Evaluation of cytotoxicity and radiation enhancement using 1.9 nm gold particles: potential application for cancer therapy.

High atomic number (Z) materials such as gold preferentially absorb kilovoltage x-rays compared to soft tissue and may be used to achieve local dose enhancement in tumours during treatment with ionizing radiation. Gold nanoparticles have been demonstrated as radiation dose enhancing agents in vivo and in vitro. In the present study, we used multiple endpoints to characterize the cellular cytotoxic response of a range of cell lines to 1.9 nm gold particles and measured dose modifying effects following transient exposure at low concentrations. Gold nanoparticles caused significant levels of cell type specific cytotoxicity, apoptosis and increased oxidative stress. When used as dose modifying agents, dose enhancement factors varied between the cell lines investigated with the highest enhancement being 1.9 in AGO-1522B cells at a nanoparticle concentration of 100 microg ml(-1). This study shows exposure to 1.9 nm gold particles to induce a range of cell line specific responses including decreased clonogenic survival, increased apoptosis and induction of DNA damage which may be mediated through the production of reactive oxygen species. This is the first study involving 1.9 nm nanometre sized particles to report multiple cellular responses which impact on the radiation dose modifying effect. The findings highlight the need for extensive characterization of responses to gold nanoparticles when assessing dose enhancing potential in cancer therapy.

Site-specific polysialylation of an antitumor single-chain Fv fragment.

Protein pharmacokinetic modulation is becoming an important tool in the development of biotherapeutics. Proteins can be chemically or recombinantly modified to alter their half-lives and bioavailability to suit particular applications as well as improve side effect profiles. The most successful and clinically used approach to date is chemical conjugation with poly(ethylene glycol) polymers (PEGylation). Here, therapeutic protein half-life can be increased significantly while retaining biological function, reducing immunogenicity and cross-reaction. Naturally occurring alternatives to such synthetic polymers could have major advantages such as lower side effects due to biodegradability and metabolism. Polysialic acid (PSA) has been investigated as a pharmacokinetic modulatory biopolymer with many successful examples in preclinical and clinical development. Single-chain Fvs (scFvs) are a choice antibody format for human therapeutic antibody discovery. Because of their small size, they are rapidly eliminated from the circulation and often are rebuilt into larger proteins for drug development and a longer half-life. Here we show that chemical polysialylation can increase the half-life of an antiplacental alkaline (PLAP) and anticarcinoembryonic antigen (CEA) scFv (F1 and MFE-23, respectively) 3.4-4.9-fold, resulting in a 10.6-15.2-fold increase in blood exposure. Amine-directed coupling of the MFE-23 scFv reduced its immunoreactivity 20-fold which was resolved by site-specific polysialylation through an engineered C-terminal thiol residue. The site-specifically polysialylated MFE-23 scFv demonstrated up to 30-fold improved tumor uptake while displaying favorable tumor:normal tissue specificity. This suggests that engineering antibody fragments for site-specific polysialylation could be a useful approach to increase the half-life for a variety of therapeutic applications.

Fragmentation and plasmid strand breaks in pure and gold-doped DNA irradiated by beams of fast hydrogen atoms.

The results of an investigation into the damage caused to dry plasmid DNA after irradiation by fast (keV) hydrogen atoms are presented. Agarose gel electrophoresis was used to assess single and double strand break yields as a function of dose in dry DNA samples deposited on a mica substrate. Damage levels were observed to increase with beam energy. Strand break yields demonstrated a considerable dependence on sample structure and the method of sample preparation. Additionally, the effect of high-Z nanoparticles on damage levels was investigated by irradiating DNA samples containing controlled amounts of gold nanoparticles. In contrast to previous (photonic) studies, no enhancement of strand break yields was observed with the particles showing a slight radioprotective effect. A model of DNA damage as a function of dose has been constructed in terms of the probability for the creation of single and double strand breaks, per unit ion flux. This model provides quantitative conclusions about the effects of both gold nanoparticles and the different buffers used in performing the assays and, in addition, infers the proportion of multiply damaged fragments.

The radiation-inducible pE9 promoter driving inducible nitric oxide synthase radiosensitizes hypoxic tumour cells to radiation.

Driving high-level transgene expression in a tumour-specific manner remains a key requirement in the development of cancer gene therapy. We have previously demonstrated the strong anticancer effects of generating abnormally high levels of intracellular NO(*) following the overexpression of the inducible nitric oxide synthase (iNOS) gene. Much of this work has focused on utilizing exogenously activated promoters, which have been primarily induced using X-ray radiation. Here we further examine the potential of the pE9 promoter, comprising a combination of nine CArG radio-responsive elements, to drive the iNOS transgene. Effects of X-ray irradiation on promoter activity were compared in vitro under normoxic conditions and various degrees of hypoxia. The pE9 promoter generated high-level transgene expression, comparable with that achieved using the constitutively driven cytomegalovirus promoter. Furthermore, the radio-resistance of radiation-induced fibrosarcoma-1 (RIF-1) mouse sarcoma cells exposed to 0.1 and 0.01% O(2) was effectively eliminated following transfection with the pE9/iNOS construct. Significant inhibition of tumour growth was also observed in vivo following direct intratumoural injection of the pE9/iNOS construct compared to empty vector alone (P<0.001) or to a single radiation dose of 10 Gy (P<0.01). The combination of both therapies resulted in a significant 4.25 day growth delay compared to the gene therapy treatment alone (P<0.001). In summary, we have demonstrated the potential of the pE9/iNOS construct for reducing radio-resistance conferred by tumour cell hypoxia in vitro and in vivo, with greater tumour growth delay observed following the treatment with the gene therapy construct as compared with radiotherapy alone.

The tissue plasminogen activator gene promoter: a novel tool for radiogenic gene therapy of the prostate?

BACKGROUND: Radiation therapy is a treatment modality routinely used in cancer management so it is not unexpected that radiation-inducible promoters have emerged as an attractive tool for controlled gene therapy. The human tissue plasminogen activator gene promoter (t-PA) has been proposed as a candidate for radiogenic gene therapy, but has not been exploited to date. The purpose of this study was to evaluate the potential of this promoter to drive the expression of a reporter gene, the green fluorescent protein (GFP), in response to radiation exposure. METHODS: To investigate whether the promoter could be used for prostate cancer gene therapy, we initially transfected normal and malignant prostate cells. We then transfected HMEC-1 endothelial cells and ex vivo rat tail artery and monitored GFP levels using Western blotting following the delivery of single doses of ionizing radiation (2, 4, 6 Gy) to test whether the promoter could be used for vascular targeted gene therapy. RESULTS: The t-PA promoter induced GFP expression up to 6-fold in all cell types tested in response to radiation doses within the clinical range. CONCLUSIONS: These results suggest that the t-PA promoter may be incorporated into gene therapy strategies driving therapeutic transgenes in conjunction with radiation therapy.

Variation of strand break yield for plasmid DNA irradiated with high-Z metal nanoparticles.

Using agarose gel electrophoresis, we measured the effectiveness of high-Z metal particles of different sizes on SSB and DSB yields for plasmid DNA irradiated with 160 kVp X rays. For plasmid samples prepared in Tris-EDTA buffer, gold nanoparticles were shown to increase G'(SSB) typically by a factor of greater than 2 while G'(DSB) increased by a factor of less than 2. Similar dose-modifying effects were also observed using gold microspheres. Addition of 10(-1) M DMSO typically decreased damage yields by a factor of less than 0.5. Plasmid samples prepared in PBS showed significantly different damage yields compared to those prepared in Tris-EDTA (P < 0.001) with G'(SSB) and G'(DSB) increasing by factors of 100 and 48, respectively. Furthermore, addition of gold nanoparticles to samples prepared in PBS decreased G'(SSB) and G'(DSB) by factors of 0.2 and 0.3, respectively. The results show plasmid damage yields to be highly dependent on differences in particle size between the micro- and nanometer scale, atomic number (Z) of the particle, and scavenging capacity of preparation buffers. This study provides further evidence using a plasmid DNA model system for the potential of high-Z metal nanoparticles as local dose-modifying agents.

Nitric oxide--a novel therapeutic for cancer.

Much research over the past two decades has focussed on understanding the complex interactions of nitric oxide (NO(.)) in both physiological and pathological processes. As with many other aspects of NO(.) biology, its precise role in tumour pathophysiology has been the cause of intense debate and we now know that it participates in numerous signalling pathways that are crucial to the malignant character of cancer. The available experimental evidence highlights contrasting pro- and anti-tumour effects of NO(.) expression, which appear to be reconciled by consideration of the concentrations involved. This review addresses the complexities of the role of NO(.) in cancer, whilst evaluating various experimental approaches to NO(.)-based cancer therapies, including both inhibition of nitric oxide synthases, and overexpression of NO(.) using donor drugs or nitric oxide synthase gene transfer. The evidence provided strongly supports a role for manipulation of tumour NO(.) either as a stand-alone therapy or in combination with conventional treatments to achieve a significant therapeutic gain.

Tumor-selective drug activation: a GDEPT approach utilizing cytochrome P450 1A1 and AQ4N.

Drug metabolizing transgene products, which activate bioreductive cytotoxins, can be used to target treatment-resistant hypoxic tumors. The prodrug AQ4N is bioreduced in hypoxic cells by cytochrome P450s (CYPs) to the cytotoxin AQ4. Previously we have shown that intra-tumoral injection of CYP3A4 and CYP2B6 transgenes with AQ4N and radiation inhibits tumor growth. Here we examine the ability of other CYPs, in particular CYP1A1, to metabolize AQ4N, and to enhance radiosensitization. Metabolism of AQ4N was assessed using microsomes prepared from baculovirus-infected cells transfected with various CYP isoforms. AQ4N metabolism was most efficient with CYP1A1 (66.7 nmol/min/pmol) and 2B6 (34.4 nmol/min/pmol). Transient transfection of human CYP1A1+/-CYP reductase (CYPRED) was investigated in hypoxic RIF-1 mouse cells in vitro using the alkaline comet assay. There was a significant increase in DNA damage following transient transfection of CYP1A1 compared to non-transfected cells; inclusion of CYPRED provided no additional effect. In vivo, a single intra-tumoral injection of a CYP1A1 construct in combination with AQ4N (100 mg/kg i.p.) and 20 Gy X-rays caused a 16-day delay in tumor regrowth compared to tumors receiving AQ4N plus radiation and empty vector (P=0.0344). The results show the efficacy of a CYP1A1-mediated GDEPT strategy for bioreduction of AQ4N.

Effect of partition coefficient on the ability of nitroimidazoles to enhance the cytotoxicity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

The ability of three nitroimidazoles [SR-2508, misonidazole (MISO), and benznidazole] with differing octanol-water partition coefficients to enhance the cytotoxicity of the nitrosourea 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was evaluated in two mouse tumors (the KHT sarcoma and the SCC VII/St carcinoma). These results were compared with the effect on two normal tissues (bone marrow CFU-S and testis spermatogonia). When given as a large single dose, benznidazole was more effective than MISO in enhancing the cytotoxicity of CCNU to both tumors. SR-2508 had no effect. The advantage of benznidazole over MISO was lost, however, because benznidazole gave more toxicity in the normal tissues than MISO. In experiments where the nitroimidazoles were administered by multiple small injections to maintain a blood plasma level between 50 and 100 micrograms/ml, benznidazole was also more effective than MISO in enhancing CCNU cytotoxicity in the tumors. In each case, enhancement was rather less than that obtained with large single injections. Again, however, benznidazole did not produce a consistently greater therapeutic gain than MISO because it also enhanced normal tissue toxicity while MISO did not. SR-2508 was ineffective in both tumors and normal tissues. We conclude that neither SR-2508 nor benznidazole are superior to MISO in combination with CCNU.

Radiogenic therapy: novel approaches for enhancing tumor radiosensitivity.

Radiotherapy (RT) is a well established modality for treating many forms of cancer. However, despite many improvements in treatment planning and delivery, the total radiation dose is often too low for tumor cure, because of the risk of normal tissue damage. Gene therapy provides a new adjunctive strategy to enhance the effectiveness of RT, offering the potential for preferential killing of cancer cells and sparing of normal tissues. This specificity can be achieved at several levels including restricted vector delivery, transcriptional targeting and specificity of the transgene product. This review will focus on those gene therapy strategies that are currently being evaluated in combination with RT, including the use of radiation sensitive promoters to control the timing and location of gene expression specifically within tumors. Therapeutic transgenes chosen for their radiosensitizing properties will also be reviewed, these include: gene correction therapy, in which normal copies of genes responsible for radiation-induced apoptosis are transfected to compensate for the deletions or mutated variants in tumor cells (p53 is the most widely studied example). enzymes that synergize the radiation effect, by generation of a toxic species from endogenous precursors (e.g., inducible nitric oxide synthase) or by activation of non toxic prodrugs to toxic species (e.g., herpes simplex virus thymidine kinase/ganciclovir) within the target tissue. conditionally replicating oncolytic adenoviruses that synergize the radiation effect. membrane transport proteins (e.g., sodium iodide symporter) to facilitate uptake of cytotoxic radionuclides. The evidence indicates that many of these approaches are successful for augmenting radiation induced tumor cell killing with clinical trials currently underway.

Oxygen delivery to tumors.

Sensitization of hypoxic cells in tumors, by increasing their oxygen supply, has been attempted for at least 30 years. Only the use of hyperbaric oxygen has been shown unequivocally as a beneficial adjunct to radiotherapy; and even then, the number of sites sensitized is limited to head and neck and cervix. It is not clear whether this implies that all other tumors reoxygenate fully during treatment, or whether a better method would sensitize other sites. Nevertheless, the elimination of hypoxic cells is viewed by many as a worthy goal in radiobiology and many strategies have been tested in animal systems. These include: oxygen releasing chemicals, artificial oxygen carriers, inhibitors of oxygen consumption, blood flow modifiers, or the exploitation of tumor adaptation to altered oxygen availability. We must be aware that any procedure which improves tumor oxygenation will not only increase radiosensitivity, but will induce an adaptive response in the tumor such that, sensitization will be of limited duration. It is likely that in the apparent failure of measures to improve substantially the oxygen delivery to tumors, the elimination of most of the hypoxic cells, of the type accessible to them, may have been achieved. If, as has been suggested, there are two distinct types of hypoxic cells, a combination of more than one strategy may be necessary to achieve more substantial gains.

Anemia: a problem or an opportunity in radiotherapy?

Anemia may often become a problem in the treatment of the cancer patient. There are insufficient clinical data to assess the overall importance of anemia in radiotherapy, but there is clear evidence that uncorrected anemia is detrimental to local tumor control in some sites. There may be situations, however, when the transfused, previously anemic patient is at an advantage. These patients have shown a dramatically better response than non-anemic patients when radiotherapy for cancer of the cervix was given in hyperbaric oxygen. Animal experiments suggest that adaptive processes may be responsible for this effect. There is an important difference between acute and chronic anemia in their influence on the radiosensitivity of mouse tumors; while acute anemia consistently causes radioresistance, this effect is lost as the duration of the anemia prior to irradiation is prolonged. This would suggest that anemia per se should not cause tumor radioresistance in the chronically anemic patient. Blood transfusion in previously anemic animals has been shown to produce a markedly increased tumor radiosensitivity, but again this is only transient and sensitivity returns to normal when the interval between transfusion and irradiation is extended to 24 hrs. The mechanisms responsible for tumor adaptation to anemia and blood transfusion are not known, but there is evidence that changes in diffusion distances occur within tumors in response to alterations in oxygen availability and that changes in blood chemistry through the 2,3-DPG system may alter the release of oxygen to the tissues. These are complex processes and it remains to be determined what influence they have in the treatment of human cancer. However, the animal data suggest a clear benefit of blood transfusion to restore the hemoglobin level in radiotherapy, but they also emphasize the need to irradiate immediately so that adaptive mechanisms cannot erode the effect.

Calcium antagonists as radiation modifiers: site specificity in relation to tumor response.

The calcium antagonists, verapamil, nifedipine, and flunarizine, were studied for their effects on blood flow and radiation response in SCCVII/St intradermal back tumors, over a dose range of 0.05-50 mg/kg. Verapamil, at low doses, increased tumor blood flow, as measured by relative fluorescence intensity of Ho33342 stain, and increased tumor radiosensitivity. However, at doses of 20 mg/kg and above, verapamil reduced Ho33342 fluorescence intensity, and increased tumor radioresistance. Nifedipine reduced tumor radiosensitivity and Ho33342 fluroscence intensity at doses above 1 mg/kg, but below this dose increased Ho33342 intensity was observed and a small radiosensitization was apparent. Flunarizine sensitized tumors to X rays at all doses tested, although increased Ho33342 intensity was seen only at 5 mg/kg. The relative affinities of these compounds for different sites within the host may explain the variations in blood flow and radiation sensitivity in this tumor system.

Chlorophenoxy acetic acid derivatives as hemoglobin modifiers and tumor radiosensitizers.

We have studied the influence of the antilipidemia drug, clofibrate, and several structurally related analogs on the binding affinity of hemoglobin for oxygen and the radiation sensitivity of the SCCVII/St carcinoma in the mouse. Several compounds in this class reduced hemoglobin affinity in vivo; and two of these, ML1024 (etophyline clofibrate) and ML1037, were at least as effective as clofibrate at reducing hemoglobin affinity and much less toxic. When given orally at a dose of 4.1 m mole/Kg, 1/2-2 hrs before 20 Gy X rays, clofibrate gave radiosensitization in the SCCVII/St tumor equivalent to a 40-fold reduction in hypoxic fraction. ML1024 and ML1037 at a dose (3.0 m mole/Kg), which had a similar effect on hemoglobin, gave much less sensitization of the tumor. Only ML1024 produced a statistically significant effect, equivalent to a four-fold reduction in hypoxic fraction. We conclude that there are several clofibrate analogs which in relation to their toxicity are much better hemoglobin modifiers than the parent compound. They do not, however, show the same radiosensitizing effects, leading us to believe that mechanisms other than changes in hemoglobin/oxygen binding must also be involved.

The therapeutic potential of misonidazole enhancement of alkylating agent cytotoxicity.

The effect of prolonged exposure to low misonidazole (MISO) levels on the cytotoxicity of three alkylating agents was studied in mouse tumors. A concentration of 100 micrograms/ml was maintained in the plasma for 7 hr by multiple injections of MISO. Cyclophosphamide (CYC). Melphalan (L-PAM), or CCNU were given after 4 hrs of MISO exposure. In each case, prolonged low level MISO exposure enhanced tumor response as measured by regrowth delay or a cloning assay. The effect of this treatment was also studied in several normal tissues: bone marrow, white blood cell counts, and spermatogonia. In none of these was any enhancement seen after prolonged MISO exposure. These encouraging results show that clinically relevant exposures to MISO can greatly improve tumor response to alkylating agents without increased normal tissue toxicity.

Changes in misonidazole binding with hypoxic fraction in mouse tumors.

Binding of misonidazole (MISO) or a derivative to hypoxic cells in tumors has been proposed as a method for identifying tumors and measuring their level of hypoxia. We have recently shown that the hypoxic fraction of tumor cells can be altered over a wide range in vivo by acutely changing the hematocrit of the host animal by transfusion. The present study aimed to investigate the changes in binding by 14C MISO that accompanied this procedure. Tumor bearing mice were injected with 14C MISO, irradiated with a single dose of X rays (20 Gy) and their tumor excised and bisected. One half of each tumor was used to determine cell survival in vitro, the other was used for 14C scintillation counting. As previously described, tumor cell survival was dramatically increased in acutely anemic mice and this was accompanied by an increase in 14C MISO binding to the tumors. The relationship between clonogenic cell survival and binding was found to be linear on a log-log plot for each of the tumor lines studied, but the slopes of the lines were different tumor lines and generally steeper than the value of 1.0 expected for a 1:1 correspondence between cells binding radioactivity and radiobiological resistance. We attribute these differences to MISO binding to cells in the tumor which were not clonogenic.

The effects of purine nucleoside analogs on the response of the RIF-1 tumor to melphalan in vivo.

The effect of several purine nucleoside analogs on the cytotoxicity of melphalan (L-PAM) in the RIF-1 tumor in vivo was investigated. All the analogs tested--3'-deoxyguanosine (3'-dG), 3'-deoxyadenosine (3'-dA), and N6-butyryl-3'-deoxyadenosine (N6-BC)--at a dose of 100 mg/kg, enhanced the tumor cell killing by L-PAM as measured by an in vivo/in vitro procedure. This enhancement was maximal when the drugs were given simultaneously. The mean enhancement ratios (ER's) determined from the L-PAM dose response curves were 1.4 for 3'-dG, 1.3 for 3'-dA, and 1.4 for N6-BC. A similar modification of the L-PAM-induced depression of white blood cell counts was not obtained. A large single dose of L-PAM (8 mg/kg) produced a transient drop in mouse body temperature. The analog 3'-dG (100 mg/kg) increased and prolonged this hypothermic effect. In addition 3'-dG also delayed the clearance of L-PAM from the plasma of C3H mice, such that the half-life of the chemotherapeutic agent was extended from 28 minutes to 35 minutes. The enhancement of the efficacy of L-PAM by these analogs probably results from this pharmacokinetic effect.

Enhancement of melphalan cytotoxicity in vivo and in vitro by inhibitors of poly (ADP-ribose) polymerase.

In these preliminary experiments, we have found enhanced cell killing by the bifunctional alkylating agent L-phenylalanine mustard (L-PAM) in the presence of inhibitors of poly (ADP-ribose) polymerase (ADPRP) in vitro. In vivo enhancement of the tumoricidal effects of L-PAM was observed with the ADPRP inhibitor nicotinamide (1000 mg/kg), although enhanced myelosuppression was also demonstrated. Nicotinamide also increased the plasma elimination half-life of L-PAM by a factor of at least 2. This alteration of L-PAm pharmacokinetics makes it difficult to assess the role that ADPRP inhibition plays in the enhancement of L-PAM tumor cell killing in vivo.

The effect of timing on chemosensitization by clinical levels of SR-2508.

The nitroimidazole SR-2508 is currently being tested clinically as a radiosensitizer. Its relatively low toxicity allows it to be used at higher doses than misonidazole so that its potential as a chemosensitizer is also of considerable interest. Multiple injections of SR-2508 were given to SCC VII/St tumor-bearing mice to achieve a clinically realistic plasma concentration of approximately 300 micrograms/ml over 8 hrs. Single doses of melphalan (L-PAM) or cyclophosphamide (CY) were given at different times after the first SR 2508 injection. With L-PAM, a delay of at least 2 hr was necessary before enhancement of L-PAM cytotoxicity was observed. A similar result was obtained when a simulation was carried out with SCC VII/St tumor cells in vitro. Results with CY were less clear, although the most consistent enhancement was observed when a 4 to 8 hr interval elapsed between the beginning of SR 2508 exposure and the CY injection. In general, although precise timing was not essential for enhancement, an interval of at least 4 hr is recommended between the administration of SR 2508 and either alkylating agent. This is particularly important for L-PAM where no enhancement would be expected if the drugs were given simultaneously.

Modification of alkylating agent cytotoxicity by cisplatin.

The effect of cisplatin on the cytotoxicity of alkylating agents in the RIF-1 tumor both in vivo and in vitro was investigated. A single dose of cisplatin (1 mg/kg) enhanced the in vivo tumor killing by melphalan (L-PAM; 8 mg/kg). The effect was maximal when cisplatin was given between 3 hours before and 1 hour after injecting L-PAM; the enhancement was lost as the time interval increased. Similar results were obtained with cyclophosphamide (CYT; 75 mg/kg) and CCNU (50 mg/kg) although the enhancement was much less than that seen with L-PAM. The enhancement by cisplatin was constant at all L-PAM doses as measured by both cloning assay and regrowth delay. Measurements of white blood cell counts four days after drug administration suggests that a therapeutic gain can be achieved. Exponential and plateau phase RIF-1 cells grown in monolayer culture were also exposed to various L-PAM doses. A 1 hour pre-exposure to cisplatin (0.5 micrograms/ml) under aerobic conditions increased the cell killing by L-PAM. The results are discussed with reference to the possible reasons for the therapeutic gain.