| Salivary adenoid cystic carcinoma (SACC) is one of the common types of salivary gland malignancies in the head and neck. Clinically, it is described as one of the most destroyed and unpredicted malignant tumors because its propensity of perineural invasion, multiple recurrences and a high incidence of distant metastases.Currently, surgery is the most effective treatment, which is usually followed by adjuvant radiotherapy including implantation of Iodine-125 seeds.Although the 5-year survival rate is relatively high, the 10-year and longer survival rate remains low.Therefore, further understanding of the characteristic biological behavior and molecular genetics of ACC may provide new insights into the novel treatment of the disease.In recent years, along with the development of ACC molecular mechanism research, it was found that the treatment of ACC to a breakthrough, need to find more targeted new gene, by gene transfection inhibit tumor cell proliferation, inducing tumor cell apoptosis, is the tumor gene therapy strategies.Hypoxia is a common feature of solid tumors which occurs when the proliferation of the tumor outstrips the speed of blood vessels formation. Clinically, hypoxia may be deleterious for the tumor control by increasing resistance to radiation, preventing drug delivery through lack of blood vessels, and adaptation to the microenvironment through genomic instability. Hypoxia-inducible factor-1 (HIF-1) is an important heterodimeric transcriptional factor composed of a labile HIF-1α subunit and a stable HIF-1 β subunit. Under normoxia condition, HIF-la is degraded proteasomally; while under hypoxia condition, HIF-la is stabilized and moves to the nucleus and freely forms a complex with HIF-1 β which binds to the hypoxia response elements (HRE).The presence of HIF-la has been observed in the majority of the common human cancers, such as head and neck cancer, ovarian cancer and oesophageal cancer, which is associated with poor prognosis and resistance to therapy. Costa et al found that ordinary as well as high-grade transformation adenoid cystic carcinoma exhibited HIF-la expression, whereas it was observed only occasionally in normal salivary tissue adjacent to the tumor.Bcl-2/adenovirus E1B 19 kDa-interacting protein3 (BNIP3), a member of BH3-only subfamily of Bcl-2 superfamily, belongs to pro-apoptotic proteins. The expression of BNIP3 is regulated by hypoxia and other factors. BNIP3 protein is localized on the outside of the mitochondrial membrane, and once stimulated, it integrates into the mitochondrial membrane, leading to permeabilization of the transition pore and decreasing the mitochondrial membrane potential resulting in chromatin condensation, DNA-fragmentation and finally apoptosis. Studies have shown that abnormal expression of BNIP3 is correlated with the growth progression and drug resistance of many tumors in recent years. Recent studies have shown that BNIP3 is also closely related to autophagy.BNIP3, which is upregulated by the transcription factor HIF-la, plays a role in hypoxia-induced autophagic cell death. However, the role of BNIP3 in hypoxia-induced autophagy is still extensively unexplored in ACC. Furthermore, the relationship between expression of BNIP3 and clinicopathologic features of tumors has not been studied in salivary adenoid cystic carcinoma tissue.Autophagy is broadly refers to the cellular catabolic processes in which cytoplasmic target material is transported to lysosomes for degradation. The lysosome is the primary organelle for degradation through its wide array of resident acid hydrolases. Classical autophagy process can be largely divided into three major steps that require specific autophagy-related(Atg) genes, which include initiation of autophagosome formation, autophagosome membrane elongation and completion and autophagosome maturation through fusion with lysosomes and cargo degradation through lysosomal enzymes and recycling. Chloroquine(CQ) inhibits lysosomal acidification and therefore prevents by blocking autophagosome fusion and degradation. Our group previously investigated 79 consecutive patients with head and neck ACC and observed the expression of autophgy-related LC3 and Beclin 1. Our results indicated that LC3 and Beclin 1 may play an important role in the tumorigenesis of ACC.Recent studies have uncovered several links between hypoxia and the induction of autophagy. However, the molecular mechanism of hypoxia-induced autophagy in ACC is not clear.Objective:1、Cobalt Chloride was applied to construct in vitro the cells hypoxia model.Under the condition of low oxygen, cultivation of ACC- M in vitro and observation of cell survival, the expression of hypoxia related genes HIF-la and BNIP3 and autophagy related gene Beclinl and LC3 was detected and its mechanism was discussed..2、To study the effects of autophagy induced by hypoxia on invasive of ACC-M cells.And chloroquine inhibits autophagy process induced by hypoxia and affects the ACC-M cells invasive.Methods:1、ACC-M cells with exponential growth in routine culture were exposed to Cobalt Chloride to mimic hypoxic conditions. 2、MTT assay was utilized to quantify the viability of ACC-M cells to detect the inhibition effect of CoCl2。 Autophagosome was observed by a transmission electron microscopy. HIF-1α/BNIP3 signal pathway related genes and autophgy-related genes LC3 and Beclin 1 were detected using immunofluorescence staining, RT-PCR and Western blot. Data were expressed as the mean ± SEM of at least three independent experiments. Statistical comparisons between groups were performed using two-tailed Student’s t-test (SPSS 15.0). Values of p< 0.05 were considered significant.3、An invasion assay was performed using a BD BioCoat Growth Factor Reduced Matrigel Invasion Chamber according to the manufacturer’s instructions. The number of cells in seven randomly chosen fields of each filter was estimated by manual counting, and these experiments were independently performed at least three times. Data were expressed as the mean ± SEM of at least three independent experiments. Statistical comparisons between groups were performed using two-tailed Student’s t-test (SPSS 15.0). Values of p<0.05 were considered significant.Results:1、MTT assay was utilized to quantify the viability of ACC-M cells treated with CoCl2, which reduced the cell viability of ACC-M cells and the inhibition effect assumed an obvious dose-response relationship. Numerous autophagosome-like structures consisting of double membranes were observed using a transmission electron microscopy. The expression of HIF-la, BNIP3, LC3 and Beclin 1 were significantly improved by COCl2.2、Compared with untreated cells, invasive cell number under mimetic hypoxia was significantly increased (p<0.001).The number of invading ACC-M cells under mimetic hypoxia was markedly reduced with CQ treatment after 24h, and higher concentration of CQ showed a greater effect on inhibiting tumor invasion than lower concentration of CQ (p<0.01).Conclusion:1、Mimetic hypoxia by CoCl2 is capable of inducing autophagy through HIF-1α/BNIP3 pathway in ACC. HIF-1α/BNIP3 pathway may play an important role in mimetic hypoxa-induced autophagy in ACC.2、Inhibition of autophagy suppressed tumor invasion induced by hypoxia. These findings reinforce the importance of autophagy in the tumor progression and indicate that manipulation of hypoxia-induced autophagy may prevent tumor invasion in ACC. |
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