Proc Natl Acad Sci U S A. 2010 Mar 15. [Epub ahead of print]
Mechanisms of acute axonal degeneration in the optic nerve in vivo.
Axonal degeneration is an initial key step in traumatic and neurodegenerative CNS disorders. We established a unique in vivo epifluorescence imaging paradigm to characterize very early events in axonal degeneration in the rat optic nerve. Single retinal ganglion cell axons were visualized by AAV-mediated expression of dsRed and this allowed the quantification of postlesional acute axonal degeneration (AAD). EM analysis revealed severe structural alterations of the cytoskeleton, cytoplasmatic vacuolization, and the appearance of autophagosomes within the first hours after lesion. Inhibition of autophagy resulted in an attenuation of acute axonal degeneration. Furthermore, a rapid increase of intraaxonal calcium levels following crush lesion could be visualized using a calcium-sensitive dye. Application of calcium channel inhibitors prevented crush-induced calcium increase and markedly attenuated axonal degeneration, whereas application of a calcium ionophore aggravated the degenerative phenotype. We finally demonstrate that increased postlesional autophagy is calcium dependent and thus mechanistically link autophagy and intraaxonal calcium levels. Both processes are proposed to be major targets for the manipulation of axonal degeneration in future therapeutic settings.
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Eur J Cancer. 2010 Mar 13. [Epub ahead of print]
Inhibition of autophagy augments 5-fluorouracil chemotherapy in human colon cancer in vitro and in vivo model.
Although 5-fluorouracil (5-FU)-based adjuvant chemotherapy is widely used in the treatment of colorectal cancer, novel therapeutic strategies need to be explored. It has been reported that autophagy is extensively implicated in cancer. However, the function of autophagy is not fully understood. In the present study, apoptosis induced by 5-FU in 3 human colon cancer cell lines (HCT116, DLD-1, and DLD-1/5-FU (a specific 5-FU-resistant sub-line)) was measured using MTT assay, DNA fragmentation assay, Hoechst 33342 staining, and caspase-3 immunoblotting. The autophagy activation induced by 5-FU treatment was revealed by microtubule-associated protein 1 light chain 3 (LC3) immunofluorescence and immunoblotting and p62 immunoblotting. Inhibition of autophagy by 3-methyladenine (3-MA) or small interference RNA targeting Atg7 (Atg7 siRNA) significantly augmented 5-FU-induced apoptosis. This synergistic effect of 5-FU and 3-MA was further confirmed in the DLD-1 xenograft tumour model. Tumour growth was suppressed more significantly with combination treatment than 5-FU treatment alone. In conclusion, autophagy was activated as a protective mechanism against 5-FU-induced apoptosis and its inhibition could be a promising strategy for adjuvant chemotherapy in colon cancer.
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BMC Cancer. 2010 Mar 16;10(1):98. [Epub ahead of print]
Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors.
ABSTRACT: BACKGROUND: : Beclin 1, an important autophagy-related protein in human cells, is involved in cell death and cell survival. Beclin 1 mapped to human chromosome 17q21. It is widely expressed in normal mammary epithelial cells. Although down-regulated expression with mono-allelic deletions of beclin 1 gene was frequently observed in breast tumors, whether there was other regulatory mechanism of beclin 1 was to be investigated. We studied the expression of beclin 1 and explored the possible regulatory mechanisms on its expression in breast tumors. METHOD: S: 20 pairs of tumors and adjacent normal tissues from patients with sporadic breast invasive ductal cancer (IDCs) were collected. The mRNA expression of beclin 1 was detected by real-time quantitative RT-PCR. Loss of heterozygosity (LOH) was determined by real-time quantitative PCR and microsatellite methods. The protein expression of beclin 1, p53, BRCA1 and BRCA2 was assessed by immunohistochemistry. CpG islands in 5' genomic region of beclin 1 gene were identified using MethylPrimer Program. Sodium bisulfite sequencing was used in examining the methylation status of each CpG island. RESULTS: : Decreased beclin 1 mRNA expression was detected in 70% of the breast tumors, and the protein levels were co-related to the mRNA levels. Expression of beclin 1 mRNA was demonstrated to be much higher in the BRCA1 positive tumors than that in the BRCA1 negative ones. Loss of heterozygosity was detected in more than 45% of the breast tumors, and a dense cluster of CpG islands was found from the 5' end to the intron 2 of the beclin 1 gene. Methylation analysis showed that the promoter and the intron 2 of beclin 1 were aberrantly methylated in the tumors with decreased expression. CONCLUSIONS: : These data indicated that LOH and aberrant DNA methylation might be the possible reasons of the decreased expression of beclin 1 in the breast tumors. The findings here shed some new light on the regulatory mechanisms of beclin 1 in breast cancer.
Mechanisms of acute axonal degeneration in the optic nerve in vivo.
Axonal degeneration is an initial key step in traumatic and neurodegenerative CNS disorders. We established a unique in vivo epifluorescence imaging paradigm to characterize very early events in axonal degeneration in the rat optic nerve. Single retinal ganglion cell axons were visualized by AAV-mediated expression of dsRed and this allowed the quantification of postlesional acute axonal degeneration (AAD). EM analysis revealed severe structural alterations of the cytoskeleton, cytoplasmatic vacuolization, and the appearance of autophagosomes within the first hours after lesion. Inhibition of autophagy resulted in an attenuation of acute axonal degeneration. Furthermore, a rapid increase of intraaxonal calcium levels following crush lesion could be visualized using a calcium-sensitive dye. Application of calcium channel inhibitors prevented crush-induced calcium increase and markedly attenuated axonal degeneration, whereas application of a calcium ionophore aggravated the degenerative phenotype. We finally demonstrate that increased postlesional autophagy is calcium dependent and thus mechanistically link autophagy and intraaxonal calcium levels. Both processes are proposed to be major targets for the manipulation of axonal degeneration in future therapeutic settings.
====
Eur J Cancer. 2010 Mar 13. [Epub ahead of print]
Inhibition of autophagy augments 5-fluorouracil chemotherapy in human colon cancer in vitro and in vivo model.
Although 5-fluorouracil (5-FU)-based adjuvant chemotherapy is widely used in the treatment of colorectal cancer, novel therapeutic strategies need to be explored. It has been reported that autophagy is extensively implicated in cancer. However, the function of autophagy is not fully understood. In the present study, apoptosis induced by 5-FU in 3 human colon cancer cell lines (HCT116, DLD-1, and DLD-1/5-FU (a specific 5-FU-resistant sub-line)) was measured using MTT assay, DNA fragmentation assay, Hoechst 33342 staining, and caspase-3 immunoblotting. The autophagy activation induced by 5-FU treatment was revealed by microtubule-associated protein 1 light chain 3 (LC3) immunofluorescence and immunoblotting and p62 immunoblotting. Inhibition of autophagy by 3-methyladenine (3-MA) or small interference RNA targeting Atg7 (Atg7 siRNA) significantly augmented 5-FU-induced apoptosis. This synergistic effect of 5-FU and 3-MA was further confirmed in the DLD-1 xenograft tumour model. Tumour growth was suppressed more significantly with combination treatment than 5-FU treatment alone. In conclusion, autophagy was activated as a protective mechanism against 5-FU-induced apoptosis and its inhibition could be a promising strategy for adjuvant chemotherapy in colon cancer.
====
BMC Cancer. 2010 Mar 16;10(1):98. [Epub ahead of print]
Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors.
ABSTRACT: BACKGROUND: : Beclin 1, an important autophagy-related protein in human cells, is involved in cell death and cell survival. Beclin 1 mapped to human chromosome 17q21. It is widely expressed in normal mammary epithelial cells. Although down-regulated expression with mono-allelic deletions of beclin 1 gene was frequently observed in breast tumors, whether there was other regulatory mechanism of beclin 1 was to be investigated. We studied the expression of beclin 1 and explored the possible regulatory mechanisms on its expression in breast tumors. METHOD: S: 20 pairs of tumors and adjacent normal tissues from patients with sporadic breast invasive ductal cancer (IDCs) were collected. The mRNA expression of beclin 1 was detected by real-time quantitative RT-PCR. Loss of heterozygosity (LOH) was determined by real-time quantitative PCR and microsatellite methods. The protein expression of beclin 1, p53, BRCA1 and BRCA2 was assessed by immunohistochemistry. CpG islands in 5' genomic region of beclin 1 gene were identified using MethylPrimer Program. Sodium bisulfite sequencing was used in examining the methylation status of each CpG island. RESULTS: : Decreased beclin 1 mRNA expression was detected in 70% of the breast tumors, and the protein levels were co-related to the mRNA levels. Expression of beclin 1 mRNA was demonstrated to be much higher in the BRCA1 positive tumors than that in the BRCA1 negative ones. Loss of heterozygosity was detected in more than 45% of the breast tumors, and a dense cluster of CpG islands was found from the 5' end to the intron 2 of the beclin 1 gene. Methylation analysis showed that the promoter and the intron 2 of beclin 1 were aberrantly methylated in the tumors with decreased expression. CONCLUSIONS: : These data indicated that LOH and aberrant DNA methylation might be the possible reasons of the decreased expression of beclin 1 in the breast tumors. The findings here shed some new light on the regulatory mechanisms of beclin 1 in breast cancer.
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