Adam V. Patterson

Novel chimeric gene promoters responsive to hypoxia and ionizing radiation.

Despite being an adverse prognostic factor in radiotherapy, hypoxia represents a physiological difference that can be exploited for selective cancer gene therapy. In this study gene therapy vectors responsive to both hypoxia and ionizing radiation (IR) were developed. Gene expression was regulated by novel, synthetic promoters containing hypoxia responsive elements (HREs) from the erythropoietin (Epo), the phosphoglycerate kinase 1 (PGK1) and the vascular endothelial growth factor (VEGF) genes, and IR-responsive CArG elements from the early growth response (Egr) 1 gene. All chimeric promoters could be activated by hypoxia and/or IR-treatment, and selectively control marker gene expression in human T24 bladder carcinoma and MCF-7 mammary carcinoma cells. Importantly, enhancers containing combinations of HREs and CArG elements were able to respond to both triggering treatments, with the Epo HRE/CArG combination proving to be the most responsive and robust. The Epo HRE/CArG enhancer could effectively control a suicide gene therapy strategy by selectively sensitizing hypoxic and/or irradiated cells expressing the enzyme horseradish peroxidase (HRP) to the prodrug indole-3-acetic acid (IAA). These data indicate that the use of such chimeric promoters may effectively regulate therapeutic gene expression within the tumor microenvironment in gene therapy strategies aimed at addressing the problem of hypoxia in radiotherapy.

Prodrugs in genetic chemoradiotherapy.

Improvements in the radiotherapeutic management of solid tumors through the concurrent use of gene therapy is a realistic possibility. Of the broad array of candidate genes that have been evaluated, those encoding prodrug-activating enzymes are particularly appealing since they directly complement ongoing clinical chemoradiation regimes. Gene-Directed Enzyme-Prodrug Therapy (GDEPT) only requires a fraction of the target cells to be genetically modified, providing that the resultant cytotoxic prodrug metabolites redistribute efficiently (the bystander effect). This transfer of cytotoxicity to neighboring non-targeted cancer cells is central to the success of any gene therapy strategy, irrespective of the therapeutic gene employed. In the context of genetic chemoradiotherapy, efficient prodrug metabolite diffusion will be a prerequisite for efficient radiosensitization. Some, but not all GDEPT approaches have been analysed in combination with radiotherapy. Examples of prodrugs of clinically established chemotherapeutic agents currently used in conjunction with radiotherapy include: 5-fluorocytosine (5FC), cyclophosphamide (CPA), irinotecan (CPT-11), gemcitabine (dFdC), capecitabine, mitomycin C (MMC) and AQ4N. Other GDEPT paradigms, such as ganciclovir (GCV) and Herpes Simplex thymidine kinase (HSV-tk), dinitrobenzamide (DNB) mustard or aziridinyl analogs and the E. coli nitroreductase (NTR), CMDA or ZP2767P with Pseudomonas aeruginosa carboxypeptidase G2 (CPG2), and indole-3-acetic acid (IAA) activated by horseradish peroxidase (HRP) have no clinically established chemotherapeutic counterpart. Each prodrug is discussed in this review in the context of GDEPT, with a particular attention to translational research and clinical utility in combination with radiotherapy.

The relative importance of NADPH: cytochrome c (P450) reductase for determining the sensitivity of human tumour cells to the indolequinone EO9 and related analogues lacking functionality at the C-2 and C-3 positions

Analogues of EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2[1H-indole-4-7-dione]prop-2-e n-1-ol) which lack functionality at either the C-2 or C-3 position were synthesised. The aim was to establish the importance of each group towards toxicity and to give an indication as to whether substitution at either position altered activation and toxicity after metabolism by cellular NADPH: cytochrome c (P450) reductase (P450R). MDA231 breast cancer cells were transfected with the cDNA for human P450R and stable clones were isolated. These high P450R-expressing clones were used to determine the aerobic and hypoxic toxicity of EO9 and the two analogues that lacked functionality at either C-2 or C-3. The results showed that P450R was strongly implicated in the bioactivation of EO9 and its analogues under both of these conditions. This data also showed that the C-3 functionality was primarily implicated in hypoxic toxicity.

Molecular Chemotherapy for Breast Cancer

Gene therapy for breast cancer initially involves local or systemic delivery. Local delivery may be intrapleural or via direct injection to lesions. However, systemic delivery remains the greatest challenge with targeting, although methods using antibodies or growth factor receptor ligands have been demonstrated in preclinical models. This review focuses on the next step of using tissue-specific promoters such as Muc-1, CEA, PSA, HER-2, Myc, L-plastin and secretory leukoproteinase inhibitor promoters. All of these have demonstrated differential upregulation in breast cancer and additional specificity may be obtained by using physiological stimuli that are more frequently expressed in cancers, such as glucose regulated promoters and hypoxia response elements or radiation inducible elements. Amongst the later are the EGR-1, p21 and tissue type plaminogen activator promoters.Potential therapy genes include the prodrug activation system 5-fluorocytosine and other analogues of antimetabolites, but all of these need gap junctions to transfer the phosphorylated metabolites. Other approaches involving more freely diffusible products include cyclophosphamide, ifosfamide and thymidine phosphorylase to activate 5-deoxy-5-fluoruridine to fluorouracil. The bystander effect is important both for cell killing and for immunological and antivascular effects. Breast cancer is one type of tumour where a major clinical research effort is underway using local delivery methods.For prodrug activation systems, the use of human enzymes is desirable to prevent an immunological response that would eventually eliminate cells producing the prodrug activation system. The use of alkylating agents has an advantage over antimetabolites in that they are cytotoxic to cycling and noncycling cells, and the cytotoxic products can diffuse across cell membranes without the need for gap junctions. They also have a much steeper dose response curve than anti-metabolites.

3-Substituted-5-aziridinyl-1-methylindole-4,7-diones as NQO1-directed antitumour agents: mechanism of activation and cytotoxicity in vitro

Indolequinone agents are a unique class of bioreductive cytotoxins that can function as dual substrates for both one- and two-electron reductases. This endows them with the potential to be either hypoxia-selective cytotoxins or NAD(P)H:quinone oxidoreductase 1 (NQO1)-directed prodrugs, respectively. We have studied the structure-activity relationships of four novel indolequinone analogues with regard to one- and/or two-electron activation. Single-electron metabolism was achieved by exposing the human carcinoma cell line T47D to each agent under hypoxic conditions, whilst concerted two-electron metabolism was assessed by stably expressing the cDNA for human NQO1 in a cloned cell line of T47D. The C-3 and C-5 positions of the indolequinone nucleus were modified to manipulate reactivity of the reduction products and the four prodrugs were identified as NQO1 substrates of varying specificity. Two of the four prodrugs, in which both C-3 and C-5 groups remained functional, proved to be NQO1-directed cytotoxins with selectivity ratios of 60- to 80-fold in the T47D (WT) versus the NQO1 overexpressing T47D cells. They also retained selectivity as hypoxic cytotoxins with oxic/hypoxic ratios of 20- to 22-fold. Replacement of the C-3 hydroxymethyl leaving group with an aldehyde group ablated all selectivity in air and hypoxia in both cell lines. Addition of a 2-methyl group on the C-5 aziridinyl group to introduce steric hinderance reduced but did not abolish NQO1-dependent metabolism. However, it enhanced single-electron metabolism-dependent DNA cross-linking in a manner that was independent of cytotoxicity. These data demonstrate that subtle structure-activity relationship exists for different cellular reductases and under certain circumstances distinct forms of DNA damage can arise, the cytotoxic consequences of which can vary. This study identifies a candidate indolequinone analogue for further development as a dual hypoxia and NQO1-directed prodrug.

Antiangiogenic, Bioreductive and Gene Therapy Approaches to the Treatment of Hypoxic Tumours

Quinone based bioreductive drugs have, potentially, a very versatile use in cancer chemotherapy. They can be activated by DT-diaphorase and hence can be used to target tumour types rich in this (O2)-independent reductase enzyme. Small molecular modifications can substantially reduce specificity for DT-diaphorase and under these circumstances the quinones become much less toxic in air but retain their potent cytotoxic effects under hypoxic conditions. Our understanding of the reductive (bio) chemistry of indolequinones, in particular, has subsequently allowed us to develop a platform technology where almost any therapeutic entity can potentially be delivered, selectively, to hypoxic tumours. Antiangiogenic approaches are currently receiving a substantial amount of attention and this review brings their development into context in view of the hypoxia dependence for neovascularization. Lastly, the use of bioreductive drugs when combined with hypoxia-mediated gene therapy is described. Such an approach provides a unique dual level of specificity for targeting hypoxic tumours and potentially can provide substantial therapeutic benefit.