Effect Of Ginsenoside Rh₂ On SGH-44 And Proteomics Study Of Medulloblastoma And Ependymoma

The completion of Human Genome Sequence draft indict that the field of life science research entered the post-genome era. In the post-genome era, proteomics, the science to study protein composition and activitie rules, quickly become an important part of life scientific research. Proteome means all proteins expressed by a genome, a cell or an organism or organization. Proteomics is science that researchs the protein composition and the activities rules within cell in the overall level, refers to a study the full expressed protein on the individual, organization or cells in a specific physiological or pathological state of science.The technology of proteomics for tumor research has opened up new way to dynamic, holistic, quantitative observation of tumor occurrence and development process. By comparing normal and anormal cells in the number, location and modification status of protein expression, the specific protein can be found associated with the pathological changes.It can be used for the screening of tumor markers , identification of early tumor, assessment and prognosis of tumor. And it will be beneficial to explore the mechanism of tumor ,research medicine and search related drug targets. Currently,there is still rare report on intracranial tumors proteomics research,so there are still many unknown areas about intracranial tumors proteomics waiting to be explored. Ⅰ. Glioma is the most common intracranial malignant tumors,and there is no significant progress in its treatment so far. Ginsenoside Rh2 is a natural active ingredients extracted from ginseng. It can induce apoptosis of variety tumor cells and with the change of cell cycle. Based on previous experimental study, in the first part of this paper the inhibition of Rh2 to SHG-44 glioma cell proliferation was investigated, the proteins change of SHG-44 cell was detected by using proteomics methods, before and after the Rh2 was given.MTT assay proved Rh2 can significantly inhibit the proliferation of SHG-44 and Rh2 do not have cytotoxicity. Flow cytometry confirmed the Rh2 significantly reduce the proportion of SHG-44 cell cycle in G2-M phase and S phase, while there is an emergence of cell block in G1 phase clearly. And the percentage of apoptotic cells 23.81±2.12% in experimental group changed very significantly, compared with that 2.01±1.13% in control group. As a large number of cell block in the G0-G1 phase, making cell proliferation is restricted, and the occurrence of cell apoptosis, induce the reduction of the cell number overall. In this experiment, with the effect of Rh2, there is a increasing trend of number in the sub-G-phase cells. And it proved Rh2 can induce apoptosis.This study found that the number and expression of protein in the glioma cell SHG-44 has changed after 72 hours action of 32μmol/L Rh2, compared with the corresponding control group. Rh2 inhibit expression of some protein in glioma can be concluded. The MALDI-TOF-MS analysis found that the following inhibition of protein expression more obvious. 1. Heat shock protein (HSP): Result shows there is a close relationship between HSP and apoptosis. in particular, HSP70, HSP27 play a apoptosis protection role induced by heat shock, oxidative stress, ionizing radiation and TNFα. And the mechanism is related with that HSP inhibit protein kinase activated by stress, inhibit the expression of apoptosis gene Bax and P53, inhibit proteolytic enzymes in the apoptosis signal transduction, inhibit oxygen free radical formation, and protect mitochondrial function. 2. Proliferation related gene 1: The protein promotes tumor cell growth and inhibit apoptosis, express high in the tissue such as liver cancer and breast cancer.It suggest that the protein is related with development of tumor. 3. Proliferating cell nuclear antigen (PCNA): PCNA is known well by the important function in nucleic acid metabolism. As a complementary protein of DNA polymeraseδ, PCNA is essential for DNA replication. 4. Phosphoglycerate kinase (PGK): PGK is a key enzyme in glycolysis, and is also a essensial biological enzyme to every organism. A lack of this enzyme can cause metabolic disorder of organisms.In short, experimental results suggest that this protein may be related with characters change of glioma cell differentiation induced by Rh2. It speculates that the treatment of Rh2 to glioma through inhibition to many kinds of proteins, so that apoptosis occurs and proliferation was inhibited. And some proteins,which have higher difference of expression abundance, were identificated by MALDI-TOF-MS. The above experimental results provide some phenomenon that occur in the process of glioma cell differentiation induced by Rh2, and to a certain protein research base. But the interaction among various proteins is still unknown, and in the subsequent study, the study on some proteins should to do deeply.Ⅱ. Medulloblastoma is a neurepithelial tumor derived from the embryonic residual organization. It occurs in children under 10 years of age often, and can occurs in any part of the brain tissue, is one of the most common intracranial malignant tumor in children. Ependymoma is neurepithelial tumor that originated in the epithelial cells of ependymal ventricular surface, occurred in the ventricular system often. Total resection of medulloblastoma and ependymoma is not easy by surgery. Metastasis and recurrence of the two tumors occur often. Now the two tumors are diagnosed by the imaging examination mainly, and misdiagnosed as other tumor often. And early diagnosis and molecular biology of the two tumors is blank. Based on the above reasons, in the second part of the experiment, intracranial common malignant tumor medulloblastoma and ependymoma were studied by proteomics technology. And we are looking forward to use emerging and gradually perfect proteomics technology and find specific protein of the two tumors as indicators of early diagnosis, treatment and prognosis track and monitor,as the related targets of drug, and at the same time, provide a theoretical basis for the development mechanism of intracranial malignant tumors.(A) Medulloblastoma:Compared with normal brain tissue, there is a total of around 1480 points in medulloblastoma. And 56 point rise, 12 points down, a total of 13 points express only in medulloblastoma. There are 15 points express in normal brain tissues. Points, that express obvious different, is analysed by mass spectrometry. 1. ER-60: only in medulloblastoma expression. ER-60: called the endoplasmic reticulum-60 protease, is a inherent endoplasmic reticulum protease in eukaryotic cells. Now, ER-60-has been proven to be a cysteine protease, including seven kinds of semi-acid residues, the four of which include two CGHC sequence. It has been demonstrated that activity units of ER-60 is based on the CGHC the sequence. Overexpression of ER-60 induce directly deformation of cofactor protein B in liver cells, in result secretion of HEPG2 cells from cofactor protein B reduced. 2. Phosphatidylethanolamine binding protein (PEBP): compared with normal brain tissue, expression of PEBP is missing in medulloblastoma completely. Database search showed that the amino acid sequence of PEBP and other proteins is no obvious sequence similarity, suggesting that in both structure and function PEBP is unique.PEBP is not only involved in biofilm construction and remodeling process, while also play a role in signal transduction, growth and development of the nervous system, such as physiological and pathological processes.(B) ependymoma:Compared with normal brain tissue, we found a variety of high- expression protein in ependymoma. There are 1,700 points in ependymoma totally. Compared with normal brain tissue, 32 point rise, 18 points down in ependymoma.There are 14 points only in ependymoma. There are 9 points in normal brain tissues. 1. Tropomyosin 1 alpha: is an important protein in muscle contraction regulation process, and it play an important role in the regulation of cell cytoplasm mobile and endothelial cell apoptosis, and so on.In the non-muscle cells, TM1 play a role in cell transformation inhibition. Overexpression of the tropomyosin will lead to malignant transformation of cells, abnormal expression of tropomyosin are discrete molecular basis of cells. Studies showed that TM1 play an important role in incidence of many human cancer, including breast cancer, prostate cancer, pancreatic and gastric cancer. And TM1 expression was significantly higher in cancer than that in normal tissue. This study confirmed TM1 expression is significantly higher in ependymoma than in normal brain tissue. And this is related likely with the character of the implanted transfer of ependymoma, can be used as a diagnosis protein in the early transfer and the drug targets of ependymoma. 2. Alpha B-crystallin: is a small molecular chaperone protein, a family member of small heat shock protein, and binding non-folding protein, inhibit their coalescence. Apart from the role of the molecular chaperone, it has capabilities of stable cytoskeleton protein and skeleton network, that help cells to oppose apoptosis induced lack oxygen, high temperature, radiation and drug. It also play a role in shear mechanisms in the cell. In recent years, it was found play a role in neurodegenerative diseases.In short, the results of this part suggest that there is great relevance between the incidence and transfer characteristics of medulloblastoma and ependymoma and abnormal and deficiencies expression of protein. But the biological characteristics and the specific mechanism should be further study.In sum, using proteomics technology,this paper detected proteomics changes in the SHG-44 cells before and after treatment by Rh2, and test intracranial malignant tumor initially, at the same time. These results suggest that there is relationship between the incidenc, growth and metastasis of intracranial tumor and the characteristics of the abnormal expression, deletion expression of protein in this tissue. And the results shows proteomics technology is a advantageous way in exploration the markers and drug targets of intracranial tumor, will provide important theoretical basis to reveal the origin and prognosis of tumor.