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ObjectiveTo investigate the effect of different oxygen concentration on the proliferation and autophagy of colon cancer cells and to explore the effect of Yangyin Huayu Jiedu Preseription （YHJP） on autophagy and apoptosis of colon cancer cells under hypoxia based on phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway. MethodHCT-116 cells were divided into normoxia group， 1% O2 group， and 5% O2 group. Cell viability was detected by cell proliferation assay （MTS）， and autophagy was observed based on monodansylcadaverine （MDC） staining. HCT-116 cells were treated with YHJP in 5% O2 microenvironment. The cells were divided into normal group， blank serum group， and low-， medium-， high-dose YHJP groups （5%， 15%， 25% serum containing YHJP）. Cell inhibition rate in each group was calculated by MTS， and changes in the rate of autophagy were detected based on MDC staining. Annexin V-fluorescein isothiocyanate （FITC）/propidium iodide （PI） was employed to detect the apoptosis rate of each group. Western blotting was applied to measure the expression of autophagy proteins microtubule-associated protein 1 light chain 3 （LC3Ⅱ/Ⅰ）， yeast Atg6 homolog （Beclin-1）， ubiquitin-binding scaffold protein p62 （p62）， apoptosis-related proteins B-cell lymphoma-2 （Bcl-2）， Bcl-2/adenovirus E1B interacting protein 3 （BNIP-3）， and Bcl-2 associated X protein （Bax）， cleaved cysteine-aspartic acid protease-3 （Caspase-3）， hypoxia-inducible factor-1α （HIF-1α） and pathway proteins PI3K， phosphorylated （p）-PI3K， Akt， and p-Akt. ResultCell survival rates of the 1% O2 and 5% O2 groups were increased compared with that in the normoxia group， particularly the 5% O2 group （P<0.01）. The fluorescence intensity for autophagy in 1% O2 and 5% O2 groups was significantly increased compared with that in the normoxia group， especially the 5% O2 group. In the presence of 5% O2， compared with the blank serum group， medium-dose and high-dose YHJP groups showed high cell inhibition rate， low autophagy rate， high apoptosis rate （P<0.01）， and low expression of Beclin-1 protein （P<0.05）. Compared with low-dose YHJP group， high-dose YHJP group demonstrated low expression of Beclin-1 protein （P<0.05）. Compared with the blank serum group， the three YHJP groups had low expression of LC3Ⅱ/Ⅰ protein （P<0.05， P<0.01）. Compared with the blank serum group， medium-dose and high-dose YHJP groups showed high expression of p62 protein （P<0.01）. Compared with low-dose YHJP group， high-dose YHJP group showed high expression of p62 protein （P<0.05）. Compared with the blank serum group， high-dose YHJP increased the expression of BNIP-3 and Bax and decreased the expression of Bcl-2 （P<0.01）. The expression of Bax protein in the high-dose YHJP group was increased compared with that in the low-dose YHJP group （P<0.05）. The expression of HIF-1α in the medium-dose and high-dose YHJP groups was decreased （P<0.01） and the expression of p-PI3K/PI3K and p-Akt/Akt in the high-dose YHJP group was increased （P<0.05， P<0.01） compared with that in the blank serum group. The expression of p-Akt/Akt was higher in the high-dose YHJP group than in the medium-dose YHJP （P<0.05）. ConclusionHypoxic microenvironment can significantly promote autophagy and proliferation of colon cancer cells. YHJP can significantly inhibit autophagy and proliferation and promote apoptosis of colon cancer cells in 5% O2 environment by up-regulating the PI3K/Akt signaling pathway.
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Hypoxic microenvironment is a common phenomenon of liver diseases and runs through the whole process of the development and progression of liver diseases. In recent years, the research on liver fibrosis and hypoxic microenvironment of hepatocellular carcinoma has attracted more and more attention. Hypoxia-inducible factor (HIF) is the most important hypoxic stress factor found at present. This article reviews the characteristics of hypoxic microenvironment in the liver and liver diseases and further elaborates on the association of HIF overexpression with hepatocyte damage, formation of fibrosis, and malignant transformation of hepatocytes.
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Objective: Adiponectin (APN) is an endogenous cytokine that mediates the development and progression of various tumors through its receptors (AdipoRs). The present study aimed to detect the expression and distribution of APN and its receptors (AdipoR1 and AdipoR2) in tongue squamous cell carcinoma (TSCC). Moreover, we explored whether the locoregional expression of APN was reg-ulated by HIF-1α in the hypoxic microenvironment. Methods: The expression and distribution of APN and its receptors in TSCC tissues were analyzed by immunohistochemical. Lentiviral expression vector for HIF-1α shRNA was constructed and stably transfected in TSCC cells to knock down HIF-1α expression. The mRNA and protein expression levels of APN and its receptors were detected using RT-PCR and Western blot, respectively, after hypoxic treatment. Results: The locoregional expression of APN and AdipoR1, but not AdipoR2, was upregulated at the early stages of T1, T2, and/or N0 stage, respectively, in tumor tissues compared to that in control paracancer-ous tissues (P<0.05 or P<0.01). The expression of APN and AdipoR1, but not AdipoR2, in TSCC cells was up-regulated on hypoxic treat-ment. Moreover, the expression of APN and AdipoR1 was down-regulated after shRNA knockdown of HIF-1α under hypoxia. Conclu-sions: The APN-AdipoR1 signaling pathway was activated and regulated by HIF-1α in the hypoxic environment of TSCC tissues.
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@#Objective To investigate the effects of hypoxic three-dimensional culture microenvironment on the proliferation of bone marrow mesenchymal stem cells and its mechanism. Methods P5 generation mouse bone marrow mesenchymal stem cells and P (3HB-co-4HB) were co-cultured under normoxic three-dimensional (20%) and hypoxic three-dimensional microenvironment (4%) respectively. After 24 hours, the proliferation of the two groups was determined by CCK-8 method. The expression of HIF-1α gene was detected by real-time quantitative PCR after 12 hours. Western blotting was used to detect the expression of HIF-1α protein after 24 hours. Results After 24 hours, the CCK-8 method showed that the OD value of the hypoxia group was significantly higher than that of the normoxia group (0.455±0.027 vs. 0.352±0.090, n=12, P<0.05). After 12 hours of hypoxic culture, the expression level of HIF-1α mRNA in the hypoxia group was significantly higher than that in the normoxia group (P<0.05). Compared with the normoxia group (0.47± 0.05), the relative expression level of HIF-1α protein in the hypoxia group (0.63±0.06) significantly increased in the Western blotting after 24 hours (n=3, P<0.05). Conclusion The hypoxic three-dimensional microenvironment can promote the proliferation of bone marrow mesenchymal stem cells, which may be related to the activation of HIF-1α signaling pathway.
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Objective To investigate the correlationbetween thecarcinoembryonic antigen ( CEA) level and hypoxic status of breast cancer, and itspotential of being a prognostic factor in breast cancer. Methods Breast cancer cell line MDA-MB-231 was cultured under hypoxic or normoxic condition to determine the protein level of CEA using Western blotting.30 breast cancer tissue sections were collected in Tianjin Medical University Cancer Institute and Hospital from April 2013 to June 2015.and were detected the expression of CEA, hypoxia-inducible factor-1α( HIF-1α) and carbonic anhydrase-9 ( CA-9 ) by immunohistochemistry.Serum CEA were detected by immunofluorescence assay of 166 breast cancer patients collected from April 2009 to June 2011.The correlation between the expression levels of CEA and breast cancer patients'clinical data was analyzed by Logistic univariate analysis.Kaplan-Meier method was employed to calculate the progression-free survival of patients with breast cancer. Log-rank test was conducted to compare the difference between groups.Results CEA levels were significantly higher in breast cancer cells under hypoxic condition in a time-dependent manner, compared to normoxiccontrol. Immunohistochemical staining indicated that CEA was coexpressed with HIF-1αand CA-9 in breast cancer and linear regression analysis revealed that CEA expression correlated with HIF-1α( P =0.0096 );Preoperative serum levels of CEA were closely related with tumor size ( P =0.015 ) andLymph node metastasis(P=0.032); CEA positive patients achieved a progression-free survival of 29.85months(versus 39.08 months in CEA negative patients, P=0.003).Conclusions Hypoxia upregulates CEA expression in breast cancer and the levels of preoperative serum CEA is a potential prognostic factor ofbreast cancer.