RESUMO
Photodynamic therapy has been considered ineffective for melanomas because of the competition between the absorbance of melanin from the melanoma and the absorbance of photosensitizers at the photosensitizer excitation light wavelength. Melanomas show considerable heterogeneity and resistance to phototherapy. The effectiveness of photodynamic therapy could be intensified by electroporation for enhanced transport of a photosensitizer by transient pores in the membrane. In this study, photodynamic therapy combined with electroporation was tested in vitro on the human melanoma cell lines melanotic melanoma (MeWo) and amelanotic melanoma (C32). Control experiments were conducted on human keratinocytes (HaCaT). Photofrin was used as a photosensitizer. Photosensitizer distribution, cloning efficacy test, comet assay, and assessment of apoptotic proteins were performed. Melanin levels were determined before and after photodynamic therapy. The experiments indicated that electroporation effectively supports the photodynamic method. It was found that photodynamic therapy with electroporation efficiently induces apoptosis in melanotic and amelanotic melanoma cells.
Assuntos
Antineoplásicos/farmacologia , Éter de Diematoporfirina/farmacologia , Eletroquimioterapia , Melanoma Amelanótico/tratamento farmacológico , Melanoma/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Absorção Fisiológica/efeitos da radiação , Antineoplásicos/efeitos adversos , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Caspases/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Ensaio Cometa , Dano ao DNA , Éter de Diematoporfirina/efeitos adversos , Éter de Diematoporfirina/metabolismo , Eletroquimioterapia/efeitos adversos , Eletroporação , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Melaninas/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Melanoma Amelanótico/metabolismo , Melanoma Amelanótico/patologia , Proteínas de Neoplasias/metabolismo , Fotoquimioterapia/efeitos adversos , Fármacos Fotossensibilizantes/efeitos adversos , Fármacos Fotossensibilizantes/metabolismo , Fatores de TempoRESUMO
Cell recurrence in cancer photodynamic therapy (PDT) is an important issue that is poorly understood. It is becoming clear that nitric oxide (NO) is a modulator of PDT. By acting on the NF-κB/Snail/RKIP survival/anti-apoptotic loop, NO can either stimulate or inhibit apoptosis. We found that pheophorbide a/PDT (Pba/PDT) induces the release of NO in B78-H1 murine amelanotic melanoma cells in a concentration-dependent manner. Low-dose PDT induces low NO levels by stimulating the anti-apoptotic nature of the above loop, whereas high-dose PDT stimulates high NO levels inhibiting the loop and activating apoptosis. When B78-H1 cells are treated with low-dose Pba/PDT and DETA/NO, an NO-donor, intracellular NO increases and cell growth is inhibited according to scratch-wound and clonogenic assays. Western blot analyses showed that the combined treatment reduces the expression of the anti-apoptotic NF-κB and Snail gene products and increases the expression of the pro-apoptotic RKIP gene product. The combined effect of Pba and DETA/NO was also tested in C57BL/6 mice bearing a syngeneic B78-H1 melanoma. We used pegylated Pba (mPEG-Pba) due to its better pharmacokinetics compared to free Pba. mPEG-Pba (30 mg/Kg) and DETA/NO (0.4 mg/Kg) were i.p. injected either as a single molecule or in combination. After photoactivation at 660 nM (fluence of 193 J/cm(2)), the combined treatment delays tumor growth more efficiently than each individual treatment (p<0.05). Taken together, our results showed that the efficacy of PDT is strengthened when the photosensitizer is used in combination with an NO donor.
Assuntos
Clorofila/análogos & derivados , Melanoma Amelanótico/tratamento farmacológico , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Clorofila/farmacologia , Feminino , Citometria de Fluxo , Melanoma Amelanótico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NG-Nitroarginina Metil Éster/farmacologia , Compostos Nitrosos/farmacologia , Neoplasias Cutâneas/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
The electrical resistance breakdown assay provides a novel approach for the quantification of cytotoxic activity of platinum based anticancer drugs. It is a functional assay system for cancer cell invasion that detects nanoscale alterations of an epithelial test barrier prior to microscopic morphometric changes. We measured changes in transepithelial electrical resistance (TEER) of a tight epithelial MDCK-C7 monolayer in response to highly invasive amelanotic melanoma cells (A7-clone) in combination with different platinum complexes (cis-, oxali- and carboplatin). The efficiency of the electrical resistance breakdown assay was compared a standard method for measurement of cytostatic activity, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The MTT-assay utilizes mitochondrial enzymatic activity to draw conclusions from a functional cell metabolism to the number of living cells in a sample. When human melanoma cells were seeded on top of an electrically tight MDCK-C7 monolayer, electrical leakage occurred within 48 h of co-culture. Electrical resistance breakdown was effectively prevented by cisplatin and its analogs (no significant difference between 100 microM cisplatin and corresponding controls with non-invasive cells). The results of the electrical resistance breakdown and MTT-assay were linearly dependent. Significance of both tests was equivalent, but the electrical resistance breakdown assay gave additional functional information. Compared to oxali- and carboplatin, cisplatin was more effective in preventing TEER-breakdown than reducing the number of tumor cells, giving rise to the assumption that cisplatin can reduce tumor cell number as well as invasiveness. In conclusion the electrical resistance breakdown assay provides a sensitive, continuous and cell-based assay system for the quantification of cancer cell invasiveness and evaluation of chemotherapeutics under physiological conditions.
Assuntos
Antineoplásicos/toxicidade , Avaliação Pré-Clínica de Medicamentos/métodos , Compostos de Platina/toxicidade , Animais , Bioensaio/instrumentação , Linhagem Celular Tumoral , Cães , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Humanos , Melanoma Amelanótico/química , Melanoma Amelanótico/metabolismoRESUMO
The human nucleolar protein p120 is highly expressed in human cancers. Its high expression in breast cancer correlates with a poor prognosis, and its overexpression in 3T3 mouse fibroblasts causes malignant transformation. This study reports that a combination of monoclonal anti-p 120 antibody (MAbp120), liposomes (Lipo), and hyperthermia (HT) resulted in enhanced antitumor effects in cultured human breast adenocarcinoma (MCF-7) and human amelanotic melanoma (LOX) cells. Monoclonal antibody uptake and intracellular localization of the protein p120 were monitored by double labeling indirect immunofluorescence. Cell growth inhibition by the combination of MAbp120 + Lipo + HT was 65% for MCF-7 cells and 96% for LOX cells. When tested on LOX cells, monoclonal antibodies (MAbB23, MAbC23) to other nucleolar proteins (B23, C23) produced only slight cytotoxicity with similar treatment protocols.