A.M. Rocha-Gonsalves

Hypoxia-induced redox alterations and their correlation with 99mTc-MIBI and 99mTc-HL-91 uptake in colon cancer cells

Colorectal cancer is one of the most common malignancies in the Western world and is an example of a solid tumour in which hypoxia is a common feature and develops because of the inability of the vascular system to supply adequate amounts of oxygen to growing tumours. Hypoxia effects on tumour cell biology can be detected and characterized using different methods. The use of imaging with gamma-emitting radionuclides to detect hypoxic tissue was first suggested by Chapman in 1979 [N Engl J Med 301 (1979) 1429-1432]. (99m)Tc-4,9-diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime, also known as (99m)Tc-HL-91, has been among the most studied hypoxia markers. The objective of this study was to correlate the uptake of (99m)Tc-HL-91 and (99m)Tc-MIBI in colon cancer cells under normoxic and hypoxic conditions and to compare this information with some parameters such as oxidative stress and mitochondrial dysfunction of the cells analyzed by flow cytometry. Our results show that the in vitro (99m)Tc-HL-91 uptake is higher in hypoxic conditions, which is confirmed by the decreased uptake of (99m)Tc-MIBI. Flow cytometry results demonstrate that hypoxic conditions used are not enough to induce cellular death, but are responsible for the alterations in the intracellular redox environment, namely, increase of ROS production, proteic pimonidazol-derived adduct formation and alteration in the mitochondrial membrane permeability. Therefore, these results confirm that (99m)Tc-HL-91 is a radiopharmaceutical with favourable characteristics for detecting hypoxia.

2-Bromo-5-hydroxyphenylporphyrins for photodynamic therapy: Photosensitization efficiency, subcellular localization and in vivo studies

BACKGROUND Photodynamic therapy (PDT) is a therapeutic modality capable of inducing cell death by oxidative stress through activation of a sensitizer by light. Aryl-porphyrin with hydroxyl groups are good photosensitizers and presence of bromine atoms can enhance the photodynamic activity through heavy atom effect. These facts and our previous work made pertinent to compare the photodynamic capacity of tetraaryl brominated porphyrin (TBr4) with the corresponding diaryl (BBr2) derivative. METHODS Cell cultures were incubated with the sensitizers, ranging from 50nM to 10μM and irradiated until 10J. Cell proliferation was analysed by MTT assay. Flow cytometry studies evaluated cell death pathways, mitochondrial membrane potential and ROS. For in vivo studies Balb/c nu/nu mice were injected with 4×10(6)cells. After PDT, monitoring was carried out for 12 days to establish Kaplan-Meier survival curves. Tumours were excised and histological analysis was performed. RESULTS Both sensitizers seem to accumulate in the mitochondria. The molecules have no intrinsic cytotoxicity or in non-tumour cells at therapeutic concentrations. Both sensitizers induced a significant decrease of cell proliferation and growth of xenografts of melanoma and colorectal adenocarcinoma. Diaryl BBr2 is more efficient than tetraaryl TBr4, concerning intracellular ROS production, mitochondrial disruption and induction of cell death. The main cell death pathway is necrosis. CONCLUSIONS TBr2 and BBr4 are promising sensitizers with good photodynamic properties and have the ability to induce cell death in human melanoma and colorectal adenocarcinoma in vitro and in vivo. We consider that BBr2 is a molecule that should be the subject of extensive studies towards clinical use.

Tumour Hypoxia and Technetium Tracers: In Vivo Studies

INTRODUCTION Hypoxia is a biochemical condition where reduced oxygen partial pressure at tissue level occurs. This metabolic situation can lead to resistance to radio and chemotherapy. In malignant solid tumours, hypoxia is a common characteristic, having a great impact at biological level, being of tremendous importance for complete understanding of tumour progression. OBJECTIVES We studied the behavior of 99mTc-HL91 in vivo, using an animal model based on Balb-c nu/nu mice with a xenotransplant of the human colorectal adenocarcinoma cell line, WiDr. MATERIAL AND METHODS In vivo studies using an animal model of xenograft on Balb/c nu/nu nude mice were carried out. This model, allowed us to evaluate the radiopharmaceutical biodistribution and to calculate tumour/muscle ratio, acquired after 99mTc-HL91 injection. We also performed ex vivo studies, using the excised tumours to access viability and to characterize the intracellular production of reactive oxygen species and the status of mitochondrial membrane potential through flow cytometry. RESULTS AND DISCUSSION The biodistribution after 99mTc-HL91 injection showed urinary and hepatobilliary excretion in similar proportions and tumour uptake around 4.4% of administered activity. This uptake was higher at the bigger tumours. Through flow cytometry we observed that larger tumours have a higher amount of reactive oxygen species and a decrease in mitochondrial membrane potential. CONCLUSIONS 99mTc-HL91 allowed a non-invasive evaluation of the solid tumours oxidative state by nuclear medicine functional imaging. This information can be of high importance at the pre-treatment estimation of this type of tumours.

Evaluation of a 99mTc‐labelled meso‐bisphenylporphyrin as a tumour image agent

Porphyrins are excellent agents for photodynamic treatment of various types of cancer and also good metal chelators that form highly stable metallo-complexes with different radionuclides. Therefore, radiolabelled porphyrins could also be potentially used as tumour imaging agents. In this context, the aim of this work was the radiolabelling of meso-bis[3,4-bis(carboxymethyleneoxy)phenyl]porphyrin, 2CPP, with Technetium-99 m ((99m) Tc) and the evaluation of its radiochemical and biological properties in vitro and in vivo. The labelling procedure was optimized resulting in an efficiency of 92.52 ± 0.48%. The complex (99m) TC-2CPP remained stable for more than 4 h. The biodistribution showed that (99m) Tc-2CPP is eliminated by gastrointestinal and urinary pathways. The tumour/muscle ratio increases over time, being 3.33 ± 1.22 and 3.55 ± 1.29 in WiDr-bearing tumours mice and in H1299-bearing tumours mice, respectively, 6 h post-injection, showing the tumour specificity of the (99m) Tc-2CPP complex. The favourable tumour/muscle ratio of (99m) Tc-2CPP shows that this complex could potentially be used as tumour imaging agent. Moreover, it could be used to follow the progression or regression of tumours before, during and after the radiotherapy, chemotherapy and photodynamic therapy.

A new therapeutic proposal for inoperable osteosarcoma: Photodynamic therapy

BACKGROUND Osteosarcoma, a malignant tumor characterized by bone or osteoid formation, is the second most common primary bone neoplasm. Clinical symptoms include local and surrounding pain, unrelieved by rest or anesthesia. Osteosarcoma has a poor chemotherapeutic response with prognosis dependent on complete tumor excision. Therefore, for inoperable osteosarcoma new therapeutic strategies are needed. The present study aimed to develop murine models of cranial and vertebral osteosarcoma that facilitate simple clinical monitoring and real-time imaging to evaluate the outcome of photodynamic therapy based on a previously developed photosensitizer. METHODS Balb/c nude mice were divided into two groups: the cranial and vertebral osteosarcoma groups. Each group was further subdivided into the photodynamic therapy-treated and untreated groups. Images were obtained by scintigraphy with 99mTc-MIBI and radiography. Tumor growth, necrotic area, osteoid matrix area, and inflammatory infiltration were analyzed. RESULTS Cranial and vertebral tumors could be macroscopically observed and measured. Radiographic and scintigraphic images showed tumor cells present at the inoculation sites. After photodynamic therapy, scintigraphy showed lower tumoral radiopharmaceutical uptake, which correlated histologically with increased necrosis. Osteoid matrix volume increased, and tumor size decreased in all photodynamic therapy-treated animals. CONCLUSION Cranial and vertebral osteosarcoma models in athymic mice are feasible and facilitate in vivo monitoring for the development of new therapies. Photodynamic therapy is a potential antitumoral treatment for surgically inoperable osteosarcoma.