Gold nanoparticles as novel agents for cancer therapy.

Gold nanoparticles are emerging as promising agents for cancer therapy and are being investigated as drug carriers, photothermal agents, contrast agents and radiosensitisers. This review introduces the field of nanotechnology with a focus on recent gold nanoparticle research which has led to early-phase clinical trials. In particular, the pre-clinical evidence for gold nanoparticles as sensitisers with ionising radiation in vitro and in vivo at kilovoltage and megavoltage energies is discussed.

Molecular biology: the key to personalised treatment in radiation oncology?

We know considerably more about what makes cells and tissues resistant or sensitive to radiation than we did 20 years ago. Novel techniques in molecular biology have made a major contribution to our understanding at the level of signalling pathways. Before the "New Biology" era, radioresponsiveness was defined in terms of physiological parameters designated as the five Rs. These are: repair, repopulation, reassortment, reoxygenation and radiosensitivity. Of these, only the role of hypoxia proved to be a robust predictive and prognostic marker, but radiotherapy regimens were nonetheless modified in terms of dose per fraction, fraction size and overall time, in ways that persist in clinical practice today. The first molecular techniques were applied to radiobiology about two decades ago and soon revealed the existence of genes/proteins that respond to and influence the cellular outcome of irradiation. The subsequent development of screening techniques using microarray technology has since revealed that a very large number of genes fall into this category. We can now obtain an adequately robust molecular signature, predicting for a radioresponsive phenotype using gene expression and proteomic approaches. In parallel with these developments, functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) can now detect specific biological molecules such as haemoglobin and glucose, so revealing a 3D map of tumour blood flow and metabolism. The key to personalised radiotherapy will be to extend this capability to the proteins of the molecular signature that determine radiosensitivity.

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.

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.

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.

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.

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.

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.

p21((WAF1))-mediated transcriptional targeting of inducible nitric oxide synthase gene therapy sensitizes tumours to fractionated radiotherapy.

Cancer gene therapy that utilizes toxic transgene products requires strict transcriptional targeting to prevent adverse normal tissue effects. We report on the use of a promoter derived from the cyclin dependent kinase inhibitor, p21((WAF1)), to control transgene expression. We demonstrate that this promoter is relatively silent in normal cells (L132, FSK, HMEC-1) compared to the almost constitutive expression obtained in tumour cells (DU145, LNCaP, HT29 and MCF-7) of varying p53 status, a characteristic that will be important in gene therapy protocols. In addition, we found that the p21((WAF1)) promoter could be further induced by both external beam radiation (up to eight-fold in DU145 cells), intracellular-concentrated radionuclides ([(211)At]MABG) (up to 3.5-fold in SK-N-BE(2c) cells) and hypoxia (up to four-fold in DU145 cells). We have previously achieved significant radiosensitization of tumour cells both in vitro and in vivo by using inducible nitric oxide synthase (iNOS) gene therapy to generate the potent radiosensitizer, nitric oxide (NO(.-)). Here, we report that a clinically relevant schedule of p21((WAF1))-driven iNOS gene therapy significantly sensitized both p53 wild-type RIF-1 tumours and p53 mutant HT29 tumours to fractionated radiotherapy. Our data highlight the utility of this p21((WAF1))/iNOS-targeted approach.

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.

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.

Potential use of the alkaline comet assay as a predictor of bladder tumour response to radiation.

Bladder tumours show a variable response to radiotherapy with only about 50% showing good local control; currently there is no test to predict outcome prior to treatment. We have used five bladder tumour cell lines (T24, UM-UC-3, TCC-SUP, RT112, HT1376) to investigate the potential of the alkaline comet assay (ACA) to predict radiosensitivity. Radiation-induced DNA damage and repair were compared to clonogenic survival. When the five cell lines were irradiated and initial DNA damage was plotted against cell survival, at all doses (0-6 Gy), a significant correlation was found (r2=0.9514). Following 4 Gy X-irradiation, all cell lines, except T24, showed a correlation between SF2 vs half-time for repair and SF2 vs residual damage at 5, 10, 20 and 30 min. The T24 cell line showed radioresistance at low doses (0-2 Gy) and radiosensitivity at higher doses (4-6 Gy) using both cell survival and ACA end points, explaining the lack of correlation observed for this cell line. These data indicate that initial DNA damage and residual damage can be used to predict for radiosensitivity. Our data suggest that predictive tests of radiosensitivity, appropriate to the clinical situation, may require the use of test doses in the clinical range.

Relationship between clonogenic radiosensitivity, radiation-induced apoptosis and DNA damage/repair in human colon cancer cells.

The intrinsic radiation sensitivity of normal and tumour tissue is a major determinant of the outcome of radiotherapy. There is currently no established test that can be used routinely to measure the radiosensitivity of the cells in an individual patient's cancer in a manner that can inform treatment planning. The purpose of this study was to evaluate, in four human colorectal adenocarcinoma cell lines, two possible end points as surrogate markers of radiation response--apoptosis and induction of DNA single-strand breaks--and to compare the results with those of a conventional clonogenic assay. Cell lines (SW707 SW480, SW48 and HT29) known to differ in radiosensitivity were exposed to single doses of X-rays ranging from 0.5 to 5 Gy and cell survival was measured using the clonogenic assay. Apoptosis was determined on the basis of morphology under fluorescent microscopy and DNA damage/repair was measured, as tail moment, using an adaptation of the alkaline comet assay. The relationship between surviving fraction at 2 Gy (SF2) and the percentage of apoptotic cells 24 h after the same dose was complex, but apoptosis accurately predicted the order of radiosensitivities as measured by SF2. Initial damage measured after 2 Gy using the alkaline comet assay gave a close correlation with SF2 (r2=0.95), whereas there was no correlation between initial DNA damage repair rate and SF2.

CT virtual intravascular endoscopy of abdominal aortic aneurysms treated with suprarenal endovascular stent grafting.

BACKGROUND: We investigated the clinical applications of virtual intravascular endoscopy (VIE) in patients with abdominal aortic aneurysms (AAA) treated by endovascular stent grafting with a suprarenal component. METHODS: Thirty-four patients with AAA undergoing endovascular stent grafting were included in the study (28 male, six female; mean age = 76 years). Helical computed tomography (CT) scanning was performed within 1 week after stent graft implantation. All patients received a Zenith/AAA endovascular graft with uncovered suprarenal struts 2.5 cm long placed around the level of the renal arteries. VIE images were created for each patient. The follow-up periods ranged from 3 to 18 months (mean = 8.3 +/- 3.7 months). RESULTS: Three of 34 celiac arteries, 22 of 34 superior mesenteric arteries, 32 of 34 right renal arteries, and 30 of 35 left renal arteries were affected by the suprarenal stent struts (wires) to different extents. VIE was able to demonstrate the struts, arterial ostia, and the strut/ostia configuration. Follow-up CT showed that all of these aortic branches were patent. CONCLUSION: Our preliminary experience has demonstrated that VIE is a novel imaging technique to visualize the three-dimensional intralumenal relationship of the aortic stent struts to the arterial ostia in patients with AAA after suprarenal stent graft placement.

Tumour cell radiosensitization using constitutive (CMV) and radiation inducible (WAF1) promoters to drive the iNOS gene: a novel suicide gene therapy.

Nitric oxide (NO(*)) has many characteristics including cytotoxicity, radiosensitization and anti-angiogenesis, which make it an attractive molecule for use in cancer therapy. We have investigated the use of iNOS gene transfer, driven by both a constitutive (CMV) and X-ray inducible (WAF1) promoter, for generating high concentrations of NO(*) within tumour cells. We have combined this treatment with radiation to exploit the radiosensitizing properties of this molecule. Transfection of murine RIF-1 tumour cells in vitro with the iNOS constructs resulted in increased iNOS protein levels. Under hypoxic conditions cells were radiosensitized by delivery of both constructs so that these treatments effectively eliminated the radioresistance observed under hypoxic conditions. In vivo transfer of the CMV/iNOS construct by direct tumour injection resulted in a delay (4.2 days) in tumour growth compared with untreated controls. This was equivalent to the effect of 20 Gy X-rays alone. Combination of CMV/iNOS gene transfer with 20 Gy X-rays resulted in a dramatic 19.8 day growth delay compared with controls. Tumours treated with the CMV/iNOS showed large areas of necrosis and abundant apoptosis. We believe that iNOS gene transfer has the potential to be a highly effective treatment in combination with radiotherapy.

The chemopotentiation of cisplatin by the novel bioreductive drug AQ4N.

AQ4N is a bioreductive drug that can significantly enhance the anti-tumour effect of radiation and cyclophosphamide. The aim of this study was to examine the ability of AQ4N to potentiate the anti-tumour effect of cisplatin and to compare it to the chemopotentiation effect of tirapazamine. In the T50/80 murine tumour model, AQ4N (50-100 mg/kg) was administered 30 min, 2.5 or 6 h prior to cisplatin (4 mg/kg or 8 mg/kg); this produced an anti-tumour effect that was approximately 1.5 to 2 times greater than that achieved by a single 4 or 8 mg/kg dose of cisplatin. Tirapazamine (25 mg/kg) administered 2.5 h prior to cisplatin (4 mg/kg) resulted in a small increase in anti-tumour efficacy. AQ4N was also successful in enhancing the anti-tumour effect of cisplatin in the SCCVII and RIF-1 murine tumour models. This resulted in an increased cell kill of greater than 3 logs in both models; this was a greater cell kill than that observed for tirapazamine with cisplatin. Combination of cisplatin with AQ4N or tirapazamine resulted in no additional bone marrow toxicity compared to cisplatin administered alone. In conclusion, AQ4N has the potential to improve the clinical efficacy of cisplatin.

Regulation of FOS by different compartmental stresses induced by low levels of ionizing radiation.

We irradiated different cellular compartments and measured changes in expression of the FOS gene at the mRNA and protein levels. [(3)H]Thymidine and tritiated water were used to irradiate the nucleus and the whole cell, respectively. (125)I-Concanavalin A binding was used to irradiate the cell membrane differentially. Changes in FOS mRNA and protein levels were measured using semi-quantitative RT-PCR and SDS-PAGE Western blotting, respectively. Irradiation of the nucleus or the whole cell at a dose rate of 0.075 Gy/h caused no change in the level of FOS mRNA expression, but modestly (1.5-fold) induced FOS protein after 0.5 h. Irradiation of the nucleus at a dose rate of 0.43 Gy/h induced FOS mRNA by 1.5-fold after 0.5 h, but there was no significant effect after whole-cell irradiation. FOS protein was transiently induced 2.5-fold above control levels 0.5 h after a 0. 43-Gy/h exposure of the nucleus or the whole cell. Irradiation of the cell membrane at a dose rate of 1.8 Gy/h for up to 2 h caused no change in the levels of expression of FOS mRNA or protein, but a dose rate of 6.8 Gy/h transiently increased the level of FOS mRNA 3-fold after 0.5 h. These data demonstrate the complexity of the cellular response to radiation-induced damage at low doses. The lack of quantitative agreement between the transcript and protein levels for FOS suggests a role for post-transcriptional regulation.

The effect of nicotinamide on spontaneous and induced activity in smooth and skeletal muscle.

BACKGROUND AND PURPOSE: Nicotinamide (NA) is currently undergoing clinical trials as a tumour radiosensitizer. The dose that can be administered is currently 80 mg/kg per day, but this may be restricted to 60 mg/kg per day by the high incidence of nausea and vomiting. To investigate some of NA's underlying mechanisms of action, we have used an ex vivo system to study the direct effect of this drug, over a wide range of concentrations, on isolated spontaneously active rat ileum. Effects on the gut were compared with the action of NA on skeletal and vascular smooth muscle. MATERIALS AND METHODS: Isolated rat ileum rings were perfused with oxygenated Krebs' solution in an organ bath. NA (1 microM to 10 mM) was introduced to the perfusate and the change in amplitude of spontaneous peristaltic activity recorded. Dissected frog sartorius muscle was bathed in modified oxygenated Ringer's solution in an organ bath. The muscle was electrically stimulated to generate isometric contractions. Tension was then measured before and after the addition of a range of NA concentrations (8.2-24.6 mM) to the organ bath. RESULTS: NA inhibited peristalsis in the ileum in a dose-dependent manner. At a drug concentration of 1 mM the amplitude of contractions was reduced to <50% of the initial control value. NA had no effect on the electrically induced contractions in the isolated frog sartorius muscle. CONCLUSIONS: Gut smooth muscle is highly sensitive to the relaxant effect of NA producing 50% relaxation at a concentration approximately 10 fold lower than that required in rat arterial smooth muscle, while having no effect on non-mammalian skeletal smooth muscle. This may provide explanations for the occurrence of emesis in patients undergoing combined nicotinamide therapies and highlight possible alternatives available to counter this unwanted side-effect.

Modification of vascular tone using iNOS under the control of a radiation-inducible promoter.

It may be therapeutically advantageous to alter tumour blood supply specifically. Nitric oxide is a potent vasodilator which is produced in many tissues by the enzyme nitric oxide synthase (NOS). We have transfected cDNA for the inducible isoform of this enzyme (iNOS), under the control of the radiation-inducible promoter WAF1. The activity of the promoter was initially assessed using green fluorescent protein (GFP) in both endothelial cells and rat tail artery segments. Induction of protein expression by 9.5- and 4.5-fold respectively, was observed after a radiation dose of 4 Gy. Artery sections were then transfected with the WAF1/iNOS construct; this gave five-fold induction of iNOS protein after a dose of 4 Gy. The transfected artery was also tested functionally for relaxation, indicative of NO production. One hour after exposure to 4 Gy there was a significant (65%) relaxation of artery segments that had been preconstricted with phenylephrine. This could be partially reversed by the NOS inhibitor nitro-L-arginine. This study demonstrates that we can regulate vascular tone using an X-ray inducible promoter.