| Part1Hypoxia induces cell death in the form of nuclear condensation and its molecular expression patterns[Purpose]A special type of cell death called linearly patterned programmed cell necrosis (LPPCN) may serve as the spatial foundation for capillary-like structure formation. The previous study of our group has showed that hypoxia induced the formation of LPPCN. Followed by blood flowing into the capillary-like structure, vasculogenic mimicry (VM) was formed. The formation of LPPCN was correlated with Bcl-2family. To investigate the possibility that tumor cells form LPPCN-like cells with condensed nuclei and to determine its relationship with Bcl-2, hypoxic microenvironment was mimicked to investigate the time-dependent expression of Bcl-2family.[Methods]Both hypoxia mimetic CoCl2and GasPak Pouch are used to mimic the microenvironment. B16cells were used as the model to observe the formation of nuclear chromosomal condensation; Wound healing assay and SRB assay were used to detect the effect of hypoxia on migration ability and proliferation of tumor cells; Western Blot and Realtime-PCR were used to analyze the effect of hypoxia on protein and RNA expression of apoptosis-related gene. Immunofluorescence was used to determine the effect of hypoxia on the subcellular location of Bcl-2and bax.[Results]1) After36-48h of hypoxia, nuclear staining with Hoechst33342revealed chromosomal condensation in more than30%of tumor cells. Most of the nuclei were abnormally shaped and deeply stained. However, the typical morphologic characteristics of apoptosis such as nuclear fragmentation were rarely observed.2) Hypoxia significantly inhibited the proliferation of tumor cells compared with cells under normoxic condition.3) The number of cells in early apoptosis was significantly higher than that in late apoptosis among the cells treated with hypoxia mimetic CoC12. However, the number of cells in late apoptosis was significantly higher than that in early apoptosis among the cells treated with the GasPak Pouch.4) When cells were cultured in hypoxia,the expression of Bcl-2and Bax showed time-dependent expression patterns. There was a Bcl-2expression peak at36h, followed by decreased expression. On the other hand, Bax expression decreased with the duration of hypoxia to its lowest point, followed by an increase.5) When caspase-3inhibitor Ac-CHO-DEVD was added under hypoxia, the protein and RNA expression of Bcl-2and Bax showed the opposite trends of those tumor cells under hypoxia alone.[Conclusions]1) Hypoxia induced the nuclear condensation of tumor cells with the same characteristics with LPPCN cells;2) Tumor cells survived in hypoxia showed enhanced potential of motility,invasion and metastasis.3) Bcl-2family protein expression is time-dependent under hypoxic conditions. The maximum expression peak of Bcl-2may play a critical role in the formation of nuclear condensation.4) Ac-CHO-DEVD treatment has a significant protective effect on the expression of apoptosis-related protein effected by hypoxia.Part2Clinic pathologic analysis:the correlation of Bcl-2and EMT[Purpose]Clinical specimens of human hepatocellular carcinoma of97patients with complete follow-up data were studied in this part to observe the correlation of Bcl-2 and EMT and their effect on clinical prognosis. We analyze preliminarily the relationship between the nuclear (Nu) and cytoplasmic (Cyt) expression of Bcl-2and the EMT regulators Twist-1, Twist-2, and Snail and the effect of their coexpression on tumor metastasis,angiogensis and prognosis. Screening the co-expression combination that have the the greatest impact on the prognosis of HCC patients to provide a clue for the effect of the interaction of Bcl-2and EMT regulators on tumor shaping, invasion and metastasis.[Methods]Resected specimens of97patients identified as hepatocellular carcinoma by pathologists with complete follow-up data were obtained from General Hospital and Cancer Institute and Hospital of Tianjin Medical University between February2001and December2005to analyze the medical records and clinical follow-up data; Immunohistochemical staining was performed to observe the expression of Bcl-2and EMT related proteins. The relationship of them and their effect on tumor metastasis, VM and prognosis was analyzed with statistical methods.[Results]1) VM was found in18out of97HCC samples(19%) and metastasis was found in49out of97HCC samples(51%).2) Bcl-2was observed in both the nucleus and cytoplasm. A higher incidence of nonmembranous expression of P-catenin was found in patients positive for nuclear Bcl-2(Bcl-2-Nu) than in patients positive for cytoplasmic Bcl-2expression (Bcl-2-Cyt). In contrast, E-cadherin positive expression was lower in patients positive for Bcl-2-Nu expression than in patients positive for Bcl-2-Cyt expression.3) Survival analysis showed that patients positive for Bcl-2-Nu, Twist-1-Nu, Twist-1-Cyt or Snail had higher hazard ratios compared with patients with negative expression, whereas Bcl-2-Cyt and Twist-2positive expression did not.4) Coexpression of Bcl-2-Nu with any individual EMT regulator (Twist-1-Nu, Twist-1-Cyt, Twist-2, and Snail) was significantly correlated with metastasis, and the coexpression of Bcl-2-Cyt with Snail was significantly correlated with metastasis. Coexpression of Bcl-2-Nu with any individual EMT regulator was significantly correlated with VM, whereas coexpression of Bcl-2-Cyt with them did not.5) Patients expressing Bcl-2-Nu with Twist-1-Cyt showed the highest hazard ratio (HR=3.824), and the hazard ratio was higher than in cases expressing either protein alone (HR=2.563and HR=2.686).6) The coexpression of Bcl-2-Nu and Twist-1-Nu was the final state of the nuclear transport of Twist-1after EMT. The expression of Bcl-2-Nu was significantly correlated with Twist-l-Nu,VE-cad,VEGF,HIF and MMP-9.[Conclusions]1) The nuclear expression of Bcl-2was correlated with EMT characterizing protein. It was aslo significantly correlated with tumor metastasis and VM. The nuclear expression of Bcl-2induce a worse prognosis, suggesting that its association with malignant behavior of HCC;2) Coexpression of Bcl-2and EMT regulators was significantly correlated with metastasis, VM and prognosis, suggesting the interaction between Bcl-2and EMT regulators;3) Bcl-2-Nu(+)/Twist-l-Cyt(+) could be an independent predictor of prognosis. The interaction between them may induce more biological effects on tumor invasion,metastasis and angiogenesis, result in a worse prognosis;4) The nuclear transportation is a continuous dynamic process. The coexpression of Bcl-2-Nu(+)/Twist-1-Nu(+) may be an final state in the process of development of EMT.Part3The molecular mechanism of nuclear transportation of [Purpose]To investigate the molecular mechanism of interaction of Bcl-2and Twist1, we mimicked the hypoxic environment to observe the time-dependent expression patterns of Bcl-2and Twist-1; Eukaryotic expression system was constructed to transfect Bcl-2and Twist-1separately or both of them in the same time to analyze the associationship between Bcl-2and Twist-1; the ChIP sequence technology was then used to observe the effect caused by interaction between Bcl-2and Twist-1.[Methods]In vitro,GasPak was used to mimic the hypoxic condition. Western Blot and Real time-PCR were performed to detect the expression of Bcl-2and Twist-1on protein and RNA level. Plasmids of Bcl-2and Twist-1were constructed, and then they were transfected into HCC cells separately or co-transfected with both. The ChIP sequence assay and miRNA chip assay were used to observe the effect caused by co-expression of Bcl-2/Twist-1.[Results]1) The proliferation of cells cultured under hypoxia showed a significant decrease compared to those cultured under normoxia.When ells were returned to normal oxygen conditions after24h of hypoxia (hypoxia-normoxia group, H-N),the proliferation rate at the terminal phase (72h) of the H-N group significantly increased compared with the normoxia alone control group. Migration and invasion assays showed similar responses.2) The expression levels of Bcl-2and Twist following hypoxia and after returning to normoxia were assessed using quantitative PCR and Western blot. mRNA and protein levels showed expression peaks for Bcl-2and Twist1about24h after cell hypoxia. The expression levels gradually decreased to undetectable levels at later time points. When hypoxia was relieved after24h by returning to normoxia, Bcl-2and Twist1still had high expression levels. Taken together, these observations suggested that return to normoxia after hypoxia may trigger an increase in cell proliferation, movement, and molding.3) Co-transfection of Bcl-2and twist-1led to an increase of multiple genes involved in embryonic development, transcription, cell adhesion, and may others involved biological functions in normal and cancer development[Conclusions]1) Expression peaks of Bcl-2and Twist-1induced by hypoxia had consistencies;2) Co-expressed Bcl-2and Twist-1could cause changes in wide signal transduction.Conclusions for whole manuscriptBased on the studies as the followings:role of hypoxia in inducing cell death in the form of nuclear condensation and its molecular expression patterns, clinicopathologic analysis--the correlation of Bcl-2and EMT, and the molecular mechanism of nuclear transportation of Bcl-2,we studied the new function of Bcl-2besides its role in anti-apoptosis to identify its role in tumor angiogenesis and metastasis. In the present study, we studied mechanism of the interaction between Bcl-2and Twist-1leading to nuclear relocation of Twist-1and evaluated the effect on the cell functions caused by the interaction. Studies about the association between Bcl-2and EMT could explain better on the effect caused by Bcl-2on tumor behavior and provide a new angel to understand the function of Bcl-2. At the same time, the theory provides a new idea and an access for basic research of tumor cells differentiation. It plays an important role in further understanding the biological behavior of tumor and in clinical diagnosis. It also provides new drug targets in antitumor treatment. |
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