|Year : 2014 | Volume
| Issue : 3 | Page : 665-670
Synergistic anti-tumor effects of nitroreductase mutants and p53
Mahboobeh Razmkhah1, Mojtaba Habibagahi2, Fatemeh Alizadeh3, Ahmad Hosseini1, Abbas Ghaderi4, Peter F Searle5, Mansooreh Jaberipour1
1 Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences, Shiraz, Iran
2 Department of Immunology, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran
3 Shiraz Institute for Cancer Research; Shiraz Pharmacy School, Shiraz University of Medical Sciences, Shiraz, Iran
4 Shiraz Institute for Cancer Research; Department of Immunology, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran
5 School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
|Date of Web Publication||14-Oct-2014|
Shiraz Institute for Cancer Research, Medical School, Shiraz University of Medical Sciences, Shiraz
Source of Support: None, Conflict of Interest: None
Introduction: The p53 gene therapy showed promising results for treatment of numerous cancers particularly in combination with chemotherapy or radiotherapy. Gene therapy combining two or more treatment options may lead to the synergistic effects between diverse therapies and provide many opportunities in our fight against cancer.
Aim: This study focused on the effects of p53 combining with the suicide gene therapy, nitroreductase (NTR)/5-(aziridin-1-yl)-2,4 dinitrobenzamide, on different cancer cell lines.
Materials and Methods: Effects of adenoviral expressing p53 alone or in combination with wild type (WT) NTR, NTR single mutant, F124N and two NTR double mutants, T41L/N71S and T41L/F70A on survival rate of A549, QU-DB, MCF-7, MDA-MB-468 and DU145 cancer cell lines were determined by MTT assay. Expressions of MDM2 and TP53 transcripts were then assessed by quantitative real-time polymerase chain reaction in p53, NTR and combination of p53 with NTR infected cell lines.
Results: According to the results, combination of p53 with NTR double mutant, T41L/F70A or NTR single mutant F124N, showed statistically significant decrease in vitality of all cancer cell lines studied compared with status of IC 50 from p53 or WT NTR and other NTR mutants alone (P < 0.05). Expressions of TP53 and MDM2 were downregulated in all T41L/F70A infected cells except for MCF-7.
Conclusion: Combination of T41L/F70A NTR with p53 may have more advantages for treatment of different types of cancers compared to the other NTRs and p53 alone. The present study results may open new windows for getting desired outcome in gene therapy of different types of cancer.
Keywords: Adenovirus vector, cancer, gene therapy, nitroreductase, p53
|How to cite this article:|
Razmkhah M, Habibagahi M, Alizadeh F, Hosseini A, Ghaderi A, Searle PF, Jaberipour M. Synergistic anti-tumor effects of nitroreductase mutants and p53. J Can Res Ther 2014;10:665-70
|How to cite this URL:|
Razmkhah M, Habibagahi M, Alizadeh F, Hosseini A, Ghaderi A, Searle PF, Jaberipour M. Synergistic anti-tumor effects of nitroreductase mutants and p53. J Can Res Ther [serial online] 2014 [cited 2019 Jun 20];10:665-70. Available from: http://www.cancerjournal.net/text.asp?2014/10/3/665/139155
| > Introduction|| |
It has long been documented that in contrast to the progress in cancer therapy such as chemotherapy and surgery, most of the patients suffered from cancer recurrence and finally surrender to death. Several recently reported clinical trials showed that gene therapy can be considered as a great alternative for the treatment of various types of cancer. ,,,,, In this connection different approaches such as suicide gene therapy, , enhancement of the immune response against tumor cells, mutation correction and tumor suppressing gene therapies  can be introduced. Furthermore, combination gene therapy against cancer is a novel therapeutic approach combining two or more treatment options, which may lead to maximize the number of tumor cells killed and provide important synergistic effects between diverse therapies. 
The p53 protein is well-known as a transcription factor and a tumor suppressor gene which is controlled by MDM2 as a negative regulator. The TP53 gene is inactivated by point mutations in approximately 50% of all human cancers. Interestingly, tumors with wild type (WT) p53 even show a non-functional form of p53 due to overexpressed MDM2. ,,,, Considering the central role of p53 and its abrogation in cancer, obtaining normal p53 function is one of the promising approaches for effective cancer therapy. The p53 gene therapy, Gendicine in China and its counterpart Advexin in the United States, showed suitable results for treatment of several cancers specially lung cancer and head and neck cancer alone or in combination with chemotherapy or radiotherapy. ,,
A variety of enzyme/prodrug combinations have been recently described among which nitroreductase (NTR)/5-(aziridin-1-yl)-2,4 dinitrobenzamide (CB1954) shows a great therapeutic index. Expression of NTR from a replication-defective adenovirus can increase the sensitivity of carcinoma cells to CB1954 by 1000-fold and this combination has entered clinical trials  for treatment of prostate cancer.  As the kinetics of CB1954 activation by NTR is relatively slow, Searle et al. analyzed a series of NTR mutants with amino acid substitutions around the active site. They reported mutants that activated CB1954 more efficiently and at lower concentrations.  Similarly, Jaberipour et al. engineered NTR in order to further improve the ability of NTR to activate CB1954 by combinations of the beneficial single mutants identified previously.  They firstly screened 53 double mutants in Escherichia More Details coli and then inserted 7 most promising double mutants into an adenovirus vector and compared for sensitizing SKOV3 human ovarian carcinoma cells to CB1954. Results showed that T41L/N71S and T41L/F70A had 14-17-fold more efficiency than WT NTR at sensitizing the cancer cells to CB1954. 
Based on the importance of p53-therapy of cancer and the advantages of using NTR, the present study further investigated the effect of combination use of adenoviral vectors expressing p53 and NTR mutants on human mammary, lung and prostate cancer cell lines, in vitro.
| > Materials and Methods|| |
Cell lines and culture conditions
The experiments were carried out using different human cancer cell lines with WT or mutant TP53 including two lung cancer cell lines, A549 and QU-DB (with WT TP53 and mutant TP53, respectively), breast cancer cell lines, MCF-7 and MDA-MB-468 (with WT and mutant TP53, respectively) and DU145 as a prostate cancer cell line with mutant TP53. The cell lines were cultured in Roswell Park Memorial Institute 1640 (Biosera, UK) containing 10% Fetal Bovine Serum (GIBCO, USA), 100 units/ml penicillin (Biosera, UK) and 100 mg/ml streptomycin (Biosera, UK) at 37°C and 5% CO 2 . Growth medium was replaced every 3 days and cultures were passaged using 0.02% trypsin 7 days post-culture.
Propagation and titration of adenovirus vectors
Adenoviruses encoding green fluorescent protein (GFP), TP53 and WT NTR, NTR single mutant, F124N and two NTR double mutants, T41L/N71S and T41L/F70A, which described previously  were propagated in human embryonic kidney 293 cells and the viral infection titers were determined by flow cytometry. The cells infected with adenovirus expressing GFP used as the control.
Cells were harvested and then resuspended in culture medium and their viability was determined. The cells were then infected with the adenoviruses that expressed p53, mutants and WT NTR at a range of multiplicities of infection and calculated as plaque forming units per cell (pfu/cell). After 2 days post-culture, the medium of the NTR infected cells was replaced with 150 μl of medium containing CB1954 in concentration of 10 μM. After 24 h, the medium containing pro-drug was removed and fresh medium was added. At 5 th day of culture, medium was removed and MTT assay was performed for estimating the 50% of cell survival (IC 50 ). Cells infected with the adenovirus expressing GFP were used as the control for cell viability. For determining the synergistic effects of WT and mutant NTRs with p53, both vectors were used together and IC 50 value was assessed by MTT assay. For this purpose the culture media were completely removed and 150 μl of 0.1% MTT solution in complete culture medium were added. The cells were incubated for 5 h at 37°C in a 5% humidified CO 2 incubator. After incubation, the formazan crystals were dissolved in 150 μl dimethyl sulfoxide and the absorbance was read at 490 nm after 24 h. Each assay was performed in triplicate.
Ribonucleic acid (RNA) isolation and complementary deoxyribonucleic acid (cDNA) synthesis
Total RNA was isolated from infected cells after lysis with TRizol reagent (Invitrogen, Paisley, UK). Then cDNA was synthesized from 5 μg of extracted RNA, using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania).
Quantitative real-time polymerase chain reaction (qRT-PCR)
The expression of MDM2 and TP53 gene transcripts were determined by qRT-PCR using an ABI system (real-time PCR Detector, ABI, USA) with SYBR Green PCR Master Mix kit (Applied Biosystems, USA). Expression of 18S rRNA housekeeping gene was used as a reference for the level of target gene expression. Each PCR reaction was performed in a final volume of 25 μl which contained 0.5 μg of the cDNA products, 10 pmol of reverse and forward primers and 1 × reaction mixture. Primers were designed by primer 3 open source software (SourceForge, USA). Thermal cycling for both genes was initiated with an initial denaturation step at 95°C for 10 min, followed by 50 cycles with denaturation at 95°C for 20 s, annealing at 56°C for 20 s and extension at 60°C for 34 s when fluorescence appeared. The qRT-PCR amplification products were analyzed by melting curve analysis.
The relative amounts of TP53 and MDM2 transcripts were determined using 2−ΔΔCt method. Expressions of TP53 and MDM2 gene transcripts in infected cells were compared to the corresponding values from control (none infected) samples using Wilcoxon matched pairs test. Relative expression was plotted by means of GraphPad Prism 5 (Inc; San Diego CA, USA, 2003) and P < 0.05 was considered as significant in all statistical analyses.
| > Results|| |
The cytotoxicity of Ad-P53 in cancer cell lines
After 5 day-incubation of each cell line with Ad-p53 the cytotoxicity of the adenovirus vectors was determined at 0, 1, 3, 10, 30 and 100 pfu/cell. No cytotoxicity was observed at 0-30 pfu/cell in all cell lines examined, except for the MDA-MB-468. The IC 50 values for each cell line was as follows, A549 (39.02 pfu/cell), QU-DB (34.53 pfu/cell), MCF-7 (31.07 pfu/cell), MDA-MB-468 (14.27 pfu/cell) and DU145 (43.52 pfu/cell) [Figure 1].
|Figure 1: Growth-inhibitory of adenovirus TP53 delivery in various cancer cell lines at different plaque forming units per cell. Cells (15,000 cells/ well) were infected with adenoviruses expressing TP53 and cultured in 96 well plates, in triplicate, for 5 days. The mean values of the cell counts from triplicate measurements are plotted|
Click here to view
The cytotoxicity of Ad-NTRS in cancer cell lines
In order to check the cytotoxicity of the NTR expressing adenoviral vectors, 15,000 cells/well were infected with adenoviruses at concentrations of 0, 1, 3, 10, 30 and 100 pfu/cell in triplicate for 5 days. Infection with the WT NTR expressing vectors resulted in an IC 50 with a range between 52.87 pfu/cell for A549 and 14.99 pfu/cell for DU145. The lowest IC 50 s for both lung cancer cells (A549 and QU-DB) and MCF-7 were achieved with double mutant T41L/F70A; whereas the lowest IC 50 values for MDA-MB-468 and DU145 were observed when single mutant F124N was used. The IC 50 values of different cancer cell lines, for each WT and mutant NTRs were summarized in [Table 1].
|Table 1: IC50 of WT and mutant adenoviral expressing NTRs in different cancer cell lines |
Click here to view
Treatment of cell lines with Ad-NTRs showed that T41L/F70A was the most effective mutant with 2.2-, 2.6-, 3.2, 1.3 and 1.7-fold more efficiency than WT NTR for sensitising the A549, QU-DB, MCF-7, MDA-MB-468 and DU145 to CB1954, respectively.
Combination of adenoviral vectors expressing NTRS and p53
To study the synergistic effects of NTRs and p53, the effectiveness of the combined treatment of the cell lines with IC 50 concentrations of p53 and NTRs were evaluated. As displayed in [Figure 2], the simultaneous use of p53 with NTRs, T41L/F70A or F124N, showed the greatest result to decrease the cell vitality compared with status of IC 50 from p53 or other NTRs alone [Figure 2]. The synergistic effects of T41L/F70A or F124N with p53 were statistically significant in all cell lines compared to each case alone (P < 0.05).
|Figure 2: The graphical presentation of the viability of cancer cell lines when TP53 expressing vector used in combination with nitroreductase mutants, T41L/F70A or F124N. The percent of cell vitality is shown in untreated, green fluorescent protein-, TP53-, and NTR-treated cell lines. As it is shown, results of combination use of TP53 and T41L/F70A or F124N are statistically significant in all cell lines compared to each case alone (P < 0.05). Y axis shows cell viability and X axis shows the virus type. **Statistical significant|
Click here to view
Expressions of TP53 and MDM2 transcripts in cancer cell lines treating with p53, T41L/F70A NTR or combination of p53 and T41L/F70A-NTR
The qRT-PCR assay was employed to determine the expressions of TP53 and MDM2 transcripts when adenoviral expressing p53, T41L/F70A or combination of p53 and T41L/F70A-NTR were used in different cancer cell lines.
Expressions of TP53 and MDM2 were downregulated in all T41L/F70A treated cells except for MCF-7 cell line. According to the results, TP53 transcript showed 90% decrease in A549, 10% in QU-DB and 60% in MDA-MB-468 and DU145 infected by T41L/F70A-NTR. Expression of MDM2 showed similar results; it had 75% decrease in A549 and 30% decrease in T41L/F70A treated QU-DB, MDA-MB-468 and DU145 [Figure 3].
|Figure 3: Expressions of TP53 and MDM2 transcripts in cancer cell lines studied. As depicted expressions of both TP53 and MDM2 were downregulated in all T41L/F70A treated cells except for MCF-7-T41L/F70A|
Click here to view
| > Discussion|| |
Tumor suppressor gene therapy, suicide gene therapy, cytokine based therapy and stimulation of apoptosis hold most promising therapeutic approaches for treating different kinds of malignancies.  It has recently been defined that combination therapy may have more advantages because of the expected synergistic effects between different strategies. ,, Accordingly, the p53 gene therapy, showed most appropriate outcomes for treatment of several cancers when used in combination with chemotherapy or radiotherapy particularly in tumors with mutant p53. ,,
Apoptosis but not necrosis was defined as the mechanism of mammary cell death by NTR/CB1954 through DNA cross-linking without important inflammatory effects on the mammary glands. ,, Apoptosis induced by activated CB1954 mainly depends on activation of caspase signalling pathway. Studies by Palmer and Zheng et al. showed that using the caspase-8 and -9 inhibitors can effectively reduce levels of apoptosis induced by activated CB1954 indicating that NTR/CB1954 may predominantly act through caspases-8 and -9. , In fact NTR/CB1954 mediated apoptosis does not depend on a functional p53 signalling pathway,  thus it provides additional support for using NTR/CB1954 with other cancer gene therapy approaches such as WT p53, particularly against tumours with deleted WT TP53 gene.
Further, the high frequent loss of p53 in human cancers is an outstanding motivation for restoring WT p53 into cancer cells.  It has been shown that reintroducing WT p53 to tumors could potentially contribute to the effect of chemotherapy and radiotherapy on the tumor response.  These data impose us to check the efficiency of combination between two different gene therapy strategies, WT p53 and E. coli NTR, in human mammary, lung and prostate cancer cell lines, in vitro.
Based on the results of this study, expression of WT tumor suppressor p53 in lung and breast cancer cell lines with mutant TP53, QU-DB and MDA-MB-468, was more effective than other cell lines studied, suggesting that gene therapy based on restoration of WT p53 function may be more feasible option in breast and lung cancer therapy than prostate cancer. Accordingly, Fujiwara et al. demonstrated the in vitro and in vivo human lung cancer growth inhibition after reintroducing p53 into lung cancer cells using retroviruses.  In another study expression of p53 has been led to the growth inhibition of breast cancer cells in a SHP-1-mediated manner. 
In addition, treatment of cell lines with NTR mutants showed that the double mutant T41L/F70A was the most effective mutant with more potent efficiency than WT NTR for sensitising all cancer cells to CB1954. Consistently, Jaberipour et al. have found that T41L/N71S and T41L/F70A were more efficient than WT NTR for sensitising the SKOV3 ovarian cancer cells to CB1954. 
The simultaneous use of p53 with different NTR mutants demonstrated that T41L/F70A NTR double mutant with p53 and F124N NTR single mutant with p53 had the greatest effects to decrease the cell vitality compared to status of IC 50 from p53 or other NTRs alone. However, the highest cell vitality decreasing effect of combination of T41L/F70A NTR with p53 was achieved in breast cancer cell lines, MCF-7 and MDA-MB-468. Thus, it is concluded that simultaneous use of NTR double mutant T41L/F70A with p53 may have more advantages for treatment of cancer particularly breast cancer compared to the other combinations and also NTR WT and p53 alone.
For evaluating apoptosis mechanisms, we assessed the expressions of MDM2 and TP53 messenger ribonucleic acid transcripts in infected cell lines. Results confirmed previous reports as Cui et al. showed that NTR mediated cell death by CB1954 is independent of p53 but dependent on caspase signalling pathway.  Thus appraising different caspase molecules especially in protein level may contribute to stronger interpretation about apoptosis mechanisms.
Collectively, the data of this study may lead to the further development in the future gene therapy clinical trial designs for various types of malignancies particularly breast cancer using combination of NTR double mutant/CB1954 and p53. However, the current in vitro data need to be established in preclinical and clinical settings as Jaberipour et al. have found T41L/N71S and T41L/F70A as more efficient mutant in ovarian cancer cells but they showed that adenoviral-mediated transfer of T41L/N71S NTR had greater anti-tumour activity in human ovarian or prostate carcinoma tumour xenograft models.  Furthermore, a clinical trial with replication-defective adenovirus expressing NTR/CB1954 in patients with prostate cancer has shown reduction of the tumor marker prostate specific antigen in some patients,  but the efficiency was inadequate and greater efficacy remains to be achieved. Thus, the present data needs to be confirmed by preclinical studies and clinical trials for getting desired outcome in gene therapy of different types of cancer in future.
| > Acknowledgment|| |
The authors thank Dr. Grove for construction of F124N NTR. This work was financially supported by a grant from Shiraz Institute for Cancer Research (Grant No 100-505) and Shiraz University of Medical Sciences (Grant No. 91-6438). This research was done in partial fulfilment of the requirements for the Pharmaceutical thesis defended by Fatemeh Alizadeh.
| > References|| |
Guise CP, Grove JI, Hyde EI, Searle PF. Direct positive selection for improved nitroreductase variants using SOS triggering of bacteriophage lambda lytic cycle. Gene Ther 2007;14:690-8.
Searle PF, Chen MJ, Hu L, Race PR, Lovering AL, Grove JI, et al
. Nitroreductase: A prodrug-activating enzyme for cancer gene therapy. Clin Exp Pharmacol Physiol 2004;31:811-6.
Jaberipour M, Vass SO, Guise CP, Grove JI, Knox RJ, Hu L, et al
. Testing double mutants of the enzyme nitroreductase for enhanced cell sensitisation to prodrugs: Effects of combining beneficial single mutations. Biochem Pharmacol 2010;79:102-11.
Taghavi MH, Davoodi J, Mirshahi M. The effect of wild type P53 gene transfer on growth properties and tumorigenicity of PANC-1 tumor cell line. Iran Biomed J 2007;11:1-6.
Cui W, Gusterson B, Clark AJ. Nitroreductase-mediated cell ablation is very rapid and mediated by a p53-independent apoptotic pathway. Gene Ther 1999;6:764-70.
Combination therapy. Available from: http ://www.merckmanuals.com/pethealth/index.html.
Vassaux G, Martin-Duque P. Use of suicide genes for cancer gene therapy: Study of the different approaches. Expert Opin Biol Ther 2004;4:519-30.
Duarte S, Carle G, Faneca H, de Lima MC, Pierrefite-Carle V. Suicide gene therapy in cancer: Where do we stand now? Cancer Lett 2012;324:160-70.
Fang B, Roth JA. Tumor-suppressing gene therapy. Cancer Biol Ther 2003;2:S115-21.
Chung-Faye GA, Kerr DJ, Young LS, Searle PF. Gene therapy strategies for colon cancer. Mol Med Today 2000;6:82-7.
Miyake H, Yamanaka K, Muramaki M, Hara I, Gleave ME. Therapeutic efficacy of adenoviral-mediated p53 gene transfer is synergistically enhanced by combined use of antisense oligodeoxynucleotide targeting clusterin gene in a human bladder cancer model. Neoplasia 2005;7:171-9.
Wang Z, Sun Y. Targeting p53 for novel anticancer therapy. Transl Oncol 2010;3:1-12.
Vazquez A, Bond EE, Levine AJ, Bond GL. The genetics of the p53 pathway, apoptosis and cancer therapy. Nat Rev Drug Discov 2008;7:979-87.
Bouchet BP, Caron de Fromentel C, Puisieux A, Galmarini CM. p53 as a target for anti-cancer drug development. Crit Rev Oncol Hematol 2006;58:190-207.
Peng Z. Current status of gendicine in China: Recombinant human Ad-p53 agent for treatment of cancers. Hum Gene Ther 2005;16:1016-27.
Patel P, Young JG, Mautner V, Ashdown D, Bonney S, Pineda RG, et al
. A phase I/II clinical trial in localized prostate cancer of an adenovirus expressing nitroreductase with CB1954 [correction of CB1984]. Mol Ther 2009;17:1292-9.
Vachani A, Moon E, Wakeam E, Haas AR, Sterman DH, Albelda SM. Gene therapy for lung neoplasms. Clin Chest Med 2011;32:865-85.
Nielsen LL, Lipari P, Dell J, Gurnani M, Hajian G. Adenovirus-mediated p53 gene therapy and paclitaxel have synergistic efficacy in models of human head and neck, ovarian, prostate, and breast cancer. Clin Cancer Res 1998;4:835-46.
Clark AJ, Iwobi M, Cui W, Crompton M, Harold G, Hobbs S, et al
. Selective cell ablation in transgenic mice expression e. coli
nitroreductase. Gene Ther 1997;4:101-10.
Melcher A, Gough M, Todryk S, Vile R. Apoptosis or necrosis for tumor immunotherapy: What's in a name? J Mol Med (Berl) 1999;77:824-33.
Steinman RM, Turley S, Mellman I, Inaba K. The induction of tolerance by dendritic cells that have captured apoptotic cells. J Exp Med 2000;191:411-6.
Palmer DH, Milner AE, Kerr DJ, Young LS. Mechanism of cell death induced by the novel enzyme-prodrug combination, nitroreductase/CB1954, and identification of synergism with 5-fluorouracil. Br J Cancer 2003;89:944-50.
Zheng TS, Hunot S, Kuida K, Momoi T, Srinivasan A, Nicholson DW, et al
. Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation. Nat Med 2000;6:1241-7.
Fujiwara T, Grimm EA, Mukhopadhyay T, Cai DW, Owen-Schaub LB, Roth JA. A retroviral wild-type p53 expression vector penetrates human lung cancer spheroids and inhibits growth by inducing apoptosis. Cancer Res 1993;53:4129-33.
Montano X. Repression of SHP-1 expression by p53 leads to trkA tyrosine phosphorylation and suppression of breast cancer cell proliferation. Oncogene 2009;28:3787-800.
[Figure 1], [Figure 2], [Figure 3]