| Research Background:Osteosarcoma is a primary malignant tumor commonly seen in adolescents. Its incidence rate accounts for20%of all bone tumors. Osteosarcoma cells proliferate rapidly to damage the surrounding normal tissue with its invasive growth. Distant metastasis is found at the early stage, among which, lung metastasis is most common with a poor prognosis. Traditional treatment methods include mainly amputation and radiotherapy, but5-year survival rate is less than20%. In recent years, the use of adjuvant chemotherapy, limb salvage surgery has seen the significant improvement of5-year survival rate and limb salvage success rate, but it still finds no striking progress in terms of the long-term efficacy and long-term survival in patients. More and more scholars these years are focusing on the study of the molecular mechanism of the occurrence, development and metastasis of osteosarcoma in the hope of finding new methods for the treatment of osteosarcoma from the gene level. With the progress of the study, it has been found that the occurrence, development and metastasis of osteosarcoma are a complex process. In addition to a series of pathophysiological reactions, many genes and transcription factors are involved and implicated in a variety of regulatory mechanisms, wherein the expression of multiple genes induced by hypoxia and regulatory mechanisms between them play an important role in the process of tumor proliferation.It has been confirmed that, in the process of the growth of malignant tumors, tissues growing too fast would inevitably causes the local tissue hypoxia and an imbalance between energy consumption and energy supply, thus leads to tumor cell apoptosis. In order to adapt to hypoxic environment, tumor cell phenotype undergoes a series of changes, such as inducing angiogenesis/vascularization and increasing glycolysis in cells, contributing enormously to the regulation of cell apoptosis. It is believed that there are now two major pathways associated with osteosarcoma:death receptor-ligand pathway and mitochondrial pathway. The mitochondrial pathway is stimulating mitochondria to release cytochrome c (Cyt c) with some apoptosis stimulating factors. Cytochrome c (Cyt c) released into the cytoplasm combines with apoptosis activating factor1in the presence of dATP, generating its polymer, and prompts caspase-9to combine with the polymer to generate apoptotic bodies. Thus, caspase-9is activated, and the activated caspase-9activates other caspases such as caspase-3, and caspase-3activates DNA fragmentation factors, resulting in the activation of endonuclease in the resting state, and ultimately causes DNA rupture. It is a common biochemical pathway for apoptosis from the mitochondrial dysfunction to DNA rupture. Since the caspase can be mutually activated, so the caspase protease cascade is a major part leading to changes in the structure of cell apoptosis. Mitochondrial dysfunction plays a key role in the pathogenesis of osteosarcoma apoptosis.It has been proved that apoptosis-related factors such as Bcl-2, inhibitors of apoptosis proteins (IAPs), p53, caspases, etc., play an important role in the process of regulating osteosarcoma cells apoptosis. Among the regulatory genes related to apoptosis, Bcl-2is the one most closely connected to apoptosis and also has been studied most in-depth, including mainly the anti-apoptosis protein Bcl-2class (Bcl-2, the Bel-x1, Ced, Bcl2w, Mcl-1-9, etc.) and the pro-apoptotic protein Bax class (including Bax, the Bid with only the BH3domain structure, Bim, PUMA, etc). Studies on apoptosis-inhibiting genes in terms of osteosarcoma focus on Bcl-2, which can encode2kinds of proteins, α and β in the mitochondrial outer membrane and part of the endoplasmic reticulum. Now it is generally believed that the pathology that the Bcl-2in osteosarcoma playing its part in inhibiting apoptosis is realized by blocking the mitochondrial pathway, i.e. blocking the release of cytochrome c into mitochondria, whereby, inhibiting the activation of downstream effect factors, and thus exerts apoptosis inhibition; The Bcl-2protein can also inhibit apoptosis through combination with the pro-apoptotic protein Bax. The expression of Bcl-2and its relationship with prognosis vary in different tumor. Some scholars studied the Bcl-2genes in osteosarcoma via immunohistochemical method to, and it was shown that the staining positive rate and the extent of staining of Bcl-2in osteosarcoma tissues were higher than they were in normal bone tissues, which revealed that the occurrence of osteosarcoma was associated with abnormal apoptosis. Vitro experiments also confirmed that osteosarcoma cells showed high expression of Bcl-2, and Bcl-2suggested inhibitory effect on apoptosis of osteosarcoma cell line. Ferrari’s research showed that in most cases with pulmonary metastasis of osteosarcoma recurrence, the expression of Bcl-2is stronger than a primary tumor, but the protein expression of Bcl-2does not rise with the rise of osteosarcoma in installment, therefore, its expression doesn’t indicate the malignant degree of osteosarcoma. The pro-apoptotic protein Bax are often found in the cytoplasm, transferring into mitochondria when cells are stimulated by apoptosis signals, and they promote apoptosis through the mitochondrial pathway. Eliseev identified that specific transcription factor RUNX2in bone cells, with Bax as targets, enhancing the sensitivity of osteosarcoma cell, Saos-2to apoptosis by increasing its expression. A vast array of studies has stated that the ratio of Bcl-2and Bax determines whether there is apoptosis or not. Now its mechanism has been agreed that, the Bax-Bax dimer induces apoptosis, but once the Bcl-2protein content increases, Bcl-2conbines Bax to form Bcl-Bax dimer more stable than the Bcl-Bax dimer, which compromises the effect on promoting apoptosis of Bax. Kaseta et al also believes that the ratio of Bcl-2and Bax and the inhibition of apoptosis in osteosarcoma were positively correlated.Glutamine transaminase, also known as transglutaminase, is an enzyme that undergoes transamidating activity between primary amine and acylamino-Y in catalytic protein or glutamine residues in polypeptide chains. It has been discovered that transglutaminase is widely distributed in the blood, extracellular matrix and intracellular subgroups and it is involved in many biochemical processes, such as blood coagulation, wound healing, tissue reconstruction, cell differentiation and death, etc. It is now known to contain eight transglutaminase types in humans. Ⅱ glutamine transaminase (transglutaminase2, TG2), also tissue transglutaminase (tTG, e. c.2.3.2.13), is a member of the TG family of enzymes which is most extensively studied in depth. This enzyme is highly multifunctional, involved in many physiological and pathophysiological processes, including fibrosis, atherosclerosis, neural-degenerative diseases, diarrhea and tumor metastasis. Over the past few years, a growing number of studies have proved that the enzyme is closely related to the occurrence and development of tumor.TG2exists in the form of dimers with each monomer consisting of686amino acids, relative molecular mass of about80kd, each monomer containing four unique structure domains:an p sandwich structure domain at amino-terminal (1-139), core structure domain for catalyzing transamination (147～460) and2β tubular structure domain at the c-terminal (472～583,591～687). TG2is a unique member of the transglutaminase family, which has both the activity of catalyzing Ca2+-dependent protein crosslinking and the function of GTP-dependent G protein. Moreover, it has an activity of protein-disulfide isomerase and the function of the protein kinase. Although TG2is mainly located in the cytoplasm, it can also be secreted to extracellular and cross-linking extracellular matrix proteins to stablize the extracellular matrix. The secretion of TG2relies on its active conformation and (or) correct tertiary structure. It can also be transferred into the nucleus, cross-linked histones, PRb and SP1transcription factors with the help of the input protein (importin)-a3, to regulate cell cycle and apoptosis. Likewise, TG2is trans-located to the membrane, together with integrin, to promote the interaction between integrin on cell surface and fibronectin. Therefore, TG2may have great impact on the occurrence, differentiation and invasion of tumors.Many research results have confirmed the role of TG2in promoting apoptosis, since a full expression of TG2is usually found in apoptotic cells, and the inhibited expression of TG2significantly reduces the number of deal cells. Under extreme circumstances (such as hypoxia, the lack of growth factors, the chemotherapy treatment), intracellular Ca2+homeostasis is damaged. The sudden change of Ca2+in cytoplasm transfers TG2into the conformation with crosslinking activity, leading to the extensive crosslinking and multimerization of intracellular proteins, thus forms an acid-insoluble structure, stabilizes the apoptotic cells and prevents inflammation and scarring. However TG2expression and apoptosis are not completely consistent. TG2knockout mice did not show the apoptosis-induced disorders, which didn’t rule out the compensation of other isomerase on TG2deficiency. In addition, while several kinds of rapidly dividing cancer cells expressed a high level of TG2, they didn’t end up in apoptosis. The latest direct evidences showed that the rising TG2prolonged the survival of the cells by preventing apoptotic cell death. Moreover, though the epidermal growth factor inhibits the doxorubicin-induced apoptotic cell death of breast cancer, but increases TG2expression. Antonyak et al have proved that in retinoic acid’s treatment of some cell lines, the increase in the levels of retinoic acid-induced TG2cells protects cells from (N-HPR)-induced apoptosis. These findings suggest that TG2not only promots apoptosis, but also resists apoptosis. The seemingly contradictory role is dictated by the cell type, the stimulation type and the positioning of TG2in the cell and its activity type. TG2promotes apoptosis through transamination in the cytoplasm, while, it protects cells from apoptosis through G protein function or (and) transamidating activity in nucleus; Likewise, on the cell membrane, TG2, as the auxiliary integrin receptor, promotes the adhesion of the integrin and the extracellular matrix and signal transduction, protecting cells from apoptosis.The latest researches have shown that TG2displays nonenzymatic functions when it generates compounds with cellular proteins based on noncovalent interactions. In HEK293cells, through the consumption of Bax and the reduction of caspase-3and caspase-9, TG2prevents cytochrome c in the cell nucleus from being released into the cytoplasm as well as the mitochondrial membrane depolarization when Ca2+increases, which in turn contributes to anti-apoptosis. The same mechanism was found in hypoxic tumor cells that caspase-3is consumed through cross binding with caspase-3, and thus plays the role of resistance to apoptosis. It suggested that in hypoxia environment, TG2may get involved in regulating the apoptosis in osteosarcoma cells through the mitochondrial pathway, but it has not been reported about the role of TG2in the process of apoptosis in osteosarcoma and its regulatory mechanism. This study is to develop osteosarcoma cells in hypoxia environment and to measure the expression of cytokines in the mitochondrial pathway as well as the change of apoptosis rate through transfection of TG2siRNA to inhibit TG2expression, with an aim to explore anti-apoptotic effect of TG2in osteosarcoma MG-63cells as well as the possible mechanism of inhibiting apoptosis in tumor cells by TG2through regulating the expression of Bcl-2, Bax, cytochrome c and caspase-3.Objective:Under hypoxic conditions, cultivate osteosarcoma MG-63cells in vitro to explore the role of transglutaminase Ⅱ in its anti-apoptosis in osteosarcoma MG-63cells, as well as its mechanism of inhibiting apoptosis in tumor cells through regulating the expression of the Bcl-2, Bax, cytochrome c and caspase-3.Methods:1) Design TG2-specific siRNA, and carry out the transfection in accordance with the steps defined in Dharmafect3kit.2) The experimental groups:(1) normoxic group:cells cultured under normal oxygen;(2) hypoxia group:cells cultured in hypoxia box;(3) control siRNA hypoxia group:si RNA cultured in hypoxia box after transfection and control;(4) TG2siRNA hypoxia group:TG2siRNA cultured in hypoxia box after transfection.3) Establish culture models of osteosarcoma cells in vitro under hypoxic conditions, place the cells in hypoxia incubators for cultivation, and treat respectively the MG-63cells according to different time periods (6,12,24,48,72h).4) Determine intracellular TG activity using microtiter plate method.5) Detect TG2, Bcl-2and Bax mRNA level using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) method.6) Understand the distribution of proteins in cells using immunohistochemical method (SP); detect the expression changes of TG2, Bcl-2, Bax, cytochrome c and caspase-3using West blotting (Western blot).7) Detect the rate of cell apoptosis with flow cytometry. Results1) TG2activity:The in situ TG2activity was not detected in normoxic group, and the activity of TG2in hypoxia group was found an increased expression compared with that in normoxic group (P<0.01), and it was statistically significant comparing the difference between the various time points;the TG2activity of TG2siRNA hypoxia group decreased significantly, compared with hypoxia group and control siRNA hypoxia group (P<0.01).2) RT-PCR method for detecting the mRNA level of TG2, Bcl-2and Bax:TG2, Bcl-2, Bax in normoxic group were restricted to the low level expression of mRNA; TG2mRNA level, in hypoxia group and control siRNA hypoxia group, increased compared with its expression in normoxic group. With the prolonged hypoxia, TG2mRNA level gradually improved, and it was statistically significant comparing the difference between the various time periods (P<0.01); the mRNA activity Bax showed no remarkable enhancement (P>0.05); while the mRNA activity of Bcl-2was signicantly increased, and gradually enhanced with prolonged hypoxia (P<0.01), and there was significant difference between the respective time periods (P<0.01). Compared with hypoxia group and control siRNA hypoxia group, in the TG2siRNA hypoxia group, the mRNA level of both TG2and Bcl-2was significantly reduced in each time period when inhibiting the expression of TG2(P<0.01); the mRNA level of Bax was slightly enhanced, however, not statistically significant when compared with the other three groups(P>0.05).3) Immunohistochemical results showed that the TG2, Bcl-2, and Bax protein in osteosarcoma MG-63cells were localized in the cytoplasm. In normoxic group, TG2protein expression was negative. In hypoxia group and control siRNA hypoxia group, the rate of TG2positive cells increased significantly(P<0.01), and the staining intensity was enhanced strikingly with prolonged hypoxia; the rate of TG2positive cells TG2siRNA hypoxia group reduced considerably when compared with hypoxia group and control siRNA hypoxia group(P<0.01).4) Western blot detection of the protein expression of TG2, Bcl-2, Bax:The result of western blot showed no obvious protein expression of TG2, Bcl-2in normoxic group, but clearly visible Bax protein bands. The protein expression of TG2, Bcl-2in hypoxia group and control siRNA hypoxia group was significantly higher than that that in normoxic group (P<0.01), and the expression intensity increased obviously with prolonged hypoxia; While, Bax protein bands were gradually weakened (P<0.01), and the expression intensity decreased with hypoxia prolonged. In TG2siRNA hypoxia group, the protein expression of TG2and Bcl-2was obviously lower than that in hypoxia group and in control siRNA hypoxia group (P<0.01), whereas Bax protein bands were enhanced obviously, significantly higher than that in hypoxia group and in control siRNA hypoxia group (P<0.01).5) Cytochrome c:The results of Western blot showed:In normoxic group, only a small amount of cytochrome c were found in the cytoplasm; In hypoxia group and control siRNA hypoxia group, the cytochrome c content in the cytoplasm and mitochondria was only slightly affected. In TG2siRNA hypoxia group, the cytochrome c content in the cytoplasm increased significantly when inhibiting the expression of TG2, while cytochrome c content in mitochondria decreased remarkably (P<0.01). It suggested the inhibition of cytochrome c in osteosarcoma MG-63from releasing into the cytoplasm by TG2under the hypoxic condition.6) Caspase-3:Caspase-3activity:Caspase-3activity in hypoxia group and control siRNA hypoxia group was shown no obvious increase (P>0.05) compared with that in normoxic group; While, in the TG2siRNA hypoxia group, caspase-3activity gradually increased with prolonged hypoxia (P<0.01).The results of Western blot showed weak caspase-3protein bands in hypoxia group; While, caspase-3protein bands obviously increased in TG2siRNA hypoxia group (P<0.01), and its expression intensity grew significantly with prolonged hypoxia (P<0.01).7) The results of flow cytometry showed that:The apoptosis rate in hypoxia group and control siRNA hypoxia group increased slightly compared with that in normoxic group; while, the apoptosis rate in TG2siRNA hypoxia group was improved significantly, compared with that in the first three groups (P<0.01).Conclusions:1) Under hypoxic conditions, the expression of TG2in osteosarcoma MG63cells was strikingly impoved;2) TG2increases the Bel-2/Bax ratio, prevents the cytochrome c from releasing into the cytoplasm, and reduces caspase-3activity by promoting the expression of Bel-2and the depletion of Bax; 3) Crosslinked polymers of TG2generated in the cytoplasm form insoluble compounds with caspase-3, reducing the expression and activity of caspase-3, thus contribute to anti-apoptosis and inhibit the apoptosis of tumor cells.4) When the TG2siRNA inhibits the expression of TG2, it facilitates the release of cytochrome c into the cytoplasm, enhances the expression and activity of caspase-3, and promotes apoptosis of tumor cells by decreasing the expression of Bcl-2and increasing the expression of Bax.5) This study reveals a new anti-apoptotic pathway of TG2, suggesting that inhibiting the expression of TG2promotes apoptosis in osteosarcoma cells and providing a new effective way for the treatment of osteosarcoma. |
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