| BackgroundBiological basis of tumorigenesis is loss of regulation of cell proliferation. The growth potential of the cells is affected by many carcinogenic factors, such as physical, chemical, biological and others factors, the activation of the normal cells in the proto-oncogene and their cancer had cancer cells. And then the cell obtained continued proliferation ability. After the occurrence of tumor, tumor cell’s ability to obtain continuous proliferation, in the process of its rapid proliferation tumor tissue growth. In this process, the tumor growth rate faster than the growth of blood vessels, within the tumor will appear blood supply shortage caused by inadequate supply of oxygen within the tumor that is hypoxia. Hypoxia is one of the typical characteristics include liver cancer, breast cancer, prostate cancer, pancreatic cancer and other solid tumors. It refers to the use of oxygen or oxygen pressure reducing state falls below the critical value, and it will be restricted or even termination physiological function of organs, tissues and cells.Some researchs found hypoxic microenvironment of the tumor significantly reduce the results of radiotherapy, chemotherapy and surgery. The mechanism may be excited to generate oxygen radicals, it can kill target cells, so as to achieve the purpose of treatment of cancer. However, tumor hypoxia zone due to lack of oxygen and produce fewer free radicals, reduce the therapeutic effect. Compared with normoxic area the hypoxic regions of tumor cells can survive and continue to proliferate after treatment. With further research, discovery of tumor hypoxia is much more than just its own role in the resistance to radiotherapy, and throughout the various areas of the cell. Body by intracellular Heme, redox state, mitochondrial ROS feel external oxygen signal change. By gaseous molecules NO, CO, etc., KATP plasma channels, intracellular free calcium, cyclic nucleotides, PKC, MAPK and other stress-related signaling pathways to regulate co-expression of mitochondrial-encoded subunits and breathing part of nuclear-encoded subunits, thereby changing the energy production and use. There are some researches suggestion intermittent hypoxic preconditioning can enhance expression of the heat shock proteins and antioxidant enzyme system, regulation of gene expression in mitochondria, inhibition of intracellular calcium overload, effect of nuclear membrane calcium transport, increase glucose transport capacity and activation of hypoxia signal transduction pathways.Hsp90 is one of the most important a class of proteins superfamily. Hsp90 is highly conserved during evolution. It plays an important role in the cell. So far, research findings its substrates have been more than 200 kinds of protein. More and more studies have found the incidence and treatment of cancer also involves all aspects of the body. However, the current therapeutic agents are mostly for a single signal path, treatment is not ideal. The combination therapy resulted in complicated and more side effects, therefore the role of drugs in multiple signaling pathways have good prospects. Hsp90 is a molecular target multiple signaling pathways, several studies have also found that it was highly expressed in a variety of tumors, it is potentially important target in cancer research for therapy. Since the first anti-tumor activity of the Hsp90 inhibitor geldanamycin was found the Hsp90 was as a target for therapeutic drugs have been for 20 years in US National Cancer Center (Bethesda, MD, USA). So far there have been a variety of anti-tumor activity of Hsp90 inhibitors are found, some of them with good results into the I or II clinical trials, such as geldanamycin,17-AAG,17-DMAG and so on. In particular, geldanamycin derivative 17-DMAG because of its good anti-tumor effect, better water solubility and antitumor activity has developed into a good prospect of anticancer drugs.CyclinB1 control cells from the G2 phase to M phase, is one of the cell G2/M phase of the key protein and also as one of the important substrate proteins of Hsp90. There are two important cell cycle checkpoint G1 and G2 in cell cycle progression to avoid cellular DNA replication and protein wrong in normal mammalian cells. However the G1 checkpoint defects in tumor cells. Therefore, the process of cell division in cancer tumor cells tend to rely on the G2 cell cycle checkpoint to avoid lethal tumor cell division. Tumor cell cycle G2 checkpoint of a targeted which has become an attractive target for cancer therapy. CyclinB1 as the G2/M phase of key protein plays a very important role of the cancer treatment and prognosis. Currently it has a variety of studies have confirmed that cyclinB1 abnormal expression in a variety of tumor including stomach cancer, esophageal cancer, etc. Several studies have confirmed that the expression level of cyclinB1 tumor therapy has obvious correlation. It also a growing concern due to showed close links with tumor development, diagnosis, treatment and prognosis.Although cyclinB1 Hsp90 is one of the substrate, but the specific role of the relationship between the two has not yet entirely clear. However, there no doubt that there are important links between them. Our previous studies show Hsp90 inhibitor 17-DMAG can increase the expression of cyclinB1, Some studies have confirmed that 17-DMAG by regulating cdc2 protein induces cyclinB1 accumulation and leading to cycle arrest. It is to say 17-DMAG induced cyclinBl accumulation lead to cell G2/M phase arrest and induce apoptosis. So far many studies have shown that tumor hypoxia may reduce the sensitivity to drugs and induces tumor cell cycle distribution changes. G2/M phase reduction means G2/M phase protein cyclinB1 of expression will change, but the expression of what cyclinBl changes and specific mechanisms occur study are to be further explored in hypoxia.The establishment of hypoxic cells in vitro model and application Hsp90 inhibitor 17-DMAG to explore the relationship between the expression and regulation mechanism cyclinBl and liver cancer cells to 17-DMAG resistance under hypoxia.Objectives1. Hepatocellular carcinoma cell lines established hypoxic stress model.2. Studied under normoxic and hypoxic state, the proliferation of hepatocellular carcinoma cells and its effect on cell cycle.3. Studied under normoxic or hypoxic conditions with different concentrations of Hsp90 inhibitor 17-DMAG inhibition of cell cycle and its effect on the growth of hepatoma cell lines.4. Effects of hypoxia and 17-DMAG cyclinB1 expression of cell cycle proteins.5. Under hypoxic state cyclinBl expression of HCC sensitivity to reduce the impact of 17-DMAG.6. Study the tumors in nude mice in vivo hypoxic tumor regions cyclinBl expression.Methods1. Establish hypoxic stress model:the HepG2 and Huh7 cells were cultured at different times of 1% 02 hypoxia (Oh,24,48,72h), CCK-8 assay at various time points cell proliferation. Western-blot detection of whole cell protein HIFla, observe the effect of hypoxia.2. HepG2 and Huh7 cells were cultured at different times (Oh,6h,12h,24h,48h) of hypoxia, flow cytometry detect the impact of hypoxia on cell cycle.3. HepG2 and Huh7 cells were cultured under normoxic or hypoxic state with different concentrations (0,0.01,0.05,0.1,0.5,1,5,10μM) of the Hsp90 inhibitor 17-DMAG were treated 24h or 48h, with CCK-8 cell survival assay, flow cytometry detect the impact of hypoxia on cell cycle, Western-blot detection relevant protein expression under normoxic or hypoxic conditions.4. Using plasmid transfection cyclinBl-mCherry establish stably transfected cell lines, continuously observed by confocal microscopy within 24h in the synthesis of proteins and cells cyclinB1 positioned within the cell.5. The use of Real-time PCR technology to detect cyclinB1 RNA transcription within Huh7 and HepG2 cells, the use of coimmunoprecipitation (Co-IP) technology to detect cyclinB1 be labeled ubiquitin situation.6. SiRNA technology use to interference cyclinB1 expression in HepG2 cells, the cells were divided into a control group and SiRNA interference group, in normoxic cultured and treated with different concentrations of 17-DMAG 24h, CCK-8 assay cell survival.7. HepG2 cells were injected into nude mice to form tumors, to observe the effect of 17-DMAG brown aneurysm growth. By immunohistochemistry difference intratumoral hypoxia or normoxic area and to detect the expression of the corresponding region cyclinB1.Results1. Intracellular HIF-lα expression was significantly increased after hypoxia treatment to achieve the desired effect of hypoxia. Hypoxia significantly promote hematoma cell lines HepG2 and Huh7 proliferation. Compared with normoxic group, different concentrations of 17-DMAG treatment of liver cancer cell lines HepG2 and Huh7 24h after less toxic hypoxic cell proliferation, cell G2/M phase arrest low level and cyclinBl low expression levels.2. Liver cancer cell lines HepG2 and Huh7 was treatment different time under hypoxic conditions after appears the cell cycle G1/S phase increased and there is time-dependent. Hypoxia can reduce the 17-DMAG induced liver cancer cell lines HepG2 and Huh7 G2/M phase arrest. In normoxic or hypoxia 17-DMAG can induce liver cancer cell lines HepG2 and Huh7 apoptosis.3.17-DMAG induces accumulation cyclinB1 by regulating cell cycle proteins cdc2 protein. Compared with normoxia, hypoxia decreased expression cyclinB1 and increase through the ubiquitin-proteasome degradation pathway. Compared with the control group after the expression of siRNA cyclinB1 17-DMAG decrease of inhibition rates.4.17-DMAG can inhibit tumor growth in tumor-bearing nude mice, tumors in vivo expression of HIF-1α expression in areas of high and cyclinB1 low expression.Conclusions1.In the hypoxic stress modeling process, the researchers showed that a 1% concentration of O2 can significantly promote hematoma cell lines HepG2 and Huh7 proliferation and induces liver cancer cell lines HepG2 and Huh7 cell cycle, resulting in an increase in the G1/S, reduce G2/M phase.2. Different concentrations of 17-DMAG under normoxic or hypoxic conditions treated hematoma cell lines HepG2 and Huh7, compared with normoxia, hypoxia may reduce the block G2/M extent of hepatocellular carcinoma cell line and it is reduce sensitivity liver cancer cell line to 17-DMAG.3. Under hypoxic conditions, the cancer by lowering the level of transcription cylinBland increase proteasome degradation rate to reduced 17-DMAG induced cyclinBl accumulation. Thereby reducing the sensitivity of liver cells to 17-DMAG.4. Tumor-bearing mice showed that,17-DMAG can significantly reduce the tumor size, tumor hypoxic regions exist in a significant and in hypoxic regions cyclinB1 reduce the level of transcription.In summary the hypoxia by reduced the cyclinBl expression and promote degradation to reduce the sensitivity of liver cells to the Hsp90 inhibitor 17-DMAG. |
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