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Isolation of Anticancer Compounds from Toad Skin and the Molecular Mechanism Study

Author: HeGuoPing
Tutor: WangHongYang
School: Second Military Medical University
Course: Biochemistry and Molecular Biology
Keywords: Toad skin Isolation Anticancer Proliferation Cyclin Cell cycle Nickel Lung
CLC: R285
Type: Master's thesis
Year: 2009
Downloads: 290
Quote: 1
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Abstract

Data the toad skin of the first part in the anti-tumor activity of small molecule compounds access and anti-tumor mechanism study research background and purpose of the Ministry of Health, \, especially in large cities, the mortality rate is accounted for than 28.84% of all causes of death, the great threat to the life and health of the people of our country. From a worldwide perspective, the malignancy has become the number one killer of mankind. Despite malignancy by surgery, chemotherapy, radiation therapy, and other means to partially cure or alleviate, the research and development of anticancer drugs has made considerable progress in the past ten years, but the surgical cure rate is low, put serious patients difficult side effects of chemotherapy withstand the situation has not fundamentally changed, and continue to look for specificity, efficiency and low toxicity of anticancer drugs, is still a research focus of the medical field. In recent years, due to the directional design, synthesis lead drug and precursor of anticancer drugs from the combination of compound libraries more and more difficult, and the relative toxicity of chemical drugs stronger, so people have turned to natural medicine. With a long history and concern in the medical profession of Chinese medicine effective in treating tumors, often used as an auxiliary method of radiotherapy or chemotherapy. Extract active ingredients from traditional Chinese medicine and the development of new drugs, is an effective way of traditional Chinese medicine to enter the international market. China has accumulated some experience in this area. Been developed formulations of artemisinin and its derivatives from Artemisia annua, is the best medicine to treat malaria, a typical success is the development of Chinese medicine. The monomer compounds have been isolated from toad Bufalin the Chinese toad Toad fine alcohol diene esters, more as a cardiac stimulant use in the clinical study of its mechanism is also more thorough. Modern pharmaceutical research shows that the exact toad skin and its preparations for treatment of liver cancer, lung cancer and other malignant tumors. Toads sources widely used in clinical anti-tumor agents Chinese toad injection, cinobufotalin capsule. However, because of the rough, high purity, complex composition, easily lead to toxicity and anti-tumor mechanism is unknown, its therapeutic value is limited; addition, the anti-tumor activity of the compounds have been isolated from toad skin preparations mostly fat-soluble and For the toad skin of water-soluble components yet to see the depth development. This study will be used bioassay-oriented separation strategy, by means of advanced means of separation and purification, the water-soluble components of the toad skin depth Purification and activity, to reveal the active components in the toad tumor cell signaling pathway regulatory role, a clear mechanism of action of the anti-tumor, and provides the theoretical basis for the further development of natural anticancer drugs and specificity, and effective small molecule medicinal compound / lead compounds. Experimental methods: 1. Use of industrial chromatography, high performance liquid chromatography, and pure chemical plant technology platform component of toad skin layers of depth separation and purification; 2. Antitumor component isolated at all levels of CCK-8 activity identified; 3. using flow cytometry analysis of active components of toad skin using Western Blot and RT-PCR and other molecular biology techniques, discussed the purpose of the active component of the toad skin regulates tumor cell tumor cell cycle; gene expression; 5. vivo imaging of tumor-bearing mouse model, long-term monitoring of the active components in vivo therapeutic effect. Results: 1. Laevis skin four kinds of crude industrial chromatography mention the component, Ⅳ number having anti-tumor activity; the the component 10-4mg/ml level exhibit the effect of inhibiting tumor cell proliferation, and as the concentration increased enhancement; 2. Ⅳ, crude extract component further separation out of the 11 kinds of screening component, No. 11 (HCS411) having anti-tumor activity, and strong activity in the IV, the component; 3 carcinoma lung cancer, leukemia and prostate cancer is the most sensitive on the HCS411 sensitive breast cancer, osteosarcoma, and esophageal cancer cells, cervical cancer and colon cancer cells are not sensitive; 4 compared with normal liver cells showed a certain specificity, HCS411 on hepatoma cells sex; 5. HCS411 the inhibition of tumor cell proliferation activity compared currently a common clinical anti-tumor Huachansu injection and cisplatin are strong; 6. HCS411 can inhibit tumor cell proliferation induced G2 / M phase arrest; 7. HCS411 anti- tumor activity is not dependent on P53; 8. HCS411 60 fine components separated by the pure chemical plants, seven strong anti-tumor activity; which activity most 2-SX048429; 9. vivo imaging detection of tumor-bearing animal models The results show that the ,2-SX048429 active ingredient in the body also has strong anti-tumor effect. Conclusion: In this study, bioassay-oriented separation method, for the first time from the soluble fraction of toad skin gradually isolated seven fine components have anti-tumor activity, and has laid a good foundation for further found that the anti-cancer small molecule compounds; Preliminary studies indicate that its anti-tumor mechanism, these components can be a way that does not depend on the p53 tumor cell cycle arrest at the G2 / M phase, thereby inhibiting tumor cell proliferation. The second part of the nickel compounds by the carcinogenic mechanisms of the regulation of the cell cycle research background and purpose of lung cancer is the most common malignant tumor, but also one of the fastest growing global morbidity and mortality in tumor. In most countries of the world, the prevalence and mortality of lung cancer showed an increasing trend, especially in developed countries, lung cancer common malignancy in men in the first place. Although for the diagnosis and treatment of lung cancer than in the past has greatly improved, but the difficult early diagnosis of lung cancer, recurrence and metastasis rate is still the bottleneck problem of clinical research. Is an urgent need to start from the prevention, control of the root causes of this great threat to human disease. And to take effective preventive measures, we must better understand the etiology of lung cancer. Tumorigenesis is the result of the interaction of environmental factors and genetic factors in vivo, But on the whole, the environmental factor is the main reason of the carcinogen. It is reported that 80% to 90% of human cancers and environment related to the physical and chemical factors, among which the carcinogenic chemical factors. Heavy metals nickel and its compounds is one of the major environmental carcinogens. Epidemiological data show that the rise is closely related to exposure to nickel compounds and the incidence of lung cancer, but so far, the mechanism of nickel-induced lung cancer is not clear. The researchers found in a variety of tumor tissue in the lung tissue, cyclin B1 were overexpressed state. Recent studies showed that in most of the nickel compound to stimulate the regulation of the gene transcription factor to determine the target protein by the HIF-1α-mediated hypoxia signal path. , Disorders of the cell cycle in tumor occurrence and development stage is considered to be one of the most important intracellular events. In tumorigenesis, the mutant cells are considered to be essential. In the transformation process of the G1 / S phase, cyclin D1 and cyclin E is an important regulatory factor, and many tumors occur, such as breast, lung and prostate cancer. In addition, Chinese hamster ovary cells after nickel G2 / M phase was significantly increased and cyclin A and cyclin B, which is usually. product p53 is a transcription factor p53 works at the G1 / S and G2 / M phase checkpoint, under normal circumstances, is very unstable, so that you can not accumulate to a high enough level to promote transcription. So, it is necessary to study the regulation of all cyclin in Nickel carcinogenic process, including cyclin D1, cyclin E, cyclin A and cyclin B, there are important cell cycle regulatory factors p53, as well as their biological function. Taking into account the lung nickel compounds act on the most important target organs of the body, the human bronchial epithelial cells Beas2B as a cell model to study the mechanism of nickel compounds in lung cancer, including its cell cycle in a variety of major cycle protein expression, cell cycle progression and cell proliferation. Experimental methods: 1. Flow cytometry NiCl2 stimulus Beas2B cell cycle changes; 2 by Western Blot and RT-PCR detection the NiCl2 processing Beas2B cells of cyclin D1, cyclin E, cyclin B1 and cyclin A expression levels ; 3. reporter gene assay method NiCl2 expression of cyclin D1 and P53 activation; 4 PI, cyclin A2 double staining method analysis the NiCl2 before and after Beas2B cell M phase change; 5. applications of recombinant DNA technology to build pSuper / siCyclin B1 expression plasmid; 6 CCK-8 was detected of NiCl2 on cell proliferation. Results: the 1 by NiCl2 stimulus Beas2B cell growth significantly inhibited flow cytometry cell group of stagnation in the G2 / M phase; 2. NiCl2 raised Beas2B cell expression of cyclin D1 and cyclin E; 3. HIF-1 not participate in NiCl2-induced expression of cyclin D1 and cyclin E; 4. P53 does not participate in NiCl2-induced G2 / M phase arrest; 5 by NiCl2 stimulate increased expression of cyclin B1; interference cyclin B1, NiCl2 induced M arrest and growth inhibition effect was significantly weakened. Conclusion: This research was first discovered in the bronchial epithelial cells, nickel compounds can induce the expression of cyclin D1 and cyclin E increase and promote the G1 / S phase of the cell conversion; transcription factor HIF-1 is not involved in this process. Nickel-induced accumulation of cyclin B1 can cause M phase of the cell block and eventually lead to cell growth inhibition; play an important role in the regulation of the cell cycle protein p53 is not involved. These results not only deepen our understanding of the mechanism for the carcinogenicity of nickel compounds, and also provides a new way of thinking for the prevention and treatment of nickel-induced tumor.

Isolation of Anticancer Compounds from Toad Skin and the Molecular Mechanism Study

Abstract

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