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Antitumor Activity of Hirsutanol A, a Novel Sesquiterpene Compound and Its Mechanisms

Author: YangFen
Tutor: GaoYouHeng
School: Guangzhou University of Traditional Chinese Medicine
Course: Of Pharmacy
Keywords: Hirsutanol A Apoptosis Autophagy ROS Antitumor
CLC: R285.5
Type: PhD thesis
Year: 2010
Downloads: 356
Quote: 0
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Abstract


A research background and purpose of cancer has a high morbidity and mortality, serious harm to human health and life. The world each year about 700 people die of cancer, and in recent years, cancer mortality was a clear upward trend. Chemotherapy is one of the primary means for the treatment of tumors, and therefore, the search for and development of high efficiency and low toxicity of anticancer drugs is still the main way to overcome tumor. In recent years, depending on the normal cells and tumor cell gene expression, the destination has made certain progress in targeted therapy for tumor-specific cancer gene, but due to the genetic instability and resistance gene therapy still facing great challenges. Therefore, tumorigenesis and development to find new tumor therapeutic targets has become the focus of the research and development of anticancer drugs. In recent years, the specific biochemical changes in the environment of the tumor cells become a new target for anticancer drugs. Reactive oxygen species (ROS) are a class of chemically active oxygen-containing functional groups of substances collectively. It consists of hydrogen peroxide (H202), hydroxyl radical (· OH), superoxide anion radical (O2 ·), singlet oxygen, ozone (03), nitrogen oxides (NOx), hypochlorous acid (HClO), etc. . Under physiological conditions, the generation of ROS is required for cell growth, under physiological conditions in ROS regulation of cell proliferation and differentiation, but the abnormally elevated ROS can promote tumor formation. Since cancer cells in ROS high-level state for a long time, the redox system in the tumor cells relative to normal cells is more fragile and less to ROS regulation ability, when ROS inducing agents induced ROS elevated can selectively kill the cancer cells while no effect on normal cells. Cell redox system is considered to be a new target of the anticancer drug research. With the shrinking of the the terrestrial the Medicinal biological resources development range, people have their eyes on the ocean. The marine biological survival of the special nature of the environment (high-pressure, low-oxygen, high salt, etc.), often contain a large number of new structures and peculiar physiological activity significantly metabolites. Therefore, from the ocean to seek efficiency and low toxicity natural medicine has attracted increasing interest in the international community. Hirsutanol A isolated from the endophytic fungus Chondrostereum sp SCS coral Sarcophyton tortuosum, a sesquiterpene-based compound, the compound is isolated from a new genus, and the chemical structure of the compound is reported only once, pharmacological The activity was not reported. Cytotoxicity screening, six compounds were isolated from the marine endophytes found a half times terpenoids Hirsutanol A strong cytotoxicity its anti-tumor effect and its explore the mechanism from in vivo and in vitro. Two research methods MTT test screening compounds having anti-tumor activity and detecting Hirsutanol A growth inhibitory effects on different tumor cell lines. DHE. CM-H2DCF-DA staining flow cytometry ROS superoxide anion and hydrogen peroxide levels. AnnexinV / PI double staining flow cytometry Hirsutanol A induction of tumor cell apoptosis rate. Cells transfected with GFP-LC3 confocal laser autophagy. Immunoblotting (Western Blot) detects changes in JNK and Akt signaling pathways, apoptosis-related proteins caspase-3 and PARP fault zone and autophagy-related protein LC3 Ⅰ - Ⅱ changes. Akt kinase activity assay kit for detection Hirsutanol A Akt activity. Mitochondrial Isolation Kit mitochondria and cytoplasm separation, immunoblot method for detection of mitochondrial and cytosolic cytochrome C expression. The the application strong antioxidant NAC detect ROS inhibition of cell proliferation induced Hirsutanol A inhibition and apoptosis and JNK, Akt phosphorylation levels. Application-specific small molecule inhibitor SP600125 and siRNA transient transfection method to detect the JNK signaling pathway to inhibit the induction of apoptosis Hirsutanol A and ROS levels. The application of small molecule inhibitors 3-MA Bafilomycin Al and siRNA transient transfection method inhibits autophagy, detect autophagy Hirsutanol A-induced cell death. Building Hep3B/myr-Aktl, Hep3B-vector stably transfected cell lines, the MTT assay Hirsutanol A Hep3B/myr-Aktl, Hep3B-vector growth inhibition. Nude mice the tumor the experimental observation HirsutanolA of SW620 in nude mice transplanted tumor growth inhibition. Screening of the active compounds of the three findings Hirsutanol A cytotoxic effect on different tumor cell lines of the six compounds screened, found the variety of tumor cells, Hirsutanol A significant cytotoxicity, and presents significant time - dose-response relationship. 2 Hirsutanol A molecular mechanism of the anti-tumor effect (1) the impact of Hirsutanol A tumor cells ROS levels were used DHE and CM-H2DCF-DA staining detected Hirsutanol A tumor cell superoxide anion and peroxide hydrogen levels, the results show that Hirsutanol A did not affect the level of superoxide anion but significantly increased tumor cell hydrogen peroxide level and showed obvious time - dose-response relationship. (2) Hirsutanol A raised ROS-induced tumor cell apoptosis ① Hirsutanol A-induced SW620, MDA-MB-231, MCF-7 tumor cell apoptosis AnnexinV / PI double staining induced by Hirsutanol A S W620, MDA-MB-231 apoptosis of MCF-7 cells. The results show Hirsutanol A significantly induced SW620, MDA-MB-231 and MCF-7 cells undergo apoptosis, apoptosis rates were: SW620 (2.8%, 12.1%, 45.0%, 65.6%), MDA-MB-231 (2.6%, 10%, 22.7%, 30.6%), MCF-7 (1.7%, 10%, 20,8%, 31.3%) and showed a significant dose-dependent relationship; detected by Western blot, caspase-3, PARP shear fragment visible in 17KD and 85KD obvious fault zone. The above results show that Hirsutanol A-induced SW620, MDA-MB-231 and MCF-7 cells undergo apoptosis. ② Hirsutanol A induces apoptosis through the mitochondrial pathway To investigate whether Hirsutanol A can induce apoptosis through the mitochondrial pathway were detected SW620, MDA-MB-231 cells mitochondrial membrane potential changes and mitochondrial and cytosolic cytochrome C expression. The results showed: SW620, MDA-MB-231 cells mitochondrial membrane potential showed a concentration dependent manner. Processing cells 24h after Hirsutanol A mitochondrial cytochrome C expression significantly reduced cytoplasmic expression of cytochrome C increased significantly. These results suggest that Hirsutanol A induced apoptosis through the mitochondrial pathway. ③ Hirsutanol A-induced apoptosis associated with ROS we found in the previous study Hirsutanol A have a variety of tumor cell proliferation inhibition can significantly upregulated SW620, MDA-MB-231 and MCF-7 cells ROS level, In order to prove Hirsutanol A by upregulating ROS play an anti-tumor effect. Detection Hirsutanol A strong antioxidant NAC inhibits Hirsutanol A upregulation of ROS cell proliferation inhibition and apoptosis. The results showed that NAC can significantly reduce-induced proliferation inhibition of Hirsutanol A Hirsutanol A induced apoptosis. The ④ Hirsutanol A by upregulating ROS activate JNK pathway research found that ROS can regulate JNK, Akt, NF-κB signaling pathway, and then we further investigate the activation of ROS Hirsutanol A JNK pathway. HirsutanolA processing SW620 cells for 24 h, Western blot detection of JNK and c-Jun protein phosphorylation levels seen JNK and c-Jun protein phosphorylation levels were significantly increased. Pretreatment of the cells with ROS inhibitors NAC, NAC Hirsutanol A can inhibit the activation of JNK. Activate JNK pathway Hirsutanol A role of ROS raised related. ⑤ JNK pathway activation with JNK inhibitor SP600125 inhibits JNK pathway, apoptosis was detected by MTT assay cell activity and AnnexinV / PI double staining, visible SP600125 can significantly increase Hirsutanol A-induced cell death; alone Hirsutanol A cell apoptosis was 35.6%, A induced apoptosis Hirsutanol SP600125 blocked JNK pathway rate rose to 48.3%, blocking the JNK pathway can increase Hirsutanol A induced apoptosis. That activation of the JNK pathway is not only unable to induce apoptosis, but also protect cells from death. Next, we further investigate the role of JNK pathway activation of ROS, JNK inhibitor SP600125 pretreatment of the cells or siRNA blocking JNK pathway, flow cytometry ROS level, the results showed that blocking the JNK pathway can significantly increase Hirsutanol A-induced ROS levels. The activation of the JNK pathway does not induce apoptosis but negative feedback regulation of ROS levels, the cells of an oxidative stress protection mechanisms. (3) Hirsutanol A raised ROS-induced tumor cells occurs from bite ① The Hirsutanol A raised ROS-induced autophagy LC3 can be as self-bite body membrane landmark protein in tumor cells, we first used the green fluorescent protein (GFP) combined with the GFP-LC3 to detect autophagy, Hep3B transfected with GFP-LC3 plasmid for 24 h, and treatment with 20μmol / L HirsutanolA 24h and observed under a confocal laser microscope, visible Hirsutanol A treatment group had a large number of LC3 punctate gathered, while the control group LC3 was dispersed. Next, we further by Western blot detection LC3 protein expression, visible prompted Hirsutanol A tumor cells can be induced autophagy, LC3 conversion increased from type I to type Ⅱ. Hirsutanol A upregulated ROS levels and induced autophagy, next we further explore HirsutanolA to raise ROS levels and induced correlation between autophagy. Respectively detected by Western blot LC3 protein expression and confocal laser scanning LC3 punctate aggregation results show that adding NAC cells were pretreated with, Hirsutanol A-induced LC3 significantly reduced by the type I to type Ⅱ conversion LC3 punctate aggregation has been reduced significantly. These results suggest that Hirsutanol A tumor cells by upregulating ROS induced autophagy. ② Hirsutanol A by upregulating ROS inhibit Akt pathway Akt / mTOR is a major pathway for the regulation of autophagy, we found in previous studies HirsutanolA can induce tumor cells to generate ROS in induction of tumor cell autophagy. So we Hirsutanol A further explore the Akt pathway. With the Hirsutanol A-treated cells after 24 h, Western blot detection of Akt the P-Akt473 and protein expression levels of P-FKHR found that Akt expression levels unchanged but the P-Akt473 and P-FKHR expression levels were significantly lower, indicating that inhibition of Akt Hirsutanol A pathway. Next, we further explore Hirsutanol A Akt pathway inhibition raised ROS the first Akt kinase activity in vitro, the results show that Akt kinase activity assay detects Hirsutanol A Hirsutanol A in vitro can not be directly inhibiting Akt activity. Then added with the NAC pretreatment Hep3B cells 1h Hirsutanol A for 24 h, Western blot protein expression levels of P-Akt473 Visible NAC to partially reversed of P-Akt473 the expression level. Description AKT pathway inhibition with ROS raised related. ③ Hirsutanol A non-apoptotic cell death induced Hep3B cells using Annexin V / PI double staining to detect apoptosis found Hep3B cells treated with 5,10,20 μmol / L HirsutanolA 48 h, the highest Annexin V single-positive rate of only 0.5 % PI-positive rate of 5.5%, 15.9%, 49.7%, respectively. Western blot analysis Caspase-3, PARP expression, the results are displayed in 17KD and 85KD were no obvious fault zone. The results show that the induced Hep3B cell Hirsutanol A non-apoptotic cell death. ④ Hirsutanol A induced Hep3B cell autophagic cell death To prove Hirsutanol A Hep3B cells induced autophagic cell death, we were used to blocking autophagy since autophagy inhibitor 3-MA and siRNA-Beclinl. MTT assay blocking autophagy Hirsutanol A on cell growth inhibition and death. The results show that blocking autophagy can significantly reduce Hirsutanol A-induced cell death. The ⑤ Hirsutanol A Hep3B cells autophagic death and ROS upregulation and Akt pathway induced inhibition the related order to verify Hirsutanol A Hep3B cells induced autophagic death and ROS hike relationship we 1mmol / L NAC pretreatment cell 1h, then add Hirsutanol A treatment for 72 h, respectively, using the MTT assay and AnnexinV / PI double staining cells proliferation inhibition and death situation, the results show that NAC can significantly reduce Hirsutanol A-induced inhibition of cell proliferation and death. We also constructed Hep3B/myr-Aktl Hep3B-vector Cell Lines, detects the relationship between the Hirsutanol A induced cell death and Akt pathway, the results show Hirsutanol A of Hep3B/myr-Akt1 cell growth inhibitory effect was significantly higher than Hep3B -vector cells. Description Hirsutanol A induced Hep3B cells autophagic death and ROS upregulation and Akt pathway inhibition related. (6) MCF-7 cells inhibit autophagy can promote Hirsutanol A induced death we found Hirsutanol A study either induce tumor cells MCF-7 apoptosis can also be induced autophagy induced autophagy Hirsutanol A play protective effect or promote cell death? respectively autophagy inhibition since autophagy inhibitor Bafilomycin Al and siRNA-Atg-7, MTT assay of cell death. The results showed that blocking autophagy can significantly increase cell death, Hirsutanol A induced autophagy in MCF-7 cells play a role in protecting cells from death. Inhibitory effect of 20 mg / kg dose Hirsutanol A 3 Hirsutanol A nude mice significantly inhibited the growth of nude mice, inhibition rate of 31.5%, 5 mg / kg treatment dose on the growth of the tumor tissue volume no obvious inhibition. Four Conclusion 1 Hirsutanol A raise of ROS cause mitochondrial damage, release of cytochrome C to induce tumor cell SW620, MDA-MB-231 apoptosis. 2 Hirsutanol A raise of ROS-induced inhibition of Akt pathway in tumor cells Hep3B autophagic cell death occurs. The 3 Hirsutanol A both MCF-7 apoptosis can also induce the autophagy induced tumor cells, autophagy in MCF-7 cells play a protective role. The 4 Hirsutanol A by upregulating ROS levels activate the JNK pathway and JNK pathway activation does not induce apoptosis, but negative feedback regulation of ROS levels. The 5 Hirsutanol A significantly inhibited SW620 tumor growth in nude mice.

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