Stan S. Stylli

Overexpression of hyaluronan synthase-2 reduces the tumorigenic potential of glioma cells lacking hyaluronidase activity.

OBJECTIVE The interactions of CD44 with hyaluronan are thought to be crucial for tumor cell attachment to the extracellular matrix, migration, and invasion. For migration to occur, however, the interactions between hyaluronan and cell surface receptors need to be transient. Hyaluronidases may facilitate the degradation of hyaluronan bound to the cell surface and thus reduce the interactions of the cells with the matrix, whereas the overproduction of hyaluronan in the absence of hyaluronidase activity may prevent cells from proliferating or invading normal surrounding tissue. METHODS We analyzed the effects in vitro and in vivo of hyaluronan synthase-2 (HAS2) overexpression on a murine glioma cell line that is deficient in hyaluronidase activity. In addition, we evaluated the expression levels of HAS and hyaluronidase genes in human glioma cell lines and in glioma specimens. RESULTS Increased hyaluronan synthesis had no effect on the in vitro proliferation of the cells but diminished their in vivo growth rate. Several human glioma cell lines were found to overexpress hyaluronan synthases, but they did so in conjunction with hyaluronidase Hyal2 and MGEA5 expression. Similarly, all glioblastomas multiforme expressed hyaluronidases MGEA5 and Hyal2. CONCLUSION The data suggest that an increased synthesis of hyaluronan by astrocytoma cells is only promoting tumor cell growth in vivo if the cells express hyaluronidases as well.

Imaging and quantitation of the hypoxic cell fraction of viable tumor in an animal model of intracerebral high grade glioma using [18F]fluoromisonidazole (FMISO)

We have demonstrated that FMISO uptake is significantly higher in tumor tissue in the C6 intracerebral glioma rat model compared to normal brain, and that there is persisting hypoxia in gliomas independent of tumor size. FMISO uptake was observed homogeneously throughout viable glioma tissue in tumor sizes ranging from 2mm to almost 1cm. Quantitation of uptake of FMISO showed a tumor/brain ratio of 1.9 and a tumor/blood ratio of 2.6 at 2 hours post injection.

Evaluation of tumour and tissue distribution of porphyrins for use in photodynamic therapy

A range of pure, monomeric porphyrins were synthesised and their localising capacities compared to HpD and Hp at 6 h and 24 h post injection in the mouse C6 intracerebral glioma model as well as in normal brain, skin, muscle, kidney, spleen, liver, lung and whole blood. The partition coefficients were examined between PBS and 2-octanol over the pH range 7.4-6.6 and pH profiles were established. A parabolic relationship was observed between log (porphyrin tumour concentration) at pH 7.4, with maximal tumour localisation at log (partition coefficient), pi, of approximately zero. Porphyrins with side chains with nett cationic character also exhibited up upward (parabolic) dependence on pi for most tissues studied, with maximal porphyrin localisation at pi of 0-0.5. In contrast, those porphyrins with nett anionic character exhibited a downward (negative) parabolic trend for all eight tissues studied, with minimal porphyrin localisation at pi of approximately zero. Four porphyrins (4, 11, 12, 13) exhibited similar or better tumour localisation than HpD, and two (11 and 12) offer promise as lead compounds for the design of improved porphyrins for use in PDT.

Synovial ablation in a rabbit rheumatoid arthritis model using photodynamic therapy.

Background:  At present there is no ideal minimally invasive method for ablating inflamed synovium in joints that has been unresponsive to optimal medical management in patients with rheumatoid arthritis. The aim of this study was to determine whether photodynamic therapy could be used for this purpose.

Two-photon absorption cross-sections and time-resolved fluorescence imaging using porphyrin photosensitisers

Three porphyrin systems have been characterised for use in two-photon fluorescence imaging of biological samples. We have determined the two-photon absorption cross sections (sigma(2)) of the di-cation, free-base and metallated forms of hematoporphyrin derivative (HpD), hematoporphyrin IX (Hp9) and a boronated protoporphyrin (BOPP) using the open-aperture Z-scan and the two-photon induced fluorescence (TPIF) techniques at an excitation wavelength of 800 nm. The insertion of either protons or a metal ion into the macrocycle is shown not to significantly influence the sigma(2) of the porphyrins. Two-photon time-resolved fluorescence images of C6 glioma cells transfected with a free-base form of the BOPP have been obtained as a function of the porphyrin concentration. These studies reveal a maximum useful porphyrin concentration for fluorescence imaging purposes of approximately 30 microg mL(-1).

Aluminium phthalocyanine mediated photodynamic therapy in experimental malignant glioma.

The efficacy of aluminium tetrasulfonated phthalocyanine (AISPc) as a photosensitizer for photodynamic therapy (PDT) was investigated in the C6 glioma model in Wistar rats. The depth and extent of tumour necrosis was dependent on both the dosage of intravenously (IV) administered AISPc, and the dose and time post-sensitization of 675 nm light administration. There was no effect on tumour or normal brain if the sensitizer dose was less than 0.5 mg/kg. Selective necrosis of tumour was evident 6 hours post-sensitization at an AISPc dose up to 1 mg/kg and light doses up to 200 J/cm(2). Necrosis occurred in normal brain at higher doses of light and sensitizer. There was no photosensitizer effect in animals treated 24 hours post AISPc administration. The ability of AISPc to act as a selective photosensitizer causing photonecrosis provides a basis for the generation of modified AISPc species as future agents in the PDT of brain tumours, especially as these sensitizers absorb light at a higher wavelength than those that are currently available.

Glycogen synthase kinase-3β (GSK-3β) and its dysregulation in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most frequently occurring and devastating human brain malignancy, retaining almost universal mortality and a median survival of only 14 months, even with recent advances in multimodal treatments. Gliomas are characterised as being both highly resistant to chemo- and radiotherapy and highly invasive, rendering conventional interventions palliative. The continual dismal prognosis for GBM patients identifies an urgent need for the evolutionary development of new treatment modalities. This includes molecular targeted therapies as many signaling molecules and associated pathways have been implicated in the development and survival of malignant gliomas including the protein kinase, glycogen synthase kinase 3 beta (GSK-3β). Here we review the activity and function of GSK-3β in a number of signaling pathways and its role in gliomagenesis.

The expression of antisense vascular endothelial growth factor (VEGF) sequences inhibits intracranial C6 glioma growth in vivo by suppressing tumour angiogenesis

Human gliomas, including astrocytomas, consist of highly heterogeneous populations of cells that represent different stages of malignancy. Glioblastoma multiforme is the most highly vascularised class of solid tumour. In order to develop efficacious adjuvant therapies for gliomas the growth pathway(s) targeted must be common to all of these tumours. As angiogenesis is a requirement for all solid tumour growth, we have targeted this process in order to suppress glioma growth in vivo. We have applied antisense VEGF gene expression to disrupt the VEGF/VEGF receptor paracrine pathway in C6 glioma cells and, thereby, inhibit tumour angiogenesis. C6 glioma cells which constitutively express antisense VEGF sequences were demonstrated to have significantly inhibited growth rates when implanted intracranially. Antisense VEGF expressing tumours had a markedly lower level of vascularisation which was accompanied by an increased level of necrosis compared to control tumours. Furthermore, these data support the notion that VEGF is the sole factor required for tumour angiogenesis as other potentially angiogenic factors could not compensate for the reduced level of VEGF expression in the antisense-VEGF expressing tumours. Our findings also suggest that the inhibition of angiogenesis is sufficient to significantly suppress tumour growth and is thus an effective point for therapeutic intervention for gliomas and all solid tumours.

Phthalocyanine photosensitizers for the treatment of brain tumours

In vitro and in vivo studies were undertaken utilizing an established rat glioma cell line (C6) to compare the phototoxicity characteristics of aluminium tetrasulfonated phthalocyanine (AlSPc), zinc tetrasulfonated phthalocyanine (ZnSPc) and haematoporphyrin derivative (HpD). AlSPc and ZnSPc were inherently less cytotoxic in the dark compared to HpD with 50% colony survival at 275, 355 and 14 mug/ml respectively. An in vitro phototoxicity study at equimolar concentrations demonstrated a 50% reduction of colony survival after exposure to white light at 1 minute for HpD, 10 minutes for AlSPc and 12 minutes for ZnSPc. The presence of fetal bovine serum (FBS) in the medium resulted in reduced in vitro uptake of AlSPc and increased cellular retention which was determined quantitatively by a fluorescence assay following extraction. This assay was also used to determine the in vivo uptake of AlSPc, which was maximal in the intracerebral C6 glioma model at 6 hours (12.3 mug/g tissue) post-intravenous administration of a 1 mg/kg dose of AlSPc, corresponding to a tumour: normal brain ratio of 22:1.

Tumour Localization of Boronated Porphyrins in an Intracerebral Model of Glioma

Treatment of the most common cerebral tumour, cerebral glioma, is unsatisfactory as the tumour recurs due to inadequate local control (1). Photodynamic therapy (PDT) and Boron Neutron Capture Therapy (BNCT) offer some promise as adjuvant treatments for cerebral glioma. Several clinical trials have been reported utilizing PDT (for review see reference 1) and BNCT (2) to treat the high grade glioma, glioblastoma multiforme. We have investigated the pharmacokinetic tissue distribution of the photosensitizer Haematoporphyrin derivative (HpD), the nido carboranyl porphyrin, boron tetraphenyl porphine (BTPP) and the closo carboranyl monomeric protoporphyrin (BOPP) in CBA mice bearing the intracerebral C6 glioma xenograft (3).