 � 105 cells per well and cultured for 24 hours before treatment with 0.5 mg/mL 5-FU alone or in combination with KH901 at an MOI of 4 pfu for 24, 48, and 72 hours. Control cells received only PBS for the times indicated above. Cells were collected, trypsinized, pelleted, and washed in pre-cooled PBS, followed by addition of 100% ethanol to a final concentration of 75% ethanol. Next, cells were washed twice with pre-cooled PBS, re-suspended in 300-500 �L PBS (including 20 �g/mL RNase A, 0.2% Triton X-100, and 0.2 mM EDTA), and placed in a water bath at 37�C for 15-30 minutes. A FACSCalibur flow cytometer (Becton, Dickinson and Company) was used to examine the DNA content of the cells, and cell cycle analysis was performed using the ModifitLT cell cycle analysis software.
3.5. Western blotting
The protein expression of cell cycle-regulated genes was examined by western blotting. Cells were treated with 5-FU at 0.5 mg/mL, alone or in combination with KH901 at an MOI of 4 pfu, for 24 and 48 hours. Cells treated with PBS were used as a control. The cells were lysed in cell-lysis buffer for western blotting. Samples containing equal amounts of protein were separated on 8% SDS-PAGE and transferred to nitrocellulose membranes, followed by blocking with 5% (w/v) non-fat dry milk. The membranes were then incubated with the primary antibody, followed by the appropriate secondary antibody. Signals and images were visualized by enhanced chemiluminescence, according to the manufacturer's instructions (Pierce Biotechnology, Rockford, IL). The primary antibodies used were mouse anti-human p-AKT, Bcl-2, Bax-1, and caspase-3 (Merck & Co, Darmstadt, German).
3.6. Statistical analysis
Data are displayed as means. The statistical significance of differences between various groups was analyzed by analysis of variance (ANOVA) or Student's t test, as appropriate. The statistical analysis was performed using SPSS version 13.0 software. Differences among groups were regarded as significant if p < 0.05.
4. RESULTS
4.1. Synergistic effect of the adenovirus KH901 and 5-FU on cell death
The CPE assay of KH901 and the wild-type adenovirus in HEK293 cells demonstrated that KH901 could effectively replicate in HEK293 cells and that it was more potent than the wild-type adenovirus (Figure 1). A greater level of cell death was noted by crystal-violet staining of cells treated with both KH901 and 5-FU together (Figure 2A). Cell killing was assessed using various concentrations of 5-FU, alone or in combination with various MOIs of KH901, in LoVo cells. An increase in the MOI of KH901 from 1 to 100 was accompanied by a sequential enhancement in cell killing (p < 0.01). Inhibition rates of 50%, 40%, 30%, and 20% were noted at 6, 3, 1.5, and 0.7 pfu, respectively (Figure 2B). A progressive killing ability was also noted with increasing concentrations of 5-FU alone (p < 0.01; Figure 2C). Inhibition rates of 50%, 40%, 30%, and 20% were observed at concentrations of 9.84 � 10-6 mol/L , 3.76 � 10-6 mol/L, 1.44 � 10-6 mol/L, and 5.50 � 10-6 mol/L, respectively.
A 2-factor, 4-level orthogonal experimental design was used for the assessment of the killing of LoVo cells by a combination of KH901 and 5-FU (Table 1). MOIs of KH901 were fixed at 0 (as control) and 0.7, while 5-FU at concentrations of 0, 5.50 � 10-7 mol/L, 1.44 � 10-6 mol/L, 3.76 � 10-6 mol/L, and 9.84 � 10-6 mol/L were added to each serial dose of KH901. KH901 and 5-FU had a synergistic effect in killing LoVo cells (F = 497.349, p < 0.01; Figure 2D).
4.2. KH901 induced LoVo cells to enter the S-phase
The proportions of G0/G1 and S-phase cells in the control, 5-FU-treated, KH901-treated, and 5-FU/KH901-treated groups are shown in Figure 3. Compared to the 5-FU group, the proportion of cells in the S-phase in the KH901 and combination groups was significantly higher at 24 and 48 hours (p < 0.01; Table 1)
4.3. The expression of p-AKT, Bax, and Bcl-2 was significantly elevated in cells treated with KH901
The expression of p-AKT was significantly elevated and that of bcl-2 was markedly increased in the KH901-treated and 5-FU/KH901-treated groups, as compared to the control and 5-FU groups. Expression of Bax was the highest in the KH901group, followed by the 5-FU/KH901 group, and least in the 5-FU group. There were no differences in the expression of caspase 3 among the 4 groups. Western blotting results are presented in Figure 4.
5. DISCUSSION
Oncolytic adenoviruses have been shown to exert a synergistic effect with chemotherapeutic agents, resulting in a better response and reduced toxicity of the chemotherapeutic agents in several types of cancers (16, 17).
In concurrence with previous results (16-18), we observed that KH901 markedly increased the killing ability of 5-FU in a synergistic manner. Additionally, as previously demonstrated (19), we found a significant increase in cells in the S-phase in the KH901 and the 5-FU/KH901 groups, as compared to the 5-FU group. It has been shown that the S-phase of cells facilitates the replication and proliferation of adenovirus, and that the adenovirus infection in turn induces infected cells to enter the proliferative stage (20). Retinoblastoma (RB) and RB-related proteins can bind to E2F and regulate the cell cycle, whereas the conservative region of E1A (CR1 and CR2) can bind to and displace RB and RB-related proteins from E2F. This activates the E2F gene and stimulates the cells to move from the G0 to the S-phase (21, 22). Kolli demonstrated that the conservative region of E1A can bind to and inactivate p300, which can lead to an enhanced activity of the c-myc gene, which also stimulates the cells to move from the G0 to the S-phase (23). Cherubini also showed that the ONYX-015 oncolytic virus induces cells with a normal p53 expression to bypass the G1/S and G2/M cell-cycle checkpoints, thus accelerating cell proliferation (19).
Our study showed that the expression of p-AKT was significantly upregulated in the 5-FU and 5-FU/KH901 groups as compared to the control and the 5-FU groups. Activation of AKT is known to stimulate cell proliferation through multiple downstream targets that influence cell-cycle regulation. AKT phosphorylates the cyclin-dependent kinase inhibitors p21Cip1/WAF1 and p27Kip1, promoting their cytosolic localization and preventing their cell-cycle inhibitory effects. AKT phosphorylation of other targets such as GSK3, TSC2, and PRAS40 also possibly induces cell proliferation through the regulation of the stability and synthesis of proteins involved in cell-cycle entry (24, 25). Icyuz has demonstrated that wild-type Ad5 infection promotes the expression and activation of AKT1 in normal pancreatic islets (26). They have suggested that adenovirus infection can activate cell survival and proliferation, in particular, by operating through the PI3K/AKT signaling pathway (26).
We found that the expression of Bax was markedly elevated by KH901. We suggest that the Bax gene accelerated the progression of the cells into the S-phase, and together with the activation of p-AKT, contributed to the S-phase stimulation induced by KH901.
In our study, expression of Bax was elevated in the KH901 group, but the expression of the apoptotic gene, caspase 3, was not. This could be explained by the structure of KH901. The E1B-55k gene in KH901 inhibits p53-dependent apoptosis (27), whereas E1B-19k binds to Bax, thus blocking activation of caspase-3 and -9 and the ensuing apoptosis (28). Additionally, the expression of Bcl-2, an inhibitor of apoptotic proteins, was also elevated, which may act together with E1B-19k to block p53-independent apoptosis. Furthermore, we found that 5-FU inhibited the expression of Bax. Further investigation needs to be conducted to elucidate this process.
Bcl-2 is an anti-apoptotic gene that enhances the G0 phase and delays the G0 to S transition, whereas Bax is a pro-apoptotic gene. As observed in our study, there was simultaneous elevated expression of Bcl-2 and Bax in the KH901 group. This seemingly paradoxical expression of the 2 genes could be attributed to the complexity of the Bcl family and its interplay (29-31) and needs to be further investigated. In conclusion, our study revealed that the oncolytic adenovirus KH901 had a synergistic effect on 5-FU-mediated antitumor activity, possibly due in part to an increased number of cells in the S-phase. We also demonstrated that p-AKT activation by KH901 stimulated the entry of cells into the S-phase. Our results showed in vitro evidence of the effectiveness of the combined application of KH901 and 5-FU. Because 5-FU is currently being used in chemotherapy of colorectal cancer, our study indicates that a combination of KH901 and 5-FU could have a potential application in the treatment of colorectal cancer. However, further investigation including animal studies and clinical trials is required.
6. ACKNOWLEDGEMENTS
The authors thank Shiang Jiin Leaw, M.D. for her assistance with the English editing of this manuscript.
7. REFERENCES
1. Wirth T., Zender L., Schulte B., Mundt B., Plentz R, Rudolph KL: A telomerase-dependent conditionally replicating adenovirus for selective treatment of cancer. Cancer Res 63, 3181-3188 (2003)
2. Tsukuda K., Wiewrodt R., Molnar-Kimber K., Jovanovic V. P., Amin K. M.: An E2F-responsive replication-selective adenovirus targeted to the defective cell cycle in cancer cells: potent antitumoral efficacy but no toxicity to normal cell. Cancer Res 62, 3438-3447 (2002)
3. Johnson L., Shen A., Boyle L., Kunich J., Pandey K., Lemmon M: Selectively replicating adenoviruses targeting deregulated E2F activity are potent, systemic antitumor agents. Cancer Cell 1, 325-337 (2002)
doi:10.1016/S1535-6108(02)00060-0
4. Kamizono J., Nagano S., Murofushi Y., Komiya S., Fujiwara H., Matsuishi T: Survivin-responsive conditionally replicating adenovirus exhibits cancer-specific and efficient viral replication. Cancer Res 65, 5284-5291 (2005)
doi:10.1158/0008-5472.CAN-04-2657
5. Myung S. J., Yang S. K., Chang H. S., Byeon J. S., Kim K. J., Hong S. S.: Clinical usefulness of telomerase for the detection of colon cancer in ulcerative colitis patients. J Gastroenterol Hepatol 20, 1578-1583 (2005)
doi:10.1111/j.1440-1746.2005.03877.x
6. Meyerson M., Counter C. M., Eaton E. N., Ellisen L. W., Steiner P., Caddle S. D: hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 90, 785-795 (1997)
doi:10.1016/S0092-8674(00)80538-3
7. Shen F. B., Chang J. H., Yang C., Li J., Guo Y., Yi B: Tumor-selective replication, cytotoxicity and GM-CSF production of oncolytic recombinant adenovirus in KH901 injection.Sichuan Da Xue Xue Bao Yi Xue Ban 38, 31-34 (2007)
8. Shen F. B., Chang J. H., Yang C., Li J., Guo Y., Yi B: Effects of KH901, a tumor-specific oncolytic recombinant adenovirus, on antitumor and expressing GM-CSF in xenograft tumor models. Sichuan Da Xue Xue Bao Yi Xue Ban 38, 386-390 (2007)
9. Chang J., Zhao X., Wu X., Guo Y., Guo H., Cao J: A phase I study of KH901, a conditionally replicating granulocyte-macrophage colony-stimulating factor: armed oncolytic adenovirus for the treatment of head and neck cancers. Cancer Biol Ther 8, 676-682 (2009)
doi:10.4161/cbt.8.8.7913
10. Noordhuis P., Holwerda U., Van Laar J. A., Van der Wilt C. L., Peters G. J.: A non-radioactive sensitive assay to measure 5-fluorouracil incorporation into DNA of solid tumors. Nucleosides Nucleotides Nucleic Acids 23, 1481-4 (2004)
doi:10.1081/NCN-200027699
11. Peters G. J., van der Wilt C. L., van Moorsel C. J., Kroep J. R., Bergman A. M., Ackland S. P.: Basis for effective combination cancer chemotherapy with antimetabolites. Pharmacol Ther 87, 227-53 (2000)
doi:10.1016/S0163-7258(00)00086-3
12. Hecht J. R., Bedford R., Abbruzzese J. L., Lahoti S., Reid T. R., Soetikno R. M: A phase I/II trial of intratumoral endoscopic ultrasound injection of ONYX-015 with intravenous gemcitabine in unresectable pancreatic carcinoma. Clin Cancer Res 9, 555-61 (2003)
13. Pan Q., Liu B., Liu J., Cai R., Wang Y., Qian C.: Synergistic induction of tumor cell death by combining cisplatin with an oncolytic adenovirus carrying TRAIL. Mol Cell Biochem 304, 315-23 (2007)
doi:10.1007/s11010-007-9514-6
14. Wu Y. M., Zhang K. J., Yue X. T., Wang Y. Q., Yang Y., Li G. C: Enhancement of tumor cell death by combining cisplatin with an oncolytic adenovirus carrying MDA-7/IL-24. Acta Pharmacol Sin 30, 467-477 (2009)
doi:10.1038/aps.2009.16
15. Lei N., Shen F. B., Chang J. H., Wang L., Li H., Yang C: An oncolytic adenovirus expressing granulocyte macrophage colony-stimulating factor shows improved specificity and efficacy for treating human solid tumors. Cancer Gene Ther 16, 33-43 (2009)
doi:10.1038/cgt.2008.46
16. Khuri F. R., Nemunaitis J., Ganly I., Arseneau J., Tannock I. F., Romel L: A controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer. Nat Med 6, 879-885 (2000)
doi:10.1038/78638
17. Raki M., Kanerva A., Ristimaki A., Desmond R. A., Chen D. T., Ranki T: Combination of gemcitabine and Ad5/3-Delta24, a tropism modified conditionally replicating adenovirus, for the treatment of ovarian cancer. Gene Therapy 12, 1198-1205 (2005)
doi:10.1038/sj.gt.3302517
18. Zhang J., Ramesh N., Chen Y., Li Y., Dilley J., Working P., Yu D. C.: Identification of human uroplakin II promoter and its use in the construction of CG8840, a urothelium-specific adenovirus variant that eliminates established bladder tumors in combination with docetaxel. Cancer Research 62, 3743-3750 (2002)
19. Cherubini G., Petouchoff T., Grossi M., Piersanti S., Cundari E., Saggio I.: E1B55K-deleted adenovirus (ONYX-015) overrides G1/S and G2/M checkpoints and causes mitotic catastrophe and endoreduplication in p53-proficient normal cells. Cell Cycle 5, 2244-2252 (2006)
doi:10.4161/cc.5.19.3263
20. Ben-Israel H., Kleinberger T.: Adenovirus and cell cycle control. Front Biosci 7, d1369-1395 (2002)
doi:10.2741/ben
21. Cobrinik, D.: Pocket proteins and cell cycle control. Oncogene 24, 2796-2809 (2005)
doi:10.1038/sj.onc.1208619
22. Frolov M. V., Dyson N. J.: Molecular mechanisms of E2F-dependent activation and pRB-mediated repression. J Cell Sci 117, 2173-2181 (2004)
doi:10.1242/jcs.01227
23. Kolli S., Buchmann A. M., Williams J., Weitzman S., Thimmapaya B.: Antisense-mediated depletion of p300 in human cells leads to premature G1 exit and up-regulation of c-MYC. Proc Natl Acad Sci U S A 98, 4646-4651 (2001)
doi:10.1073/pnas.081141998
24. Liang J., Zubovitz J., Petrocelli T., Kotchetkov R., Connor M. K., Han K: PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 8, 1153-60 (2002)
doi:10.1038/nm761
25. Viglietto G., Motti M. L., Bruni P., Melillo R. M., D'Alessio A., Califano D: Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer. Nat Med 8, 1136-44 (2002)
doi:10.1038/nm762
26. Icyuz M., Bryant S. M., Fortinberry H. K., Molakandov K., Siegal G. P., Contreras J. L: Adenovirus infection activates akt1 and induces cell proliferation in pancreatic islets. Transplantation 87: 821-4 (2009)
doi:10.1097/TP.0b013e318199c686
27. Zhang X., Turnell A. S., Gorbea C., Mymryk J. S., Gallimore P. H., Grand R. J.: The targeting of the proteasomal regulatory subunit S2 by adenovirus E1A causes inhibition of proteasomal activity and increased p53 expression. J Biol Chem 279, 25122-33 (2004)
doi:10.1074/jbc.M403287200
28. Cuconati A., White E.: Viral homologs of BCL-2: role of apoptosis in the regulation of virus infection. Genes Dev 16, 2465-2478 (2002)
doi:10.1101/gad.1012702
29. Zhu ZH, Gao YS, Zeng BF, Zhang CQ. The effect of dexamethasone and hypoxic stress on MC3T3-E1 cells. Front Biosci 17, 2747-2755 (2011)
doi:10.2741/3883
30. Walia V, Kakar S, Elble R. Micromanagement of the mitochondrial apoptotic pathway by p53. Front Biosci 16, 749-758 (2011)
doi:10.2741/3717
31. Zinkel S., Gross A., Yang E.: BCL2 family in DNA damage and cell cycle control. Cell Death Differ 13, 1351-1359 (2006)
doi:10.1038/sj.cdd.4401987
Abbreviations: 5-FU: 5-fluorouracil; TERT: telomerase reverse-transcriptase; GM-CSF: granulocyte macrophage colony-stimulating factor; RB: Retinoblastoma;
Key Words: KH901, 5-fluorouracil, p-AKT, LoVo cells, antitumor efficacy, S phase
Send correspondence to: Jin Li, Department of Medical Oncology, Fudan University Shanghai Cancer Center, 270 Dongan Rd, Shanghai 200012, China, Tel: 086 02164175590, Fax: 086 02164170366, E-mail:fudanlijin@163.com