Strategies for enzyme/prodrug cancer therapy.

The selective activation of prodrug(s) in tumor tissues by exogenous enzyme(s) for cancer therapy can be accomplished by several ways, including gene-directed enzyme prodrug therapy (GDEPT), virus-directed enzyme prodrug therapy (VDEPT), and antibody-directed enzyme prodrug therapy (ADEPT). The central part of enzyme/prodrug cancer therapy is to deliver drug-activating enzyme gene or functional protein to tumor tissues, followed by systemic administration of a prodrug. Although each approach (GDEPT, VDEPT, and ADEPT) has been tested in clinical trials, there are some potential problems using the current delivery systems. In this article, disadvantages and advantages associated with each approach (GDEPT, VDEPT, and ADEPT) and future perspective for improving current systems are discussed.     

A mammalianized synthetic nitroreductase gene for high-level expression

Background  

The nitroreductase/5-(azaridin-1-yl)-2,4-dinitrobenzamide (NTR/CB1954) enzyme/prodrug system is considered as a promising candidate for anti-cancer strategies by gene-directed enzyme prodrug therapy (GDEPT) and has recently entered clinical trials. It requires the genetic modification of tumor cells to express the E. coli enzyme nitroreductase that bioactivates the prodrug CB1954 to a powerful cytotoxin. This metabolite causes apoptotic cell death by DNA interstrand crosslinking. Enhancing the enzymatic NTR activity for CB1954 should improve the therapeutical potential of this enzyme-prodrug combination in cancer gene therapy.     

射线联合嵌合启动子介导的自杀基因对肿瘤细胞的靶向杀伤作用

目的 探讨放射线联合新型嵌合启动子介导辣根过氧化物酶/吲哚乙酸(HRP/IAA)自杀基因系统特异高效的抗肿瘤作用.方法 构建含有4个串联放射反应元件CArG、含或不含巨细胞病毒(CMV)启动子的人端粒酶逆转录酶嵌合启动子,筛选肿瘤特异性及放射诱导性强的嵌合启动子,下游连接自杀基因辣根过氧化物酶(HRP),检测放射线联合基因治疗对肿瘤细胞HeLa、A549和MHCC97增殖及凋亡的影响.结果 人胚肺成纤维细胞MRC-5中C4-hTC嵌合启动子的活性(0.1±0.0)明显低于肿瘤细胞株HeLa、A549和MHCC97(0.6±0.0、1.1±0.1和1.0±0.1,P＜0.01),经过6 Gy射线诱导后,这种差异更加显著(活性分别为0.2±0.1、1.7±0.2、2.3±0.2和2.3±0.1,P＜0.01).在肿瘤细胞株HeLa、A549和MHCC97中,嵌合启动子C4-hTC-HRP携带的自杀基因系统SER值分别为2.64、2.75和2.82,显著高于单纯hTERT启动子.在肿瘤细胞中,除阴性对照质粒pGL3-control-Luc外,自杀基因与放射治疗相联合对肿瘤细胞的生长抑制明显高于单一治疗方法,而联合组中pC4-hTC-HRP/IAA系统与放射联合的生长抑制作用最显著,对HeLa、A549和MHCC97细胞的生长抑制率分别为67.3%、69.0%和64.6%.在肿瘤细胞中,除阴性对照质粒pGL3-control-Luc外,联合组诱导的早期凋亡率明显高于单一治疗方法组,其中pC4-hTC-HRP/IAA系统与放射联合诱导的凋亡率最高,HeLa、A549和MHCC97细胞凋亡率分别为39.6%、33.0%和33.2%.结论 新型嵌合启动子C4-hTC具有良好的肿瘤特异性及放射诱导性,放射线联合基因治疗对肿瘤细胞具有特异高效的杀伤作用,在肿瘤的基因放疗中具有较强的应用潜力.

Abstract：

Objective To explore the synergistic anti-tumor effect of radiotherapy and horseradish peroxidase/prodrug indole-3-acetic acid (HRP/IAA) gene therapy system using chimeric hTERT promoter responsive to ionizing radiation.Methods The synthetic hTERT promoters containing four tandem-repeat copies of radio-inducible CArG elements, and the chimeric promoter containing cytomegalovirus (CMV)early promoter were both constructed.The activities of the chimeric promoters in cancer cell lines ( HeLa,A549, and MHCC97 ) and normal cell line ( MRC-5 ) were detected using luciferase reporter gene expression analysis after a 60 Co γ-irradiation treatment at a series of doses (a single dose of 0 to 10 Gy).The anti-tumor effect of combining irradiation with HRP/IAA gene-directed enzyme prodrug therapy system controlled by the chimeric promoter was tested by colony formation assay, cell counting and apoptosis analysis.Results The chimeric promoters were ineffective in normal human cells, even after irradiation, but the expression of luciferase gene in tumor cells was significantly higher.The activity of the chimeric promoter in MRC-5 cells was 22.3%, 12.9% and 13.6% of that in HeLa, A549 and MHCC97 cells, respectively.After irradiation,the ratios were 11.7%, 8.7% and 8.8%, respectively.Furthermore, the chimeric promoters could successfully induce the expression of luciferase gene following different doses of radiation, with maximal inducible activity seen after 6 Gy irradiation.The chimeric promoter containing four tandem-repeat copies of radio-inducible CArG elements and CMV early promoter showed the highest activity with 6 Gy irradiation.The relative luciferase activities in HeLa, A549 and MHCC97 cells were 1.7 ± 0.2, 2.3 ± 0.2 and 2.3±0.1, respectively.The chimeric promoter mediated suicide gene therapy system could increase radiosensitivity in different cancer cells.Compared with the control system, it plus irradiation showed stronger cell proliferation inhibition, 67.3% vs.26.1% in HeLa, 69.0% vs.28.3% in A549, 64.6% vs.20.8% in MHCC97 cells, and also higher apoptosis-inducing effect, 39.6% vs.14.2% in HeLa, 33.0% vs.12.4%in A549, and 33.2% vs.14.2% in MHCC97 cells.Conclusions Chimeric promoter containing hTERT promoter, CArG elements and CMV promoter preserve the tumor-specificity in telomerase-positive tumor cells, and irradiation-responsive to low dose of radiation.The suicide gene therapy using this promoter plus radiotherapy show a strong anti-tumor effect in vitro.It is expected to have a good potential for future application in gene radiotherapy.     

Combined enzyme/prodrug treatment by genetically engineered AT-MSC exerts synergy and inhibits growth of MDA-MB-231 induced lung metastases

Background

Metastatic spread of tumor cells remains a serious problem in cancer treatment. Gene-directed enzyme/prodrug therapy mediated by tumor-homing genetically engineered mesenchymal stromal cells (MSC) represents a promising therapeutic modality for elimination of disseminated cells. Efficacy of gene-directed enzyme/prodrug therapy can be improved by combination of individual systems. We aimed to define the combination effect of two systems of gene therapy mediated by MSC, and evaluate the ability of systemically administered genetically engineered mesenchymal stromal cells to inhibit the growth of experimental metastases derived from human breast adenocarcinoma cells MDA-MB-231/EGFP.

Methods

Human adipose tissue-derived mesenchymal stromal cells (AT-MSC) were retrovirally transduced with fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT) or with Herpes simplex virus thymidine kinase (HSVtk). Engineered MSC were cocultured with tumor cells in the presence of prodrugs 5-fluorocytosin (5-FC) and ganciclovir (GCV). Combination effect of these enzyme/prodrug approaches was calculated. SCID/bg mice bearing experimental lung metastases were treated with CD::UPRT-MSC, HSVtk-MSC or both in combination in the presence of respective prodrug(s). Treatment efficiency was evaluated by EGFP-positive cell detection by flow cytometry combined with real-time PCR quantification of human cells in mouse organs. Results were confirmed by histological and immunohistochemical examination.

Results

We demonstrated various extent of synergy depending on tested cell line and experimental setup. The strongest synergism was observed on breast cancer-derived cell line MDA-MB-231/EGFP. Systemic administration of CD::UPRT-MSC and HSVtk-MSC in combination with 5-FC and GCV inhibited growth of MDA-MB-231 induced lung metastases.

Conclusions

Combined gene-directed enzyme/prodrug therapy mediated by MSC exerted synergic cytotoxic effect and resulted in high therapeutic efficacy in vivo.

Electronic supplementary material

The online version of this article (doi:10.1186/s13046-015-0149-2) contains supplementary material, which is available to authorized users.     

Toward targeted “oxidation therapy” of cancer: peroxidase-catalysed cytotoxicity of indole-3-acetic acids

Purpose: The study aimed to identify suitable prodrugs that could be used to test the hypothesis that peroxidase activity in cells, either endogenous or enhanced by immunological targeting, can activate prodrugs to cytotoxins. We hypothesized that prototype prodrugs based on derivatives of indole-3-acetic acid (IAA), when activated by peroxidase enzymes (e.g., from horseradish, HRP) should produce peroxyl radicals, with deleterious biological consequences.Methods and Materials: V79 hamster cells were incubated with IAA or derivatives ± HRP and cytotoxicity assessed by a clonogenic assay. To assess the toxicity of stable oxidation products, prodrugs were also oxidized by HRP without cells, and the products then added to cells.Results: The combination of prodrug and enzyme resulted in cytotoxicity, but neither indole nor enzyme in isolation was toxic under the conditions used. Although lipid peroxidation was stimulated in liposomes by the prodrug/enzyme treatment, it could not be measured in mammalian cells. Adding oxidized prodrugs to cells resulted in cytotoxicity.Conclusions: Although the hypothesis that prodrugs of this type could enhance oxidative stress via lipid peroxidation was not established, the results nonetheless demonstrated oxidatively-activated cytotoxicity via indole acetic acid prodrugs, and suggested these as a new type of substrate for antibody-directed enzyme-prodrug therapy (ADEPT). The hypothesized free-radical fragmentation intermediates were demonstrated, but lipid peroxidation associated with peroxyl radical formation was unlikely to be the major route to cytotoxicity.     

The adenovirus-mediated linamarase/linamarin suicide system: A potential strategy for the treatment of hepatocellular carcinoma

Suicide gene therapy, also known as gene-directed enzyme prodrug therapy, has the potential to provide a cure for hepatocellular carcinoma (HCC), which is a leading cause of cancer death worldwide. Linamarase from cassava is an enzyme that can hydrolyze the innocuous substrate linamarin into cyanide, which can block the mitochondrial respiratory chain of cells. To investigate whether linamarase and linamarin can be used for HCC treatment, a recombinant adenovirus carrying linamarase cDNA was constructed using the ViraPower™ Adenoviral Expression System. In vitro cytotoxicity assays indicated that the system has a dramatic inhibitory effect and a strong bystander effect on HCC cell lines. Application of the recombinant adenovirus with linamarin in nude mice bearing HCC xenografts also resulted in significant inhibition of the tumors, while the animals did not show any appreciable toxic effects. Our findings provide the first in vitro and in vivo evidence that this system may provide a potential strategy for HCC treatment.     

Systemic gene-directed enzyme prodrug therapy of hepatocellular carcinoma using a targeted adenovirus armed with carboxypeptidase G2

Hepatocellular carcinoma is the fifth most common cancer worldwide, and there is no effective therapy for unresectable disease. We have developed a targeted systemic therapy for hepatocellular carcinoma. The gene for a foreign enzyme is selectively expressed in the tumor cells and a nontoxic prodrug is then given, which is activated to a potent cytotoxic drug by the tumor-localized enzyme. This approach is termed gene-directed enzyme prodrug therapy (GDEPT). Adenoviruses have been used to target cancer cells, have an intrinsic tropism for liver, and are efficient gene vectors. Oncolytic adenoviruses produce clinical benefits, particularly in combination with conventional anticancer agents and are well tolerated. We rationalized that such adenoviruses, if their expression were restricted to telomerase-positive cancer cells, would make excellent gene vectors for GDEPT therapy of hepatocellular carcinoma. Here we use an oncolytic adenovirus to deliver the prodrug-activating enzyme carboxypeptidase G2 (CPG2) to tumors in a single systemic administration. The adenovirus replicated and produced high levels of CPG2 in two different hepatocellular carcinoma xenografts (Hep3B and HepG2) but not other tissues. GDEPT enhanced the adenovirus-alone therapy to elicit tumor regressions in the hepatocellular carcinoma models. This is the first time that CPG2 has been targeted and expressed intracellularly to effect significant therapy, showing that the combined approach holds enormous potential as a tumor-selective therapy for the systemic treatment of hepatocellular carcinoma.     

Appropriate subcellular localisation of prodrug-activating enzymes has important consequences for suicide gene therapy

Escherichia coli B nitroreductase (NR) has been expressed stably in MDA-MB-361 human breast adenocarcinoma cells either as the wild-type protein (wtNR), which is distributed evenly between the cytoplasmic and nuclear compartments, or targeted to the mitochondrion (mtNR). Whereas bacterial NR is active as a dimer, a proportion of wtNR is monomeric. In contrast, mtNR is mostly dimeric, suggesting that it adopts a more stable, native conformation. Despite this, when tested in gene-directed enzyme prodrug therapy cell cytotoxicity studies, cells expressing wtNR or mtNR had similar sensitivity to the prodrug CB1954 and mounted similar bystander killing effects. Furthermore, when short prodrug exposures were given, wtNR was more efficient at killing cells than mtNR. These data demonstrate that the site of enzyme expression and prodrug activation is an important variable that requires consideration in suicide gene therapy approaches.     

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10 mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10 mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis.     

Local bystander effect induces dormancy in human medullary thyroid carcinoma model in vivo

The extent of local bystander effect induced by fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD) in combination with 5-fluorocytosine (5FC) was evaluated in xenogeneic model of human medullary thyroid carcinoma (MTC). This approach to gene-directed enzyme/prodrug therapy (GDEPT) induces strong bystander cytotoxicity. Effector yCD-TT mixed with target EGFP-TT cells in a ratio 2:9 could achieve significant tumor regression and 14-fold decrease in serum marker calcitonin upon 5FC administration. Histopathological analysis unraveled that antitumor effect resulted in tumor dormancy and proliferation arrest of remaining tumor cell clusters in vivo. yCD/5FC combination represents another GDEPT approach to achieve tumor growth control in MTC.     

Gene therapy for brain tumors

‘Gene therapy’ can be defined as the transfer of genetic material into a patient’s cells for therapeutic purposes. To date, a diverse and creative assortment of treatment strategies utilizing gene therapy have been devised, including gene transfer for modulating the immune system, enzyme prodrug (‘suicide gene’) therapy, oncolytic therapy, replacement/ therapeutic gene transfer, and antisense therapy. For malignant glioma, gene-directed prodrug therapy using the herpes simplex virus thymidine kinase gene was the first gene therapy attempted clinically. A variety of different strategies have now been pursued experimentally and in clinical trials. Although, to date, gene therapy for brain tumors has been found to be reasonably safe, concerns still exist regarding issues related to viral delivery, transduction efficiency, potential pathologic response of the brain, and treatment efficacy. Improved viral vectors are being sought, and potential use of gene therapy in combination with other treatments is being investigated.     

硝基还原酶/CB1954自杀基因系统对宫颈癌Hela细胞杀伤效应的实验研究

目的:体外观察大肠杆菌硝基还原酶/[5-(1-氮丙啶)-2,4-二硝基苯甲酰胺](以下简称NTR/CB1954)自杀基因系统对宫颈癌Hela细胞的杀伤效应,探索一种新的宫颈癌基因治疗方法.方法: 利用PCR技术从Escherichia coli K12的基因组中扩增出编码NTR的基因nfsB,酶切后,连接到真核表达载体pcDNA3上,获得重组载体pcDNA3-nfsB,lipofectamineTM2000脂质体转染法将pcDNA3-nfsB转染Hela细胞,筛选稳定表达细胞株,应用RT-PCR以及SDS-PAGE检测NTR在Hela细胞中的表达,MTT法检测NTR/CB1954对Hela细胞活力的影响,流式细胞术检测亚二倍体细胞率改变,PI/Hoechest33258双染荧光显微镜下观察Hela细胞凋亡率.结果: 成功构建了真核表达载体pcDNA3-nfsB,获得稳定表达NTR的Hela细胞株,在mRNA水平以及蛋白水平检测到NTR在Hela细胞中的表达,NTR/CB1954自杀基因系统明显影响Hela细胞的活力,增加了Hela细胞的凋亡率.结论: NTR/CB1954自杀基因系统对Hela细胞在体外通过凋亡产生明显的杀伤效应.     

Effects of hypoxia and limited diffusion in tumor cell microenvironment on bystander effect of P450 prodrug therapy

Cytochrome P450 (CYP) enzyme 2B1 metabolizes the anticancer prodrug cyclophosphamide (CPA) to 4-hydroxy-CPA, which decomposes to the cytotoxic metabolites acrolein and phosphoramide mustard. We have evaluated the bystander cytotoxicity of CPA in combination with CYP2B1 gene-directed enzyme prodrug therapy using a cell culture-based agarose overlay technique. This method mimics the tumor microenvironment by limiting the diffusion of metabolites and by reducing the oxygen concentration to levels similar to those found in solid tumors. Under these conditions, the CYP activity of CYP2B1-expressing tumor cells was decreased by 80% compared to standard aerobic conditions. Despite this decrease in metabolic activity, a potent bystander effect was observed, resulting in up to 90% killing by CPA of a tumor cell population comprised of only approximately 20% CYP-expressing tumor cells. Similarly, transient transfection of a small fraction ( approximately 14%) of a human hepatoma Huh7 cell population with a CYP2B1 expression plasmid followed by short-term treatment with CPA (5 h) led to an eradication of 95% of the cells. No such bystander effect was observed without the agarose overlay. These findings suggest that the agarose overlay technique is very useful as an in vitro test system for investigation of the bystander effect of CYP/CPA and other enzyme/prodrug combinations under conditions that mimic the hypoxic conditions present in solid tumors in vivo.     

The potential of acetaminophen as a prodrug in gene-directed enzyme prodrug therapy

Acetaminophen is oxidized by human CYP1A2 to the cytotoxic metabolite N-acetylbenzoquinoneimine (NABQI). Incubation of cells transfected with human CYP1A2 (H1A2 MZ cells) with 4-20 mM acetaminophen for 6 hours at 37 degrees C caused extensive cytotoxicity (cell viability <10%). In contrast, nontransfected V79 MZ cells were unaffected (viability >95%). By mixing H1A2 MZ cells with V79 MZ cells in various proportions and incubating with 4 mM acetaminophen, it was shown that the NABQI released from H1A2 MZ cells also caused cytotoxicity of bystander cells. Thus, in a mixture containing 5% H1A2 MZ cells, exposure to 4 mM acetaminophen for 6 hours resulted in complete cell killing by 24 hours. A similar bystander effect was found by incubating the same proportion of CYP1A2-containing cells with ovarian tumor-derived SK-OV-3 cells or colon tumor-derived HCT116 cells. However, breast tumor-derived MDA-MB-361 cells displayed resistance to the cytotoxic effect of NABQI, and it was necessary to increase the proportion of H1A2 MZ cells to 50% to achieve complete cell killing. In conclusion, the use of acetaminophen as prodrug and CYP1A2 as an activating enzyme is a promising combination for gene-directed enzyme prodrug therapy.     

Bystander cytotoxicity in human medullary thyroid carcinoma cells mediated by fusion yeast cytosine deaminase and 5-fluorocytosine

In our work, we have evaluated efficiency of gene-directed enzyme/prodrug therapy (GDEPT) based on combination of fusion yeast cytosine deaminase (yCD) and 5-fluorocytosine (5FC) on model human medullary thyroid carcinoma (MTC) cell line TT. We determined the efficiency of this GDEPT approach in suicide and bystander cytotoxicity induction. We have shown significant bystander effect in vitro and 5FC administration resulted in potent antitumor effect in vivo. Furthermore, we have unraveled high efficiency of cell-mediated GDEPT, when human mesenchymal stromal cells (MSC) were used as delivery vehicles in direct cocultures in vitro. Nevertheless, effector MSC exhibited inhibitory effect on TT cell proliferation and abrogated TT xenotransplant growth in vivo. We suggest that yCD/5FC combination represents another experimental treatment modality to be tested in MTC and our data further support the exploration of MSC antitumor potential for future use in metastatic MTC therapy.     

Harnessing apoptosis for improved anticancer gene therapy

Advances in our understanding of the mechanisms by which tumor cells detect drug-induced DNA damage leading to apoptotic death have aided in the design of novel, potentially more selective strategies for cancer treatment. Several of these strategies use proapoptotic factors and have shown promise in sensitizing tumor cells to the cytotoxic actions of traditional cancer chemotherapeutic drugs. Although antiapoptotic factors are generally regarded as poor prognostic factors for successful cancer chemotherapy, strategies that use antiapoptotic factors in combination with suicide or other gene therapies can also be considered. The introduction of antiapoptotic factors that act downstream of drug-induced mitochondrial transition delays, but does not block, the ultimate cytotoxic response to cancer chemotherapeutic drugs that activate a mitochondrial pathway of cell death. Recent studies using the cytochrome P-450 prodrug cyclophosphamide exemplify how the antiapoptotic, caspase-inhibitory baculovirus protein p35 can be combined with P-450 gene-directed enzyme prodrug therapy to prolong localized, intratumoral production of cytotoxic drug metabolites without inducing tumor cell drug resistance. This model may be adapted to other gene therapies, including those that target death receptor pathways, to maximize the production of soluble, bystander cytotoxic factors and prodrug metabolites and thereby amplify the therapeutic response.     

Enhanced bystander cytotoxicity of P450 gene-directed enzyme prodrug therapy by expression of the antiapoptotic factor p35

Cytochrome P450 gene-directed enzyme prodrug therapy substantially augments intratumoral activation of anticancer prodrugs, such as cyclophosphamide (CPA), leading to a strong increase in antitumor effect without a corresponding increase in host toxicity. Attempts to additionally increase tumor cell kill by enhancing the intrinsic chemosensitivity of P450-expressing tumor cells by chemical means (depletion of cellular glutathione) or by coexpression of proapoptotic factors was shown to result in the desired increase in chemosensitivity, but with a decrease in net production of bystander cytotoxic drug metabolites because of accelerated death of the prodrug-activating tumor cells. Moreover, tumor cell P450 activity declined during the course of apoptosis induced by P450-activated CPA, limiting the potential of the tumor cell for continued production of activated drug metabolites. This limitation could be overcome by retroviral delivery of the baculovirus-encoded caspase inhibitor p35 to P450-expressing tumor cells. p35 substantially prolonged the activation of CPA by P450 "factory cells," leading to an increase in their bystander cytotoxicity toward P450-deficient tumor cells. This effect was greatest in tumor cells treated with CPA for an 8-h period, a schedule designed to model the effective time period of drug exposure in bolus CPA-treated patients in vivo. Notably, retroviral transduction of tumor cells with p35 did not induce drug resistance, as shown by the absence of long-term tumor cell survival or detectable colony formation activity after CPA treatment. These findings demonstrate that antiapoptotic factors, such as p35, can be used in a novel manner to enhance prodrug activation gene therapy by delaying tumor cell death, thereby increasing the net production of bystander cytotoxic metabolites and, hence, the overall effectiveness of the anticancer strategy.