Cationic liposomes, loaded with m-THPC, in photodynamic therapy for malignant glioma.

In this study we investigated the feasibility of mixed liposomes formed by dimyristoyl-sn-glycero-phosphatidylcholine (DMPC) and cationic gemini surfactant (Gemini 1) loaded with the chlorin m-tetrahydroxyphenylchlorin (m-THPC), in photodynamic therapy (PDT) for glioma. To this aim, an in vitro study was carried out by employing various human glioblastoma cell lines (A172, DBTRG, LN229, U118). The following liposomal formulations were tested: (i) DMPC and Gemini 1; (ii) m-THPC in DMPC in the absence or (iii) in the presence of Gemini 1 in the molar ratio 8:2; 7:3, and 6:4. The presence of Gemini 1 significantly increased the intracellular uptake of chlorin in all cell tested although with a different extent: LN229>U118>A172>DBTRG. The cytotoxicity of chlorin-loaded liposomes was then tested by cloning efficiency performed on different cultures, before and after irradiation with laser light at 652nm, at a Fluence Rate of 200mW/s for 100s, with a total Fluence of 20J/cm(-2). In the absence of irradiation, the different liposomal formulations induced a cytotoxicity in less than 30% of glioblastoma cells. On the contrary, irradiation induced total destruction of all cultures treated with m-THPC/DMPC+Gemini 1 in the ratios 8:2, or 7:3, or 6:4.

Invasive properties of multidrug resistant human melanoma cells.

Melanoma cells exhibit a high level of intrinsic or acquired resistance to the cytotoxic agents often associated with the over-expression of drug transporters such as P-glycoprotein (P-gp). In this in vitro study, we investigated the possible relationship between P-gp and CD44, the cell adhesion molecule involved in metastasis and tumor progression of melanoma cells. CD44 expression appeared to be similar in the parental sensitive M14 WT cells and in their resistant counterparts M14 ADR cells. Double-labeling of cryosectioned cells showed that P-gp and CD44 were transported from the synthesis loci to the cell periphery by different vesicles and began to coalesce in proximity of the plasma membrane; thus, P-gp and CD44 seemed to reach together the cell surface. Moreover, P-gp and CD44 appeared to be associated with ERM proteins. The invasive activities of both M14 WT and M14 ADR cells were analyzed by the "transwell chamber invasion" assay. M14 WT cells revealed low capacity to traverse the filters, both in the absence (motility) and in the presence (invasion) of a Matrigel coating. In comparison, M14 ADR cells displayed significantly higher motility and invasion. SEM observations showed that sensitive cells employed lamellar cytoplasmic extrusions to pass through the filter pores whereas resistant cells elongated along the hole through globular processes. In conclusion, the results herein reported suggest that drug resistance in melanoma cells appears associated with a more aggressive behaviour. P-gp and CD44 might cooperate to confer this more invasive phenotype.

Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells.

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells. Pgp was found abnormally expressed on the plasma membrane of tumor cells with "classical" MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100). Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.

What is the relationship between P-glycoprotein and adhesion molecule expression in melanoma cells?

A number of studies have reported that increased P-glycoprotein expression in drug-resistant tumour cells may be associated with decreased expression of a family of surface glycoproteins. However, despite its potential biological and clinical relevance, this phenomenon has not been extensively studied. In this study the phenotypic alterations that are associated with the acquisition of the multidrug-resistant phenotype in tumour cells, together with drug transporter overexpression, were investigated in human melanoma cells. The expression of cell adhesion molecules was analysed in a panel of multidrug-resistant melanoma cell lines (M14Dx) showing different degrees of resistance to doxorubicin and different levels of the expression of the drug transporter P-glycoprotein. In particular, expression of intercellular adhesion molecule-1 (ICAM-1), CD44, very late activation antigen (VLA)-5 and VLA-2 was determined by flow cytometry in the different resistant cell lines. A progressive downregulation of all the adhesion molecules examined was revealed in M14Dx cells, in parallel with an increasing level of expression of the drug transporter P-glycoprotein. The results obtained raise the question of the role of P-glycoprotein in the invasive and metastatic behaviour of tumour cells.

Induction of P-glycoprotein expression on the plasma membrane of human melanoma cells.

Melanoma cells exhibit, both in vivo and in vitro, intrinsic drug resistance to various chemotherapeutic agents. Cultured human melanoma cells (M14) intrinsically express significant amounts of multidrug resistance-related protein (MRP1) and P-glycoprotein (P-gp) in the Golgi apparatus, but do not express these drug transporters on the plasma membrane. A panel of multidrug resistant (MDR) melanoma cell lines (M14Dx), showing different degrees of resistance to doxorubicin (DOX), were isolated. In M14Dx lines, the appearance of surface P-gp, but not of MRP1 or lung resistance related protein (LRP), occurred in cells grown in the presence of DOX concentrations higher than 60 nM. Furthermore, P-gp levels appeared to be dose-dependent. Flow cytometry, laser scanning confocal microscopy and cytotoxicity studies demonstrated that the activity of the drug extrusion system was related to both surface P-gp expression and resistance to DOX. In conclusion, P-gp, but not MRP1 or LRP, might play a pivotal role in the pharmacologically-induced MDR phenotype of melanoma cells.