Objective: To investigate the role of adiponectin in human glioma cell lines against the temozolomide and the molecular regulation mechanism. Methods: Human glioma cell lines U251 and U-87MG were cultured in Dulbecco’s modified eagle medium (DMEM) containing 4500 mg/L glucose. MTT was used to measure cell growth ratio. Western blot was used to detect the protein levels of autophagy-related protein (Beclin 1, LC3 I/II, p62) and phosphorylated AMPK (p-AMPK) in human glioma cell lines. After AICAR and Compound C were administered, the change of p-AMPK and the autophagy level were examined by western blot. Results: While adiponectin stimulates AMPK in phosphatase and up-regulates the level of autophagy, human glioma cell lines obtain more resistance against the temozolomide, which is facilitated by AICAR and weakened by Compound C. Conclusion: As an important adipokine, adiponectin can up-regulate the glioma cell autophagy by activating the AMPK signaling pathway which increases the resistance of glioma cells to temozolomide.
Human Glioma is one of the most common malignancies in central nervous system and accounts for 20% - 40% intracranial tumors. At present, the treatment of glioma is mainly surgical resection, combined with radiotherapy, chemotherapy and other comprehensive treatments, but the overall therapeutic effectiveness is discontented with high recurrence rate and the poor prognosis [
Recombinant human full-length adiponectin/Acrp30 was purchased from R&D Systems (USA). AICAR and Compound C were received from Sigma (USA) and dissolved in Dimethyl sulfoxide (DMSO) (from sigma, USA). 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide; Thiazolyl blue (MTT) was from Sigma(UK). Temozolomide (TMZ) and antibodies against adiponectin receptor 2 were from abcom (USA). Antibodies against adiponectin receptor 1, LC3 I/II, becline 1, p62, AMPK, p-AMPK were purchased from Cell Signal Technology (CST) (USA). U-87MG and U251 cells were obtained from the American Type Culture Collection(USA) and were grown in Dulbecco’s modified eagle medium (DMEM) containing 4500 mg/L glucose, 1% penicillin/streptomycin with 10% foetal bovine serum (FBS) (all from Hyclone, USA) in 5% CO2/95% air at 37˚C. Cells were subcultured following enzymatic digestion using trypsin/EDTA solution (Hyclone, USA).
The MTT was dissolved in PBS solution at a concentration of 5 mg/ml and filtered through a 0.22μm filter to sterilize and remove insoluble residues, and then stored in the amber vials at 4˚C for a month. After 48 and 72 h incubation, 20 μl of the MTT solution was added to each well of 96-well plates and incubated for 4 h at 37˚C in a humidified atmosphere of 5% CO2. At end of the incubation period, DMSO was added to each well of 96-well plates for removing insoluble residues. The absorbance was determined at 490 nm. The A490 was taken as an index of the cell viability and the activity of mitochondria. The net absorbance from the plates of cells cultured with the control medium (not treated) was considered as 100% of the cell growth ratio and the mitochondrial activity.
The expression of adiponectin receptor 1, adiponectin receptor 2, LC3 I/II, becline 1, p62, AMPK, p-AMPK were determined by western blotting. Total protein content in each lane was determined using the bicinchoninic acid assay and then each lane containing equal amounts of protein were separated on a 10% - 15% SDSP-AGE gel. After transfer to nitrocellulose membranes, blots were blocked with 5% nonfat milk in 0.2% Tween 20 in Tris-buffered saline (TBS) for 1 h and then incubated with primary antibody at 4˚C overnight. Blots were then washed five times for 10 min in washing buffer (0.2% Tween 20 in TBS), followed by incubation for 2 h at room temperature with a specific secondary antibody. Subsequently, these specific antibodies were detected by using ECL Western blotting detection (Millipore, USA) and the fluorescence excitation was imaged on X-ray film. Normalization was based on the protein level of GAPDH and analyzed by Imagine J software.
All statistical analysis was performed using SPSS 13.0 (SPSS Inc. USA). All data were presented as mean ± SEM. All data were analyzed by analysis of t-test when normality and homogeneity of variance assumptions are satisfied. Significance was set at P < 0.05.
Adiponectin can’t display biological functions until adiponectin is combinated with adiponectin receptor (ADIPOR), locating on cell membrane surface. There are both ADIPOR1 and ADIPOR2 expression (
5 × 103 cells per well were seeded in 96 well plates in 1% FBS containing medium for 24h. Cells were subsequently treated with increasing concentrations (0 μg/ml, 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml, 3 μg/ml, 10 μg/ml) of adiponectin and incubated for a further 24 h. we assessed the cell growth ratio of the human glioma cell line, U251 and U87-MG, by MTT assay. It has been discovered that adiponectin at 3 μg/ml reliably produced near maximal stimulation and was used for subsequent mechanistic studies (P < 0.05,
starvation for 24 h, U251 and U-87MG cells were treat with 3 μg/ml adiponectin for a different time course (1 h, 6 h, 12 h, 24 h, 48 h). We find that adiponectin in the time course, 24 h, reliably produced near maximal stimulation and was used for subsequent mechanistic studies (P < 0.05,
The protein levels of autophagy in U251 and U-87MG cells were determined by Western blotting. After being incubated with 3 μg/ml adiponectin for 24 h, compared to the control group, the expression of Beclin1 and the ratio of LC3 II/I were significantly increased, the expression of p62 was sharply reduced (
value analysis by Image J showed that the expression of autophagy related proteins (Beclin1, LC3, p62) in the 3 μg/ml group compared with the untreated group has statistical difference(P < 0.05,
We detected the expression of AMPK and p-AMPK in glioma cells by Western blotting. U251 and U-87MG cells were incubated with 3 μg/ml adiponectin for a different time course. The expression of p-AMPK began to be increased at 12 h and reached the peak at 24 h (
WB showed that AICAR can increase the AMPK phosphorylation and the expression of autophagy related protein Beclin1 of glioma cell after being incubated with adiponectin, and gray value analysis showed that the expression of p-AMPK and Beclin were significantly increased in Ad + AICAR group compared to the control group; CC can reduce the AMPK phosphorylation and the expression of autophagy related protein of glioma cell after being incubated with adiponectin. Gray value analysis also showed that the expression of p-AMPK and Beclin were significantly reduced in Ad + CC group compared to the control group. The difference had statistically significant (P < 0.05,
We added different concentrations of temozolomide into culture medium after seeding plated the glioma cells
for 48 h and found that 100 μM, 1 mM temozolomide can significantly reduced the cell survival rate (P < 0.05,
Adiponectin, which is an important adipokine, is known to be a key molecule in the positive correlation between obesity and cancer, such as breast cancer, prostate cancer and colorectal cancer [
Autophagy is a homeostatic and evolutionarily conserved process, which degrades cellular contents containing useless macromolecules such as long-lived proteins, faulty aggregates or misfolded proteins, as well as damaged or redundant organelles such as mitochondria, endoplasmic reticulum, or peroxidase, in order to maintain the the cell homeostasis [
LC3 I becoming LC3 II is the specific process of autophagy, so that the ratio of LC3 II/I is regarded as an important indicator of the formation of autophagosome. The directly combination of p62 and LC3 is involved in the autolysosome’s development. If large number of autophagosome formed in cells without p62 consumption, it suggests that the formation of autolysosome was restrained and autophagy was inhibited [
Autophagy plays a double-edged sword role in the occurrence and development of tumor and has different effects in the different stages of the tumor. Autophagy limits tumor formation by preventing the accumulation of damaged proteins and organelles; after tumor formated, autophagy promotes tumor cell survival. In the process of treatment with chemotherapy drugs, autophagy makes a positive sense for the cancer cells to survive and adapt to changes in the external environment. As a result, autophagy presents a cell protection function what we don’t want to see―enhancing the tumor cells resistance to chemotherapeutic drugs [
As an important adipokine, adiponectin can up-regulate the glioma cell autophagy by activating the AMPK signaling pathway, which increases the resistance of glioma cells to temozolomide. The biological effects of adiponectin are thought to be clinically important in the pathophysiology of tumor development and progression.
Peng Sun,Feng Yan,Jinning Song,Xudong Ma, (2016) Adiponectin Supports Human Glioma Cells Survival against Temozolomide through Enhancement of Autophagic Response in Glioma Cells. Journal of Biosciences and Medicines,04,1-9. doi: 10.4236/jbm.2016.44001