Effects of Beclin 1 overexpression on aggressive phenotypes of colon cancer cells.

Beclin 1 is involved in autophagy, differentiation, apoptosis and cancer progression, and functions as a haploinsufficient tumor suppressor gene. The aim of the present study was to elucidate the function of Beclin 1 in colon cancer. A Beclin 1-expressing plasmid was transfected into HCT-15 and HCT-116 cells, and the phenotypes and associated molecules were determined. Beclin 1 transfectants were subcutaneously injected into nude mice to determine tumor growth, and proliferation and apoptosis levels using Ki-67 immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively. Beclin 1 overexpression inhibited viability as determined using a Cell Counting Kit-8 assay, inhibited migration and invasion as determined using a wound healing assay or Transwell assay, and lamellipodia formation by filamentous actin staining, induced autophagy as determined using electron microscopy, and light chain 3B (LC-3B) expression, and apoptosis as determined using Annexin V staining in the two cell lines (P<0.05). Beclin 1 induced G2 arrest of HCT-15 transfectants as determined using propidium iodide staining (P<0.05), whereas HCT-116 transfectants were arrested in G1 phase (P<0.05). The two transfectants exhibited increased expression of c-Myc, cyclin D1, ß-catenin, insulin-response element 1 and 78 kDa glucose-regulated protein compared with the control and mock cells as determined using the reverse transcription-quantitative polymerase chain reaction (P<0.05). Beclin 1 overexpression upregulated LC-3B and cyclin-dependent kinase 4 expression, but downregulated cyclin E expression of the cancer cell lines as determined using western blot analysis (P<0.05). Beclin 1 expression in vivo significantly suppressed the proliferation of colon cancer cells in xenograft models via inducing apoptosis by TUNEL, and inhibiting proliferation by Ki-67 expression (P<0.05). Beclin 1 overexpression may reverse aggressive phenotypes and suppress colon cancer tumor growth, and be employed as a target molecule for gene therapy of patients with colon cancer.

[Homer protein expression after focal brain contusion in rats].

OBJECTIVE: To investigate Homer protein expression after focal brain contusion and explore the relationship between expression and injury time. METHODS: Focal brain contusion in rats was established and Homer protein expression in brain at different injury intervals after contusion was detected by immunohistochemistry and Western blotting. RESULTS: A small amount of Homer positive expression cells were detected in control group, sham operated group and experimental group (0.5 h after contusion). The amount of Homer positive expression cells increased after 3 h and reached peak 12 h after contusion. The amount of positive cells continued to decrease 1 d after contusion and to the base level 7 d after contusion. Homer protein expression based on immunohistochemistry and Western blotting had statistical difference among adjacent groups. CONCLUSION: Expression of Homer protein near the focal contusion area shows time dependence after brain contusion in rats.

[Expression of matrix metalloproteinase-3 after brain contusion in rats].

OBJECTIVE: To investigate the expression of matrix metalloproteinase-3 after brain contusion and its applicability for estimating the age of brain contusion. METHODS: Rats had been divided into three groups: control group, sham operation group and brain contusion group. The expression of matrix metalloproteinase-3 at different time was detected by immunohistochemistry and Western blot. RESULTS: By the immunohistochemistry, no staining was observed in control and sham operation groups. The positive staining of MMP-3 appeared 6 hours after contusion, increased gradually in 24 hours and peaked 5 days after contusion, then started to decrease, 14 days after contusion still could be observed. By the Western blot analysis, no expression of MMP-3 was detected in control and sham groups. The positive staining of MMP-3 appeared 6 hours after contusion, increased gradually and maximized 5 days after contusion, then started to decrease, 14 days after contusion still could be found. CONCLUSION: Time-order expression of MMP-3 could be used for estimating the age of brain contusion in forensic pathology.

Study on changes of MMP-3 expression after brain contusion in rats.

To investigate the mechanism and to explore the applicability of MMP-3 (matrix metalloproteinase-3) to determine the age of brain contusion, expression of MMP-3 was studied by immunochemistry and Western blot techniques on model of brain contusion in rats. Brain contusion was performed by falling weight in adult male Sprague-Dawley rats after anesthetized with diethyl ether, then maintained with 2% pentobarbital sodium (30 mg/kg). Samples were collected at 6 and 12 h, 1, 3, 5, 7, 10 and 14 days after the contusion. Histopathological examination and immunostaining of MMP-3 were conducted using paraffin sections. Protein of MMP-3 bands was visualized by ECL kit in Western blot. Result showed that: No staining in control and sham operation group. The staining of MMP-3 positive appeared 6 h after contusion, and it becomes stronger 24 h after contusion, the staining reached the maximum at 5 days post-contusion, then it decrease, positive staining could still be observed at 14 days after contusion. No MMP-3 expression was detected in control and sham group by Western blot analysis. Brain contusion caused the appearance of a band of 45 ku molecular weight, which corresponds to an active form of MMP-3. Conclusions were drew that there is no expression of MMP-3 in normal brain, that the expression of MMP-3 appears 6h after TBI, and that there is a relationship between the expression of MMP-3 and the time course after TBI, and MMP-3 would be used as an indicator for estimating the age of traumatic brain contusion in forensic pathology.

[Animal model of grading skeletal muscle contusion due to blunt impact in rats].

OBJECTIVE: To establish a new animal model of grading skeletal muscle contusions that could be controllable and repetitive. METHODS: The rats' gastrocnemius was injured by a new weight-dropping device designed. The force acting on gastrocnemius with a comparatively constant duration and inducing elastic deformation of the gastrocnemius was expressed with velocity (v) and deformation (DF). Instant velocity was changed to create gastrocnemius contusions. Pathological changes of gastrocnemius were graded by the gross and histological examinations of 39 rats. RESULTS: At low level of impact (v: 2 m/s, DF: 5.5 mm), mild injuries were detected in epimysium and superficial layer of gastrocnemius. At moderate level of impact (v: 2.5 m/s, DF: 6.5 mm), the injuries were observed in epimysium and whole gastrocnemius. At high level of impact (v: 3 m/s, DF: 7.5 mm), severe injuries were seen deeper to soleus with more extensive skeletal muscle damage. CONCLUSION: Grading of skeletal muscle blunt force contusion is created by parameter of velocity and muscle deformation. The model could be used for further research on skeletal muscle contusions.