RESUMEN
A mild and efficient synthetic development of 2-arylbenzothiazoles 5 mediated by ceric ammonium nitrate (CAN) via intramolecular cyclization of N-phenyl-thiobenzamides 4 was achieved. Further compounds 5 were reduced to corresponding amines 6, and their photodynamic therapy (PDT) effect was evaluated on malignant human melanoma A375 cells. Amine 6l plus ultraviolet A (UVA) induced caspase-3 activity, poly(ADP-ribose)polymerase cleavage, M30 positive CytoDeath staining, and subsequent apoptotic cell death. Our data disclosed that treatment of A375 cells with 6l plus UVA resulted in a decrease in mitochondrial membrane potential (ΔΨmt), oxidative phosphorylation system (OXPHOS) subunits, and adenosine triphosphate (ATP) but an increase in mitochondrial DNA 4977-bp deletion via reactive oxygen species (ROS) generation. Transmission electron microscopy (TEM) observations also showed major ultrastructural alterations of mitochondria. Additionally, 6l plus UVA was also shown to reduce murine melanoma size in a mouse model. The present study supports the hypothesis that 6l-PDT may serve as a potential ancillary modality for the treatment of melanoma.
Asunto(s)
Benzotiazoles/farmacología , Melanoma Experimental/metabolismo , Mitocondrias/efectos de los fármacos , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Neoplasias Cutáneas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Benzotiazoles/efectos de la radiación , Benzotiazoles/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citocromos c/metabolismo , Femenino , Humanos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos ICR , Mitocondrias/fisiología , Fármacos Fotosensibilizantes/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Carga Tumoral/efectos de los fármacos , Rayos UltravioletaRESUMEN
Photodynamic therapy (PDT) employing exogenous photosensitizers is currently being approved for treatment of basal cell carcinoma (BCC). 2-(4-Aminophenyl)benzothiazoles (6) consist of chromophoric structure and absorb light in the UVA (315-400 nm). These results encouraged us to design and synthesize a diversity of 2-phenylbenzothiazoles (6). Studies on the apoptotic mechanism involved in photosensitive effects induced by UVA-activated 6 in BCC cells are carried out in the present article. 6-UVA-treated cells displayed several features of apoptosis, including an increase in the sub-G1 population, a significantly increased annexin V binding, and activation of caspase-3. 6-UVA induced a decrease in mitochondrial membrane potential (Deltapsi(mt)) and ATP via enhanced ROS generation and promoted phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK expression. These results suggest that 6-UVA elicits photosensitive effects in mitochondria processes which involve ERK and p38 activation, and ultimately lead to BCC cell apoptosis.
Asunto(s)
Benzotiazoles/química , Benzotiazoles/farmacología , Carcinoma Basocelular/tratamiento farmacológico , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Benzotiazoles/síntesis química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Mitocondrias/efectos de la radiación , Fotoquimioterapia , Fármacos Fotosensibilizantes/síntesis química , Rayos UltravioletaRESUMEN
A series of novel thiobenzanilides is described. These compounds have been previously found to show strong biological activity such as antimycotic and antifungal actions. This is the first demonstration on the mechanism of the anticancer effect of thiobenzanilide agents (4a-c) on human melanoma A375 cells. The cytotoxic studies of compounds 4a-c on human melanoma A375 cells indicate thiobenzanilides induced higher cytotoxicity than nitrobenzanilides (3a-c). In addition, DNA flow cytometric analysis shows that 4a-c displays a significant G2/M phase arrest, which progresses to early apoptosis as detected by flow cytometry after double-staining with annexin V and propidium iodide (PI). Because cellular apoptosis is often preceded by the disruption of mitochondrial function, the assessment of mitochondrial function in 4a-c-treated cells is worthy of investigation. Our data revealed that treatment of A375 cells with 4a-c resulted in the loss of mitochondrial membrane potential (DeltaPsi(mt)), a reduction of ATP synthesis, increased reactive oxygen species (ROS) generation, and activation of caspase-3. Thus, we suggest that 4a-c agents are potent inducers of cell apoptosis in A375 cells.
Asunto(s)
Anilidas/síntesis química , Anilidas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Benzamidas/síntesis química , Benzamidas/farmacología , Adenosina Trifosfato/metabolismo , Anilidas/química , Antineoplásicos/química , Benzamidas/química , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN Mitocondrial/química , ADN Mitocondrial/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Citometría de Flujo/métodos , Fase G2/efectos de los fármacos , Humanos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Estructura Molecular , Especies Reactivas de Oxígeno/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
BACKGROUND: To develop a simple and cost-effective method for the detection and genotyping of high-risk human papillomaviruses (HPV) using seminested polymerase chain reaction (PCR) and reverse hybridization. METHODS: Cervical swabs for HPV testing were collected from 127 women with normal cervical cytology and 57 patients with cervical lesions of various degrees. After DNA isolation, PCR amplification was first carried out using MY11 and MY09/HMB01 primers, then labeled by seminested PCR using the first PCR products and MY11/bioGP6+ primers. One fifth of the second PCR products were resolved by gel electrophoresis. Genotyping for high-risk HPV was done separately, using the remaining products, by a high-risk HPV chip, which contained 13 type-specific oligonucleotides on a nylon membrane. The final result was detected by colorimetric change on the chip under direct visualization. RESULTS: High-risk HPV DNA was detected in 19 (15%) of 127 women with normal cervical smear cytology, in 26 (89.7%) of 29 patients with cervical intraepithelial neoplasia (CIN), and in 27 (96.4%) of 28 patients with invasive cervical carcinoma. Multiple high-risk HPV infections were detected in five cases. HPV type 16 was the most frequent type of infection, comprising 34.5% and 53.6% of the patients with CIN and invasive carcinoma, respectively. The samples without a visible 190-bp band on electrophoresis exclusively showed negative hybridization results. This method could detect one to two copies of the HPV-16 genome derived from one SiHa cell. The overall sensitivity of HPV detection was 25 to 50 copies of HPV genome for each specimen. Thirteen high-risk types and twenty-four different types of HPV DNA showed specific hybridization without any cross-reaction. CONCLUSIONS: Our results demonstrated the feasibility and optimistic prospects for this simple and cheap method of high-risk HPV genotyping. This technology can be easily set up in a routine molecular laboratory and would probably be of great value in cervical cancer prevention programs.