RESUMEN

Macroautophagy/autophagy is the process of self-digestion through the lysosomes; it disassembles unnecessary or dysfunctional long-lived proteins and damaged organelles for the recycling of biomacromolecules. Unfortunately, cancer cells can hijack this mechanism to survive under metabolic stress or develop drug resistance during chemotherapy. Increasing evidence indicates that the combination of autophagy inhibition and chemotherapy is a promising cancer treatment strategy. However, effective autophagy inhibitors with satisfied potency, bioavailability, and clearly-defined drug targets are still rare. Here, we report the identification of a potent autophagy inhibitor toosendanin which can effectively block autophagosome maturation, causing the accumulation of autophagy substrates in multiple cancer cells. Toosendanin did not inhibit the fusion process between autophagosome and lysosome but elevated lysosomal pH and impaired lysosomal enzymes activity. Using rat liver lysosome fraction and purified yeast V-ATPase, we found that toosendanin directly inhibited V-ATPase activity. By applying cellular thermal shift assay (CETSA), immunoprecipitation-coupled LC-MS/MS analysis, and biotin-toosendanin pull-down assay, we confirmed the direct binding between toosendanin and V-ATPase. Furthermore, toosendanin blocked chemotherapy-induced protective autophagy in cultured cancer cells and xenograft tumor tissues to significantly enhance anti-cancer activity. These results suggest that toosendanin has the potential to be developed into an anti-cancer drug by blocking chemotherapy-induced protective autophagy.

Asunto(s)

Antineoplásicos , Neoplasias , ATPasas de Translocación de Protón Vacuolares , Adenosina Trifosfatasas/metabolismo , Animales , Antineoplásicos/farmacología , Autofagia , Cromatografía Liquida , Humanos , Neoplasias/tratamiento farmacológico , Ratas , Espectrometría de Masas en Tándem , Triterpenos , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/farmacología

RESUMEN

Autophagy, a conserved cellular degradation and recycling process, can be enhanced by nutrient depletion, oxidative stress or other harmful conditions to maintain cell survival. 6-Hydroxydopamine/ascorbic acid (6-OHDA/AA) is commonly used to induce experimental Parkinson's disease (PD) lesions by causing oxidative damage to dopaminergic neurons. Activation of autophagy has been observed in the 6-OHDA-induced PD models. However, the mechanism and exact role of autophagy activation in 6-OHDA PD model remain inconclusive. In this study, we report that autophagy was triggered via mucolipin 1/calcium/calcineurin/TFEB (transcription factor EB) pathway upon oxidative stress induced by 6-OHDA/AA. Interestingly, overexpression of TFEB alleviated 6-OHDA/AA toxicity. Moreover, autophagy enhancers, Torin1 (an mTOR-dependent TFEB/autophagy enhancer) and curcumin analog C1 (a TFEB-dependent and mTOR-independent autophagy enhancer), significantly rescued 6-OHDA/AA-induced cell death in SH-SY5Y cells, iPSC-derived DA neurons and mice nigral DA neurons. The behavioral abnormality of 6-OHDA/AA-treated mice can also be rescued by Torin 1 or C1 administration. The protective effects of Torin 1 and C1 can be blocked by autophagy inhibitors like chloroquine (CQ) or by knocking down autophagy-related genes TFEB and ATG5. Taken together, this study supports that TFEB-mediated autophagy is a survival mechanism during oxidative stress and pharmacological enhancement of this process is a neuroprotective strategy against oxidative stress-associated PD lesions.

Asunto(s)

Antiparkinsonianos/farmacología , Autofagia/efectos de los fármacos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Encéfalo/efectos de los fármacos , Curcumina/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Naftiridinas/farmacología , Estrés Oxidativo/efectos de los fármacos , Trastornos Parkinsonianos/tratamiento farmacológico , Animales , Ácido Ascórbico , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Curcumina/análogos & derivados , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Femenino , Humanos , Ratones Endogámicos C57BL , Mitofagia/efectos de los fármacos , Oxidopamina , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Transducción de Señal , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

Autophagy is the lysosome-mediated bulk degradation of cellular components for material recycling to maintain cellular homeostasis. Autophagy was initially regarded as a nonselective process, however, recent evidence indicates that this process can in fact be highly selective, especially for targeting and degrading organelles, invading pathogens and protein aggregates. Recent studies have revealed an intrinsic connection between selective autophagy and neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Given the vital roles of selective autophagy in these neurodegenerative diseases, modulation of this process is emerging as a new therapeutic strategy for neuroprotection. This review introduces the concept of selective autophagy, provides an overview of the pathological connection between selective autophagy and neurodegenerative diseases, and discusses approaches to modulate selective autophagy for therapeutic effects against neurodegenerative diseases.

Asunto(s)

Autofagia/efectos de los fármacos , Autofagia/fisiología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Animales , Humanos

RESUMEN

Autophagy is a cellular bulk degradation pathway implicated in various diseases. Inhibition of autophagy has been regarded as a new therapeutic strategy for cancer treatment, especially in combination with chemotherapy. In our study, we identified two natural compounds, dauricine (DAC) and daurisoline (DAS), as two potent autophagy blockers through a high-content screening. DAC and DAS are alkaloids isolated from traditional Chinese medicine Rhizoma Menispermi. We systematically examined the effects of DAC and DAS on autophagy function in HeLa cells and found that DAC and DAS induced massive formation of autophagic vacuoles and lipidation of LC3. The accumulation of autophagic vacuoles and LC3 lipidation are due to blockage of autophagosome maturation as evidenced by interrupted colocalization of autophagsosome and lysosome, increased GFP-LC3/RFP-LC3 ratio and accumulation of autophagic substrate p62. Moreover, DAC and DAS impaired lysosomal function, as indicated by reduced lysosomal protease activity and increased lysosomal pH values. Importantly, we showed that DAC and DAS strongly inhibited the lysosome V-type ATPase activity. For the therapeutic potential, we found that DAC and DAS blocked the campothecin (CPT)-induced protective autophagy in HeLa cells, and dramatically sensitized the multiple cancer cells to CPT-induced cell death. In conclusion, our result shows that DAC and DAS are autophagy inhibitors which inhibit the lysosomal degradation of auophagic vacuoles, and sensitize the CPT-induced cancer cell death. The study implies the therapeutic potential of DAC and DAS in the treatment of cancers in combination of chemotherapy by inhibiting autophagy.

RESUMEN

6-OHDA plus ascorbic acid (AA) has long been used to induce Parkinson's disease in rodents, while only 6-OHDA is commonly used to induce cell damage in cellular PD models. AA was believed to act as an anti-oxidant to prevent the degradation of 6-OHDA; however, some studies suggested that AA dramatically enhanced the selectivity and toxicity of 6-OHDA. To understand the mechanisms by which 6-OHDA/AA induces cell death, we established a 6-OHDA/AA cell toxicity model in human dopaminergic neuroblastoma SH-SY5Y cells. We confirmed that the toxicity of 6-OHDA was dramatically increased in the presence of AA, and the toxicity can be prevented by a flavonoid, baicalein. Mechanistically, our research reveals that 6-OHDA/AA induces cell death mainly through the interruption of intracellular calcium homeostasis, which leads to calpain activation and mitochondrial damage. Baicalein prevents 6-OHDA/AA-induced intracellular calcium elevation as well as consequent mitochondria damage. Taken together, our study confirms that 6-OHDA/AA is a more sensitive model for inducing neuronal lesion in vitro and reveals the central role of intracellular calcium in 6-OHDA/AA-induced cell death. Our studies further show that baicalein prevents 6-OHDA/AA-induced cell death by inhibiting intracellular calcium elevation.

Asunto(s)

Adrenérgicos/toxicidad , Ácido Ascórbico/toxicidad , Hormonas y Agentes Reguladores de Calcio/toxicidad , Muerte Celular/efectos de los fármacos , Neuronas Dopaminérgicas/efectos de los fármacos , Flavanonas/metabolismo , Oxidopamina/toxicidad , Calcio/metabolismo , Calpaína/metabolismo , Línea Celular , Neuronas Dopaminérgicas/fisiología , Homeostasis , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM), an ancient yet still alive medicinal system widely used in East Asia, has played an essential role in health maintenance and diseases control, for a wide range of human chronic diseases like cancers and neurodegenerative diseases. TCM-derived compounds and extracts attract wide attention for their potential application as therapeutic agents against above mentioned diseases. AIM OF REVIEW: Recent years the enthusiasm in searching for autophagy regulators for human diseases has yielded many positive hits. TCM-derived compounds as important sources for drug discovery have been widely tested in different models for autophagy modulation. Here we summarize the current progress in the discovery of natural autophagy regulators from TCM for the therapeutic application in cancer and neurodegenerative disease models, aiming to provide the direct link from traditional use to new pharmacological application. METHODS: The present review collected the literature published during the recent 10 years which studied the effect of TCM-derived compounds or extracts on autophagy regulation from PubMed, Web of Science, Google Scholar and Science Direct. The names of chemical compounds studied in this article are corresponding to the information in journal plant list. RESULTS: In this review, we give a brief introduction about the autophagy and its roles in cancer and neurodegenerative disease models and describe the molecular mechanisms of autophagy modulation. We also make comprehensive lists to summarize the effects and underlying mechanisms of TCM-derived autophagy regulators in cancer and neurodegenerative disease models. In the end of the review, we discuss the current strategies, problems and future direction for TCM-derived autophagy regulators in the treatment of human diseases. CONCLUSIONS: A number of data from in vivo and in vitro models indicated TCM derived compounds and extracts hold great potential for the treatment of human diseases including cancers and neurodegenerative diseases. Autophagy, as a novel and promising drug target involved in a wide range of human diseases, can be modulated by many TCM derived agents, indicating autophagy modulation may be an important mechanism underlying the therapeutic effect of TCM in treating diseases. Furthermore, we look forward to seeing the discovery of ideal autophagy modulators from TCM with considerably higher selectivity for the treatment of human diseases.

Asunto(s)

Autofagia/efectos de los fármacos , Medicamentos Herbarios Chinos/uso terapéutico , Neoplasias/tratamiento farmacológico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Etnofarmacología/métodos , Humanos , Medicina Tradicional China/métodos , Fitoterapia/métodos

RESUMEN

OBJECTIVE: To explore the studies and application status of sentinel lymph node biopsy (SLNB) in breast cancer in China by statistically analyzing the relevant domestic literature. METHODS: The literatures published from January 1999 to December 2005 were searched in the databases of China, Info, CBM and CNKI retrieval system with "breast tumor, SLN and SLNB" as the key words. A total of 88 manuscripts were selected with 2 new reports of SLNB. The successful rate, accuracy, false-negative rate and sensitivity of SLNB were analyzed by SPSS 10.0 statistical analysis software. RESULTS: Among a total of 6282 patients, the detection rate of SLNB was 90.82% (5705/6282) and the overall false-negative rate 9.69% (259/2671). The prediction sensitivity, specificity, false positive rate, accuracy, negative and positive predictive value of SLN for axillary lymph nodes status were 90.30%, 99.64%, 0.41%, 86.52%, 92.11% and 99.55% respectively. CONCLUSION: SLNB can accurately predict the axillary lymph node metastasis. And its detection rate is correlated with patient age and tumor location. The detection rate and false-negative rate has nothing to do with the tracer injection site. A combined regimen has a higher detection rate and a low false negative rate. Affecting the whole breast, SLN is not correlated with a particular area of breast. The validation phase of SLNB in breast cancer is currently feasible in China.

Asunto(s)

Neoplasias de la Mama/patología , Ganglios Linfáticos/patología , Biopsia del Ganglio Linfático Centinela/métodos , Adulto , Anciano , Anciano de 80 o más Años , Reacciones Falso Negativas , Femenino , Humanos , Persona de Mediana Edad , Sensibilidad y Especificidad , Adulto Joven

RESUMEN

OBJECTIVE: To study the relation between histopathologic grading and some of the cytogenetic and molecular biology characteristics of breast cancer. METHODS: On the basis of estrogen receptor (ER) expression, DNA content, S-phase fraction (SPF), bcl-2 and mutant p53 protein (mtp53) expression were examined by FCM in 121 breast cancer patients. In 66 patients with invasive ductal breast cancer, histopathologic grading was also examined. RESULTS: The aneuploidy rate and DNA index (DI) were significantly different in grade I, II and III breast cancer. SPF and mtp53 expression significantly increased with increase in histopathologic grading (P < 0.05), but bcl-2 did not show this trend. SPF and mtp53 expression were significantly more in breast cancer with negative ER than in those with positive ER (P < 0.05). Again, no such differences in bcl-2 regardless of ER expression. Correlations existed between DI vs SPF, DI vs mtp53, and SPF vs mtp53 expressions (P < 0.01) but bcl-2 did not correlate with any one of them. CONCLUSION: Cytogenetic and molecular biology studies on the basis of histopathologic grading may provide more information in prognostic prediction of breast cancer.

Asunto(s)

Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Receptores de Estrógenos/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Adolescente , Adulto , Aneuploidia , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , ADN de Neoplasias/análisis , Femenino , Citometría de Flujo , Humanos , Persona de Mediana Edad , Mutación , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores de Estrógenos/genética , Fase S , Proteína p53 Supresora de Tumor/genética