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1.  Autophagy inhibition enhances apigenin-induced apoptosis in human breast cancer cells 
Apigenin (4',5,7-trihydroxyflavone) is a member of the flavone subclass of flavonoids present in fruits and vegetables. The involvement of autophagy in the apigenin-induced apoptotic death of human breast cancer cells was investigated. Cell proliferation and viability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Flow cytometry, fluorescent staining and Western blot analysis were employed to detect apoptosis and autophagy, and the role of autophagy was assessed using autophagy inhibitors. Apigenin dose- and time-dependently repressed the proliferation and clonogenic survival of the human breast cancer T47D and MDA-MB-231 cell lines. The death of T47D and MDA-MB-231 cells was due to apoptosis associated with increased levels of Caspase3, PARP cleavage and Bax/Bcl-2 ratios. The results from flow cytometry and fluorescent staining also verified the occurrence of apoptosis. In addition, the apigenin-treated cells exhibited autophagy, as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles (AVOs) by flow cytometry. Furthermore, the results of the Western blot analysis revealed that the level of LC3-II, the processed form of LC3-I, was increased. Treatment with the autophagy inhibitor, 3-methyladenine (3-MA), significantly enhanced the apoptosis induced by apigenin, which was accompanied by an increase in the level of PARP cleavage. Similar results were also confirmed by flow cytometry and fluorescence microscopy. These results indicate that apigenin has apoptosis- and autophagy-inducing effects in breast cancer cells. Autophagy plays a cyto-protective role in apigenin-induced apoptosis, and the combination of apigenin and an autophagy inhibitor may be a promising strategy for breast cancer control.
doi:10.3978/j.issn.1000-9604.2013.04.01
PMCID: PMC3626985  PMID: 23592903
Apoptosis; autophagy; apigenin; breast cancer; 3-methyladenine
2.  Suppression of Liver Regeneration and Hepatocyte Proliferation in Glypican 3 Hepatocyte-targeted Transgenic Mice 
Hepatology (Baltimore, Md.)  2010;52(3):1060-1067.
Glypican 3 (GPC3) belongs to a family of glycosylphosphatidylinositol-anchored, cell-surface heparan sulfate proteoglycans. GPC3 is over-expressed in hepatocellular carcinomas (HCC). Loss-of-function mutations of GPC3 result in the Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by overgrowth of multiple organs, including liver. Our previous study showed that GPC3 plays a negative regulatory role in hepatocyte proliferation, and this effect may involve CD81, a cell membrane tetraspanin. To further investigate GPC3 in vivo, we engineered transgenic (TG) mice over-expressing GPC3 in liver under the control of the albumin promoter. GPC3 TG mice with hepatocyte-targeted over-expressed GPC3 develop normally compared with their non-transgenic littermates, but have a suppressed rate of hepatocyte proliferation and liver regeneration after partial hepatectomy (PHx). Moreover, gene array analysis revealed a series of changes in the gene expression profiles in TG mice, both in normal mice and during liver regeneration. In unoperated GPC3 TG mice there was over-expression of Runx3 (7.6 fold), C/EBPα (2.5 fold) and GABA A Receptor (2.9 fold) and Wnt7b (2.8 fold). There was down-regulation of IGF BP1 (8.4 fold), Rab2 (5.6 fold), beta Catenin (1.7 fold), TGF beta 1 (3.1 fold), Nodal (1.8 fold) and Yap (1.4 fold). Changes after hepatectomy included decreased expression in several cell cycle related genes.
Conclusion
Our results indicate that in GPC3 transgenic mice, hepatocyte over-expression of GPC3 suppresses hepatocyte proliferation and liver regeneration, and alters gene expression profiles in GPC3 TG mice, in which potential cell cycle related proteins and multiple other pathways are involved and affected.
doi:10.1002/hep.23794
PMCID: PMC2936713  PMID: 20812357
glypican 3; liver regeneration; partial hepatectomy; gene array; cell cycle
3.  Classification and clinical features of headache patients: an outpatient clinic study from China 
The Journal of Headache and Pain  2011;12(5):561-567.
This study aimed to analyze and classify the clinical features of headache in neurological outpatients. A cross-sectional study was conducted consecutively from March to May 2010 for headache among general neurological outpatients attending the First Affiliated Hospital of Chongqing Medical University. Personal interviews were carried out and a questionnaire was used to collect medical records. Diagnosis of headache was according to the International classification of headache disorders, 2nd edition (ICHD-II). Headache patients accounted for 19.5% of the general neurology clinic outpatients. A total of 843 (50.1%) patients were defined as having primary headache, 454 (27%) secondary headache, and 386 (23%) headache not otherwise specified (headache NOS). For primary headache, 401 (23.8%) had migraine, 399 (23.7%) tension-type headache (TTH), 8 (0.5%) cluster headache and 35 (2.1%) other headache types. Overall, migraine patients suffered (1) more severe headache intensity, (2) longer than 6 years of headache history and (3) more common analgesic medications use than TTH ones (p < 0.001).TTH patients had more frequent episodes of headaches than migraine patients, and typically headache frequency exceeded 15 days/month (p < 0.001); 22.8% of primary headache patients were defined as chronic daily headache. Almost 20% of outpatient visits to the general neurology department were of headache patients, predominantly primary headache of migraine and TTH. In outpatient headaches, more attention should be given to headache intensity and duration of headache history for migraine patients, while more attention to headache frequency should be given for the TTH ones.
doi:10.1007/s10194-011-0360-2
PMCID: PMC3173628  PMID: 21744226
Outpatient; Headache; Cross-sectional study; Clinical feature; Migraine
4.  Classification and clinical features of headache patients: an outpatient clinic study from China 
The Journal of Headache and Pain  2011;12(5):561-567.
This study aimed to analyze and classify the clinical features of headache in neurological outpatients. A cross-sectional study was conducted consecutively from March to May 2010 for headache among general neurological outpatients attending the First Affiliated Hospital of Chongqing Medical University. Personal interviews were carried out and a questionnaire was used to collect medical records. Diagnosis of headache was according to the International classification of headache disorders, 2nd edition (ICHD-II). Headache patients accounted for 19.5% of the general neurology clinic outpatients. A total of 843 (50.1%) patients were defined as having primary headache, 454 (27%) secondary headache, and 386 (23%) headache not otherwise specified (headache NOS). For primary headache, 401 (23.8%) had migraine, 399 (23.7%) tension-type headache (TTH), 8 (0.5%) cluster headache and 35 (2.1%) other headache types. Overall, migraine patients suffered (1) more severe headache intensity, (2) longer than 6 years of headache history and (3) more common analgesic medications use than TTH ones (p < 0.001).TTH patients had more frequent episodes of headaches than migraine patients, and typically headache frequency exceeded 15 days/month (p < 0.001); 22.8% of primary headache patients were defined as chronic daily headache. Almost 20% of outpatient visits to the general neurology department were of headache patients, predominantly primary headache of migraine and TTH. In outpatient headaches, more attention should be given to headache intensity and duration of headache history for migraine patients, while more attention to headache frequency should be given for the TTH ones.
doi:10.1007/s10194-011-0360-2
PMCID: PMC3173628  PMID: 21744226
Outpatient; Headache; Cross-sectional study; Clinical feature; Migraine

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