Lysosomotropism depends on glucose: a chloroquine resistance mechanism.

There has been long-standing interest in targeting pro-survival autophagy as a combinational cancer therapeutic strategy. Clinical trials are in progress testing chloroquine (CQ) or its derivatives in combination with chemo- or radiotherapy for solid and haematological cancers. Although CQ has shown efficacy in preclinical models, its mechanism of action remains equivocal. Here, we tested how effectively CQ sensitises metastatic breast cancer cells to further stress conditions such as ionising irradiation, doxorubicin, PI3K-Akt inhibition and serum withdrawal. Contrary to the conventional model, the cytotoxic effects of CQ were found to be autophagy-independent, as genetic targeting of ATG7 or the ULK1/2 complex could not sensitise cells, like CQ, to serum depletion. Interestingly, although CQ combined with serum starvation was robustly cytotoxic, further glucose starvation under these conditions led to a full rescue of cell viability. Inhibition of hexokinase using 2-deoxyglucose (2DG) similarly led to CQ resistance. As this form of cell death did not resemble classical caspase-dependent apoptosis, we hypothesised that CQ-mediated cytotoxicity was primarily via a lysosome-dependent mechanism. Indeed, CQ treatment led to marked lysosomal swelling and recruitment of Galectin3 to sites of membrane damage. Strikingly, glucose starvation or 2DG prevented CQ from inducing lysosomal damage and subsequent cell death. Importantly, we found that the related compound, amodiaquine, was more potent than CQ for cell killing and not susceptible to interference from glucose starvation. Taken together, our data indicate that CQ effectively targets the lysosome to sensitise towards cell death but is prone to a glucose-dependent resistance mechanism, thus providing rationale for the related compound amodiaquine (currently used in humans) as a better therapeutic option for cancer.

Emulsion technologies for multicellular tumour spheroid radiation assays.

A major limitation with current in vitro technologies for testing anti-cancer therapies at the pre-clinical level is the use of 2D cell culture models which provide a poor reflection of the tumour physiology in vivo. Three dimensional cell culture models, such as the multicellular spheroid, provide instead a more accurate representation. However, existing spheroid-based assessment methods are generally labour-intensive and low-throughput. Emulsion based technologies offer enhanced mechanical stability during multicellular tumour spheroid formation and culture and are scalable to enable higher-throughput assays. The aim of this study was to investigate the characteristics of emulsion-based techniques for the formation and long term culture of multicellular UVW glioma cancer spheroids and apply these findings to assess the cytotoxic effect of radiation on spheroids. Our results showed that spheroids formed within emulsions had similar morphological and growth characteristics to those formed using traditional methods. Furthermore, we have identified the effects produced on the proliferative state of the spheroids due to the compartmentalised nature of the emulsions and applied this for mimicking tumour growth and tumour quiescence. Finally, proof of concept results are shown to demonstrate the scalability potential of the technology for developing high-throughput screening assays.

Gamma irradiation and targeted radionuclides enhance the expression of the noradrenaline transporter transgene controlled by the radio-inducible p21(WAF1/CIP1) promoter.

The use of radiation-inducible promoters to drive transgene expression offers the possibility of temporal and spatial regulation of gene activation. This study assessed the potential of one such promoter element, p21(WAF1/CIP1) (WAF1), to drive expression of the noradrenaline transporter (NAT) gene, which conveys sensitivity to radioiodinated meta-iodobenzylguanidine (MIBG). An expression vector containing NAT under the control of the radiation-inducible WAF1 promoter (pWAF/NAT) was produced. The non-NAT expressing cell lines UVW (glioma) and HCT116 (colorectal cancer) were transfected with this construct to assess radiation-controlled WAF1 activation of the NAT gene. Transfection of UVW and HCT cells with pWAF/NAT conferred upon them the ability to accumulate [(131)I]MIBG, which led to increased sensitivity to the radiopharmaceutical. Pretreatment of transfected cells with γ radiation or the radiopharmaceuticals [(123)I]MIBG or [(131)I]MIBG induced dose- and time-dependent increases in subsequent [(131)I]MIBG uptake and led to enhanced efficacy of [(131)I]MIBG-mediated cell kill. Gene therapy using WAF1-driven expression of NAT has the potential to expand the use of this therapeutic modality to tumors that lack a radio-targetable feature.

Inhibition of poly(ADP-Ribose) polymerase enhances the toxicity of 131I-metaiodobenzylguanidine/topotecan combination therapy to cells and xenografts that express the noradrenaline transporter.

UNLABELLED: Targeted radiotherapy using (131)I-metaiodobenzylguanidine ((131)I-MIBG) has produced remissions in some neuroblastoma patients. We previously reported that combining (131)I-MIBG with the topoisomerase I inhibitor topotecan induced long-term DNA damage and supraadditive toxicity to noradrenaline transporter (NAT)-expressing cells and xenografts. This combination treatment is undergoing clinical evaluation. This present study investigated the potential of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP-1) inhibition, in vitro and in vivo, to further enhance (131)I-MIBG/topotecan efficacy. METHODS: Combinations of topotecan and the PARP-1 inhibitor PJ34 were assessed for synergism in vitro by combination-index analysis in SK-N-BE(2c) (neuroblastoma) and UVW/NAT (NAT-transfected glioma) cells. Three treatment schedules were evaluated: topotecan administered 24 h before, 24 h after, or simultaneously with PJ34. Combinations of PJ34 and (131)I-MIBG and of PJ34 and (131)I-MIBG/topotecan were also assessed using similar scheduling. In vivo efficacy was measured by growth delay of tumor xenografts. We also assessed DNA damage by γH2A.X assay, cell cycle progression by fluorescence-activated cell sorting analysis, and PARP-1 activity in treated cells. RESULTS: In vitro, only simultaneous administration of topotecan and PJ34 or PJ34 and (131)I-MIBG induced supraadditive toxicity in both cell lines. All scheduled combinations of PJ34 and (131)I-MIBG/topotecan induced supraadditive toxicity and increased DNA damage in SK-N-BE(2c) cells, but only simultaneous administration induced enhanced efficacy in UVW/NAT cells. The PJ34 and (131)I-MIBG/topotecan combination treatment induced G(2) arrest in all cell lines, regardless of the schedule of delivery. In vivo, simultaneous administration of PJ34 and (131)I-MIBG/topotecan significantly delayed the growth of SK-N-BE(2c) and UVW/NAT xenografts, compared with (131)I-MIBG/topotecan therapy. CONCLUSION: The antitumor efficacy of topotecan, (131)I-MIBG, and (131)I-MIBG/topotecan combination treatment was increased by PARP-1 inhibition in vitro and in vivo.

Deletion of the dual specific phosphatase-4 (DUSP-4) gene reveals an essential non-redundant role for MAP kinase phosphatase-2 (MKP-2) in proliferation and cell survival.

Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specific phosphatase (DUSP) implicated in a number of cancers. We examined the role of MKP-2 in the regulation of MAP kinase phosphorylation, cell proliferation, and survival responses in mouse embryonic fibroblasts (MEFs) derived from a novel MKP-2 (DUSP-4) deletion mouse. We show that serum and PDGF induced ERK-dependent MKP-2 expression in wild type MEFs but not in MKP-2(-/-) MEFs. PDGF stimulation of sustained ERK phosphorylation was enhanced in MKP-2(-/-) MEFs, whereas anisomycin-induced JNK was only marginally increased. However, marked effects upon cell growth parameters were observed. Cellular proliferation rates were significantly reduced in MKP-2(-/-) MEFs and associated with a significant increase in cell doubling time. Infection with adenoviral MKP-2 reversed the decrease in proliferation. Cell cycle analysis revealed a block in G(2)/M phase transition associated with cyclin B accumulation and enhanced cdc2 phosphorylation. MEFs from MKP-2(-/-) mice also showed enhanced apoptosis when stimulated with anisomycin correlated with increased caspase-3 cleavage and γH2AX phosphorylation. Increased apoptosis was reversed by adenoviral MKP-2 infection and correlated with selective inhibition of JNK signaling. Collectively, these data demonstrate for the first time a critical non-redundant role for MKP-2 in regulating cell cycle progression and apoptosis.

Should patients be manually ventilated during rapid sequence induction of anaesthesia?

Pulmonary aspiration of gastric contents has been recognized as a cause of significant morbidity and mortality during anaesthesia since the classic publication by Mendelson in 1946. The introduction of suxamethonium 5 years later allowed tracheal intubation to be achieved under complete muscle relaxation within 1 minute of induction of anaesthesia. In 1961 Sellick described the use of cricoid pressure to prevent gastric regurgitation.

Radiation quality-dependent bystander effects elicited by targeted radionuclides.

The efficacy of radiotherapy may be partly dependent on indirect effects, which can sterilise malignant cells that are not directly irradiated. However, little is known of the influence of these effects in targeted radionuclide treatment of cancer. We determined bystander responses generated by the uptake of radioiodinated iododeoxyuridine ([*I]IUdR) and radiohaloanalogues of meta-iodobenzylguanidine ([*I]MIBG) by noradrenaline transporter (NAT) gene-transfected tumour cells. NAT specifically accumulates MIBG. Multicellular spheroids that consisted of 5% of NAT-expressing cells, capable of the active uptake of radiopharmaceutical, were sterilised by treatment with 20 kBqmL(-1) of the alpha-emitter meta-[211At]astatobenzylguanidine ([211At]MABG). Similarly, in nude mice, retardation of the growth of tumour xenografts containing 5% NAT-positivity was observed after treatment with [131I]MIBG. To determine the effect of subcellular localisation of radiolabelled drugs, we compared the bystander effects resulting from the intracellular concentration of [131I]MIBG and [131I]IUdR (low linear energy transfer (LET) beta-emitters) as well as [123I]MIBG and [123I]IUdR (high LET Auger electron emitters). [*I]IUdR is incorporated in DNA whereas [*I]MIBG accumulates in extranuclear sites. Cells exposed to media from [131I]MIBG- or [131I]IUdR-treated cells demonstrated a dose-response relationship with respect to clonogenic cell death. In contrast, cells receiving media from cultures treated with [123I]MIBG or [123I]IUdR exhibited dose-dependent toxicity at low dose but elimination of cytotoxicity with increasing radiation dose (i.e. U-shaped survival curves). Therefore radionuclides emitting high LET radiation may elicit toxic or protective effects on neighbouring untargeted cells at low and high dose respectively. It is concluded that radiopharmaceutical-induced bystander effects may depend on LET of the decay particles but are independent of site of intracellular concentration of radionuclide.

A transfectant mosaic xenograft model for evaluation of targeted radiotherapy in combination with gene therapy in vivo.

UNLABELLED: For gene therapy to be efficacious in the treatment of cancer, therapeutic transgenes must be limited in their expression to tumor cells and must be expressed at sufficiently high transcriptional levels. Moreover, the inadequacy of gene delivery must be overcome by induction of toxicity to neighboring nontargeted cells. Combining targeted radionuclide therapy with gene therapy using human telomerase promoters has shown promise in these respects, and the efficacy of this scheme has been assessed in vitro using transfectant mosaic tumor spheroids. To enable the evaluation of targeted radiotherapy combined with gene transfer in vivo, we have developed a transfectant mosaic xenograft (TMX) model. METHODS: Human telomerase promoters were used to drive expression of the noradrenaline transporter (NAT) transgene in 2 human cell lines (UVW and EJ138). Promoter activity was assessed in xenografts in nude mice by determination of the uptake of the radiopharmaceutical (131)I-metaiodobenzylguanidine ((131)I-MIBG) and by measurement of tumor growth. The efficacy of (131)I-MIBG treatment was also assessed in TMXs to determine the delay in growth of tumors composed of various proportions of NAT-expressing cells-a likely clinical scenario after gene delivery in vivo. RESULTS: In terms of induction of the capacity for active uptake of (131)I-MIBG and the resultant inhibition of tumor growth in vivo, both telomerase promoters (hTR and hTERT) were similar in potency to the CMV (cytomegalovirus) promoter as controlling elements for the expression of the NAT transgene. In TMXs derived from UVW and EJ138 cells, (131)I-MIBG uptake was proportional to NAT gene expression (r(s) = 0.910, P < 0.001 for UVW; r(s) = 0.971, P < 0.001 for EJ138). Inhibition of the growth of these tumors correlated with the fraction of NAT-transfected cells (r(s) = 0.910, P < 0.001 for UVW; r(s) = 0.971, P < 0.001 for EJ138), and substantial tumor growth delay was observed when 5% of the xenograft was composed of NAT-positive cells. CONCLUSION: TMXs constitute a suitable model to measure the efficacy of cancer gene therapy strategies when <100% of the tumor mass can be targeted to express the therapeutic transgene.

[131I]meta-iodobenzylguanidine and topotecan combination treatment of tumors expressing the noradrenaline transporter.

PURPOSE: Both [(131)I]meta-iodobenzylguanidine ([(131)I]MIBG) and the topoisomerase I inhibitor topotecan are effective as single-agent treatments of neuroblastoma. The aim of this study was to investigate the efficacy of [(131)I]MIBG in combination with topotecan in vitro and in vivo. EXPERIMENTAL DESIGN: The cell lines used were SK-N-BE(2c) (human neuroblastoma) and UVW/NAT (glioma cell line transfected with the noradrenaline transporter gene). Three different treatment schedules were assessed: topotecan given before (schedule 1), after (schedule 2), or simultaneously (schedule 3) with [(131)I]MIBG. DNA strand breakage was evaluated by comet assay, and cytotoxicity was determined by clonogenic survival. Efficacy was also measured by growth delay of tumor xenografts in nude mice. RESULTS: Combination schedules 2 and 3 caused more cytotoxicity than schedule 1. Similarly, significant DNA damage was observed following treatment schedules 2 and 3 (P < 0.005) but not schedule 1. The mean number of days for a doubling in volume of SK-N-BE(2c) tumors and a 10-fold increase in volume of UVW/NAT tumors were 10.4 and 18.6 (untreated), 19.7 and 25.3 (topotecan alone), 22.8 and 31.9 ([(131)I]MIBG alone), 26.3 and 37.1 (combination schedule 1), 34.3 and 49.7 (combination schedule 2), and 53.2 and >71 (combination schedule 3), respectively. The highest rate of cure of both xenografts was observed following treatment with combination schedule 3. CONCLUSIONS: The combination of topotecan and [(131)I]MIBG compared with either treatment alone gave rise to greater than additive DNA damage, clonogenic cell kill, and tumor growth delay. These effects were dependent on the scheduling of the two agents.

[131I]MIBG and topotecan: a rationale for combination therapy for neuroblastoma.

MIBG is selectively concentrated in neuroblastoma cells, and radioiodinated MIBG has been used with some success for targeted radiotherapy. However, long-term cure remains elusive, and the topoisomerase I inhibitor topotecan may improve upon existing [131I]MIBG therapy. While synergistic killing by combinations of ionising radiation and topoisomerase I inhibitors has been reported, there is no consensus on optimal scheduling. Furthermore, there has been no attempt to demonstrate radio-potentiation by topoisomerase I inhibitors and targeted radiotherapy. We are investigating various scheduled combinations of topotecan and [131I]MIBG on neuroblastoma cells, and preliminary data suggests that topotecan induces increased accumulation of [131I]MIBG in vitro.

Application of targeted radiotherapy/gene therapy to bladder cancer cell lines.

OBJECTIVES: A targeted radiotherapy/gene therapy strategy for transitional cell carcinoma of bladder is described, using [131I]meta-iodobenzylguanidine ([131I]MIBG), a radionuclide combined with a tumour-seeking drug. The aim is to decrease side effects from radiation toxicity, while increasing radiation dose to tumour. This tumour cell kill approach is augmented by radiological bystander effects. METHODS: The bladder cancer cell line EJ138 was transfected with a gene encoding the noradrenaline transporter (NAT) under the control of tumour-specific telomerase promoters. Resulting uptake of [131I]MIBG was assessed by gamma-counting of cell lysates, and NAT transgene expression by real-time RT-PCR. Cell kill of monolayers and disaggregated spheroids, dosed with [131I]MIBG, was assessed by clonogenic assay. RESULTS: NAT gene transfected cells exhibited a significantly increased active uptake of [131I]MIBG, leading to dose-dependent cell kill. Clonogenic assay of disaggregated spheroids, a three-dimensional model, suggested cell kill via bystander effects. CONCLUSIONS: Expression of a functional NAT after in vitro transfection of bladder cancer cells with the NAT gene under the control of telomerase promoters leads to active uptake of [131I]MIBG and dose-dependent cell kill. This strategy could produce a promising new treatment option for bladder cancer.

Development of a real-time polymerase chain reaction assay for prediction of the uptake of meta-[(131)I]iodobenzylguanidine by neuroblastoma tumors.

PURPOSE: The suitability of neuroblastoma patients for therapy using radiolabeled meta-iodobenzylguanidine (MIBG) is determined by scintigraphy after the administration of a tracer dose of radioiodinated MIBG whose uptake is dependent upon the cellular expression of the noradrenaline transporter (NAT). As a possible alternative to gamma camera imaging, we developed a novel molecular assay of NAT expression. mRNA extracted from neuroblastoma biopsy samples, obtained retrospectively, was reverse transcribed, and NAT-specific cDNA was quantified by real-time PCR, referenced against the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. EXPERMENTAL DESIGN: Tumor specimens from 54 neuroblastoma patients were analyzed using real-time PCR, and NAT expression was compared with the corresponding diagnostic scintigrams. RESULTS: Forty-eight of 54 (89%) of tumors showed MIBG uptake by scintigraphy. NAT expression was found to be significantly associated with MIBG uptake (P < 0.0001, Fisher's exact test). None of the samples from the six tumors that failed to concentrate MIBG expressed detectable levels of the NAT (specificity = 1.0). However, of the 48 MIBG uptake-positive tumors, only 43 (90%) expressed NAT (sensitivity = 0.9). The real-time PCR test has a positive predictive value of 1.0 but a negative predictive value of 0.55. CONCLUSIONS: The results indicate that whereas this method has substantial ability to predict the capacity of neuroblastoma tumors to accumulate MIBG, confirmation is required in prospective studies to determine more accurately the predictive strength of the test and its role in the management of patients with neuroblastoma.