| Natural materials have been an important source of natural medicines, and also a major method of treating and preventing human diseases. The incidence of cancer is increasing every year, and is one of leading causes of death worldwide. Among various cancers, prostate cancer (PCa) is the second leading cause of cancer-related death among the male population of Western society. Although the incidence of prostate cancer was lower than the Western countries’, in recent years confirmed PCa, especially for advanced or metastatic PCa, represents an obviously rising trend in China. Currently, androgen deprivation therapy is the standard treatment for PCa patients due to its effective suppression of androgen receptor signaling. Even though androgen deprivation therapy shows its initial effectiveness on shrinking tumor size, it eventually fails to cure advanced PCa, which is determined by the occurrence of castration-resistance (HRPC) within a few years after the therapy. There are few efficacious treatment options available for curing, or even improving the survival and quality of life in patients with HRPC. Since complicated factors and signals involves in the development of HRPC, monotherapy regimens or chemotherapy drugs targeting a particular signaling pathway are difficult to obtain a satisfactory result. Thus, novel natural compounds that are able to induce prostate cancer cell death through various signal pathways are highly desirable. Millions of plants, animals, microorganisms, and marine organisms are an important source of new drug discovery. Currently discovering novel leading compounds from special biological resources has been the international mainstream direction of drug research.Part Ⅰ. Antitumoral and autophagy-inducing activity screening of natural compoundsProstate cancer (PCa) usually develops to hormone-refractory prostate cancer (HRPC), with poor prognosis and few therapeutic options. Compelling evidence suggests that progression of PCa to metastatic HRPC is associated with chemoresistant nature and dysregulation of normal apoptotic mechanisms. Naturally occurring small molecule compounds isolated from natural materials have been an important source of new drug discovery. In this study, we evaluated antitumoral and autophagy-inducing activities of 28 natural compounds extracted from liverworts, myrrh or fungus, and preliminarily studied and analyzed the biological effects of Retigeric acid B (RB) and Malformin A1 (MA1) in different types of prostate cancer cells.1. Antitumoral and autophagy-inducing activity screening of natural compoundsAntitumoral and autophagy-inducing activity screening of 28 natural compounds firstly revealed that RB and MA1 remarkably inhibited the proliferation of prostate cancer cells and induced autophagy in different degree.Antitumoral drugs screening via MTT assay showed that prostate cancer cells were the most sensitive to RB among different cancer cells; IC50 of androgen-dependent LNCaP cells is 7.9μM; IC50 of androgen-independent PC3and DU145 is about 10μM; the inhibitory effect of RB on nonneoplastic human benign prostate epithelial RWPE1 cells decrease over 6-folds; antitumoral drugs screening revealed MA1 effectively inhibited proliferation in PC3 and LNCaP cells in the range of 50-400nM in a dose-dependent manner; the benign prostate epithelial RWPE1 cells exerted a slightly lower sensitivity under the same conditions.Induced-autophagy was determined by the number of punctate dots comprising LC3B protein in U87 cells with stable GFP-LC3B expression. The results showed 12 natural sesquiterpenes extracted from myrrh had weak effect on induction of autophagy; among 12 natural triterpenes, only RB induced autophagy in a moderate manner; among 4 compounds produced from Aspergillus niger, only MA1 remarkably promoted autophagy.2. RB suppressed invasion, induced S-cell cycle arrest and apoptosis1) Transwell assay revealed that the invasiveness through matrigel was concentration-dependently decreased in RB-treated PC3 and DU145 cells when compared to vehicle treated cells.2) RB caused a dose-dependent accumulation of cells in S phase, reduced DNA synthesis, and induced apoptosis in PC3 cells. Cell cycle arrest induced by RB was revealed by flow cytometry. Treatment of PC3 cells with RB led to changes of cell regulatory molecules:cyclin A, cyclin E and cyclin B, and the phosphorylation level of retinoblastoma tumor suppressor protein (pRb). RB caused a dose-related reduction in the BrdU incorporation and the expression of PCNA; RB treatment caused a dose-related decrease in the antiapoptotic protein Bcl-2 level, and increase in Bax, causing an increase in the ratio of Bax/Bcl-2. RB treatment led to activation of caspases and cleavage of PARP; pretreatment with the pan-caspase inhibitor z-VAD-fmk partially blocked the RB induced apoptosis. 3) RB suppressed AR transcriptional activity in LNCaP cells and induced apoptosis. RB concentration-dependently inhibited the expression of AR and suppressed AR transcriptional activity detected by androgen-stimulated PSA promoter transcription. RB treatment resulted in activation of caspase-3 and the elevated cleaved PARP.3. MA1 triggered DNA damage, apoptosis and necrosis in PCa cells1) MA1 induced apoptosis. AnnexinV-FITC/PI assay showed MA1 caused apoptosis in a time-dependent manner in PC3 and LNCaP cells; western blotting showed MA1 induced activation of caspase 3 and cleavage of the nuclear protein PARP, and decrease in the expression of anti-apoptotic protein Bcl-2; pretreatment with the pan-caspase inhibitor z-VAD-fmk partially blocked the MA1-induced cell death.2) MA1 provoked cell necrosis. PI staining assay showed cell necrosis time-dependently increased following MA1 treatment; release of LDH occurs during cell necrosis; QRT-PCR assays revealed MA1 triggering a profound inflammatory response determined by the increase in mRNA levels of pro-inflammatory cytokines.Part Ⅱ Mechanisms of autophagy induced by Retigeric acid B and Malformin A1Autophagy is a catabolic pathway that maintains cellular homeostasis via the self-digestion of unwanted proteins and damaged or aging organelles, such as damaged mitochondria, by double-membrane autophagosomes. In mammals, the Atg8 homolog LC3BI is conjugated with phosphatidylethanolamine, forming LC3BII and resulting in the maturation of autophagosomes, which associate with lysosomes to form autophagolysosomes. When cells suffer extra- or intra-cellular stress, the level of autophagy could be remarkably activated as a cytoprotective response, resulting in adaptation and survival, while autophagic cell death could occur with dysregulated or excessive autophagy. Recent studies have demonstrated that autophagy, including mitophagy, is often stimulated in tumor cells in response to multiple forms of cellular stress induced by chemotherapeutic agents. In fact, the mode of action of some anticancer drugs from natural materials such as paclitaxel and camptothecin involves the induction of autophagy, which is important for the regulation of cancer development and progression and for determining the response of tumor cells to anticancer therapy.1. RB stimulated mitophagy via inhibiting the PI3K/Akt/mTOR pathway1) RB induced up-regulation of the autophagy marker protein LC3B and lipidated LC3BⅡ. Microarray and QRT-PCR assays revealed RB promoted mRNA expression of LC3B in PC3 and LNCaP cells, up to 2.5-3.5 folds; Western blotting showed RB time-dependently induced protein levels of LC3B and lipidated LC3ⅡB; MA1 induced punctate dots of LC3B via immunofluorescence analysis, indicating RB treatment caused autophagy.2) Inhibition of autophagy potentiated RB-induced cell death. Pharmacological blockade of autophagy using 3-MA or CQ or knockdown of LC3B by siRNA enhanced RB-mediated cell death, which indicated that RB induced promoted a protective autophagic process and delayed PCa cell death.3) RB induced mitochondria damage and the generation of ROS, and induced mitophagy. RB treatment led to mtΔψ decreased in a time-dependent manner, with a reduction of over 90% at 24h in PC3 and LNCaP cells, indicating that RB damaged mitochondria in PCa cells; kinetic studies revealed that RB time-dependently increased the intracellular accumulation of ROS in PC3 and LNCaP cells, up to 3 folds at 24h; electron microscopy of RB-treated PC3 cells showed dramatic mitochondrial swelling, the formation of autohagosomes and autolysosomes, and the colocalization of mitochondria and autophagolysosomes.4) RB blocked PI3K/Akt/mTOR pathway. Western blotting showed treatment with RB caused a significant decrease in the phosphorylation of Akt and mTOR, the downstream effector of mTOR; RB also inhibited the downstream effector of mTOR, phospho-p70S6K, revealing that the suppression of PI3K/Akt/mTOR signaling was involved in RB-triggered autophagy in PCa cells.2. MA1 stimulated autophagy through AMPK/mTOR pathway1) MA1 rapidly up-regulated autophagy levels determined by the accumulation of autophagy marker protein LC3B and the accumulation of lipidated LC3BII. Western blotting and QRT-PCR assays revealed MA1 time-dependently induced protein and mRNA expression of LC3B in PC3 and LNCaP cells; p62 was reduced after 2h in response to MA1 accordingly; immunofluorescence analysis confirmed MA1-induced autophagy by the number of punctate dots comprising LC3B protein in PC3 cells.2) Pharmacological blockade of autophagy attenuated MA1-mediated cell death, which indicated MA1-induced autophagic cell death.3) MA1 triggered autophagy through the activation of AMPKα/mTOR pathway. MA1 treatment led to a significant increase in the phosphor-AMPKa, and down-regulated the phosphorylation of mTOR in PC3 and LNCaP cells; MA1 had no detectable effect on AKT phosphorylation during treatment.4) MA1 activated AMPK/mTOR pathway and autophagy via causing oxidative stress. JC-1 staining assay revealed MA1 treatment led to mtΔψ decreased in PC3 and LNCaP cells in a time-dependent manner; MA1 caused rapid generation of intracellular ROS determined by Time kinetic studies; western blotting and QRT-PCR assays revealed MA1 induced up-regulation of protein and mRNA levels of the antioxidants SOD2, GSTP1 and DJ-1 respectively; Pharmacological inhibition of MA1-induced oxidative stress by the antioxidants VC and NAC partially restored MA1-triggered inhibition on cell proliferation; depletion of SOD2 by SOD2-targeting siRNA promoted MA1-mediated autophagy and dramatically enhanced cell death; transfection of pEGFP-N1-SOD2 expression plasmid attenuated MA1-induced autophagy and resulted in a partial rescue of the cell death. These indicated that MA1 induced oxidative stress and activated a pronounced autophagic cell death process via AMPKa/mTOR pathway.Due to the significant cytotoxicity of MA1 as evidenced by inducing cell necrosis and severe inflammatory response, we focused our research on the anti-cancer mechanism of RB in the following study.Part Ⅲ Retigeric acid B inhibits cell proliferation and invasion through suppression of nuclear factor-κB signaling in prostate cancer cells in vitro and in vivoThe pivotal nuclear factor κB (NF-κB), a well-documented transcriptional factor, is critically important for control of cell proliferation in mammals. In classical pathway, the typical NF-κB dimers (p50/p65) are normally sequestered by binding to IκBα in the cytoplasm. The IκBα subunit is phosphorylated by the IKK, and then degradation through the proteosomal pathway, the p50-p65-IκBα heterotrimer turning into the p50-p65 heterodimer. The nuclear localization signals of NF-κB protein are exposed and its p65 subunit is phosphorylated, leading to nuclear translocation and transcriptional activation potential, and finally inducing the expression of a large number of target genes. Part Ⅰ showed that RB possesses ability to inhibit cell growth and invasion in PCa cells. Here, we study the effect of RB on NF-κB signaling in androgen-independent PC3 and DU145 cells, and in vivo anticancer activity in C56BL/6 homografts mice.1. RB exhibited inhibitory effect on p65 phosphorylation in carcinoma cells1) RB inhibited p65 phosphorylation in PC3 and DU145 cells both dose-and time-dependently, whereas showed slight inhibitory effect on the expression of total p65.2) RB did not affect the expression of p50 and p52, suggesting RB mainly targeted to cannonical NF-κB pathway.3) RB reduced p65 phosphorylation in different carcinoma cell lines, including human liver hepatocellular HepG2 cells, human myeloid leukemia K562 cells and adriamycin-resistant K562/AO2 cells.2. RB suppressed the nuclear translocation and activation of NF-κB, and blocked IκBα degradation in PCa cells1) RB inhibited nuclear localization of p65 in PC3 cells. Western blotting and immunofluorescence analysis displayed that RB reduced nuclear localization of phosphor-p65 in a dose-dependent manner.2) RB reduced NF-κB DNA binding ability in PC3 cells in dose-dependent manner indicated by EMSA assay3) RB suppressed NF-κB transcriptional activity. The luciferase reporter gene analysis revealed that RB caused a significant decrease in NF-κB reporter activity in PC3 and DU145 cells and LPS-treated LNCaP cells as compared to the untreated cells.4) RB inhibited IκBα phosphorylation and its degradation in both PC3 and DU145 cells. Proteasome activity assay revealed that RB did not exert inhibitory effect on recombinant 20S proteasome enzyme.The data suggested that down-regulated of IκBα degradation and p65 phosphorylation may be due to the effective suppression of IKK activation by RB, which eventually lead to the inhibition of NF-κB activity.3. RB inhibited cell proliferation and migration through repressing the expression of NF-κB-regulated genes1) RB affected the target genes expression of NF-κB. Western blotting and qPCR assay showed treatment of RB significantly decreased the expression of Bcl-xL, Bcl-2, survivin, and cyclin D1 at both protein and mRNA levels in PC3 and DU145 cells in a dosage-dependent manner.2) Gene chip verified the overall effect of RB on NF-κB-regulateded genes.10μM RB treatment resulted in obviously alteration of mRNA levels of cell proliferation- and survival- genes, and invasion-associated regulatory genes.3) NF-κB p65 was essential for RB-induced cell death. Over-expression of p65 by transfecting p65 expression plasmid promoted cell survival; ablation of p65 by using p65-targeting siRNA accelerated RB-induced cell death in PC3 and DU145 cells.4. RB exerted in vivo anti-tumor activity in the tumor tissue of mice1) RB reduced RM-1 cell growth in vitro. RB and AKBA remarkably reduced RM-1 cell growth and phosphorylation of p65 in RM-1 cells at desired concentrations.2) RB inhibited cell vitality and induced apoptosis in PCa cells in vivo. PCa homografts in male C57BL/6 mice with RM-1 cells were established and treated with RB for 18 days. Compared to vehicle control group, RB-treatment significantly reduced the average tumor volume and weights. TUNEL staining demonstrated increasing numbers of TUNEL positive stained apoptotic cells in the tumor tissues from RB treated mice; H&E staining showed the morphologic changes in the tumor tissues.3) RB affected NF-κB signaling and apoptosis-related proteins expression. Western blotting assay showed that RB produced a profound inhibition of PARP, Bcl-2, and Bcl-xL expressions in tumor tissues, whereas increased Bax expression consistent with the results in culture cells. RB suppressed phosphor-p65 expression and nuclear localization revealed by western blotting and immunofluorescence analysis.Part Ⅳ Induction of DNA damage and ATF3 by Retigeric acid B, a novel Topoisomerase Ⅱ inhibitor, promotes apoptosis in prostate cancer cellsTopoisomerases are ubiquitous enzymes that are responsible for the topological interconversions of DNA by generating transient single-strand breaks (SSBs) or double-strand breaks (DSBs) during various biological events such as replication, transcription, recombination, repair and chromosome segregation. Topoisomerase Ⅱ (Topo Ⅱ), with two isoforms Ⅱα and Ⅱβ, is essential for relaxing supercoiled DNA via catalysis of DSBs, believed to be a well-documented target for many anti-tumor chemotherapeutics. Topo Ⅱ inhibitors cause SSBs or DSBs by trapping topoisomerase-DNA covalent complexes, and strongly inducing chromosome and DNA damage, subsequently leading to the cell death. Activating transcription factor 3 (ATF3) is a member of the ATF/CREB family of transcription factors. ATF3 is induced by a variety of stresses including DNA damage, oxidative stress and endoplasmic reticulum stress, to maintain cellular homeostasis, or accelerate apoptosis during lasted and overloaded stress. The present study focused on whether RB acting as a Topo Ⅱ inhibitor induced DNA damage and ATF3 expression to tigger cell death.1. RB induced DNA damage and inhibited Topo Ⅱα activity1) RB induced DNA damage in PCa cells. Neutral comet assays showed that RB treatment promoted DNA damage in a time-dependent manner; western blotting assay verified that a well-known indicator of DNA damage yH2AX was elevated without any changes for total H2AX protein levels.2) RB suppressed Topo Ⅱα activity in vitro. The decatenation of kinetoplast DNA assay showed RB treatment significantly reduced both closed circular and nicked open circular forms produced by Topo Ⅱ in a dose-dependent manner; docking method revealed the potential binding sites.3) RB had weak effect on Topoisomerase related genes. Microarray and QPCR data showed that RB treatment inhibited the mRNA levels of TOP2A and 3A in a moderate manner, and had a slight inhibition for others; western blotting assay revealed the similar results.2. RB elicited DNA damage response via activation of ATM/ATR pathways1) RB activated DNA damage ATM/ATR signaling network. Dynamic analysis indicated at early time RB activated key downstream effector proteins including p53, Chkl and Chk2 of ATM/ATR signaling; the specific inhibitor of ATM Ku55933 resulted in substantial reduction of γH2AX and phosphor-Chk2.2) ATM and ATR signaling pathways contributed to RB-mediated cell death. The inhibitor of ATM/ATR caffeine almost completely abrogated the ability of RB to promote H2AX phosphorylation and partially rescued viability rates in PC3 and LNCaP cells.3. RB inhibited DNA repair in PCa cells1) RB reduced DNA repair-related gene expression. Microarray and qPCR data showed that many genes associated with DNA damage response and DNA replication were time-dependently down-regulated by RB, most becoming significant at late time; western blotting assay showed RB activated BRCA1 at early time, decreased the expressions of Rad51 and Ku86 after 24h, whereas no significant decrease in Ku70 expression.2) RB suppressed DNA end-joining activity. DNA end-joining assays indicated that RB-treatment suppressed the catalytic activity of nuclear extract as indicated by appearance of strong monomer bands and corresponding attenuated dimer and multimer bands.4. RB-induced DNA damage associated with changes of ATF3 and other cellular stress response genes1) RB induced changes of stress response gene expressions. Microarray data explored global changes in the stress response gene expressions in response to RB treatment and qPCR was performed to validate the changes:transcription factors ATF3, ATF4, ATF6, KLF6, EGR1, and ETS1, the stress-responsive gene PTGS2, proapoptotic genes DDIT3, DDIT4, GADD45A and DR5 were predominantly upregulated. Western blotting assay showed RB time-dependently enhanced ATF3 protein level.2) RB decreased ATF3 promoter activity in PC3 and LNCaP cells into which an ATF3 promoter luciferase reporter was transfected.3) p53 contributed to the readily RB-induced ATF3 expression in LNCaP cells. RB-enhanced ATF3 was significantly abolished by activation of p53 through transfection of a mutant p53 expression plasmid into LNCaP cells.4) RB promoted nuclear localization of ATF3, whereas less ATF3 protein in cytoplasm correspondingly, which was essential for activation of ATF3 transcriptional function.5. RB-induced ATF3 promoted cell death through activating apoptosis-related genes1) The inhibition of ATF3 expression promoted cell survival. Knocking-down ATF3 expression by siRNA significantly reversed RB-mediated inhibitory effect on cell proliferation and rescued cell death. yH2AX levels remained unchanged in responseto ATF3 knockdown. The accumulation and the nuclear translocation of ATF3 were not inhibited by the broad caspase inhibitor z-VAD-fmk, indicating that ATF3 induction was not a downstream event of caspase activation.2) Depletion of ATF3 significantly led to reduction of mRNA levels of DDIT4, GADD45A and DR5, and an increase in CDC25A, important regulators of cell death and cell cycle that are transcriptionally activated by ATF3.3) KLF6 was involved in RB-induced ATF3 overexpression. An ATF3 promoter luciferase reporter was co-transfected with the expressional plasmids of EGR1, ETS1, E2F1, Spl, STAT3, KLF6, ETV1, and ERG1 in PC3 cells respectively, only KLF6 further dramatically increased the promoter activity of the ATF3 in cells exposed to RB compared to the vehicle-treated cells, suggesting that RB-induced KLF6 promoted ATF3 expression in PC3 cells.Part V Conclusions and Innovations1. Conclusions1) Natural compounds screening revealed that Retigeric acid B and Malformin A1 inhibited cell proliferation and induce DNA damage, and induced autophagy in a moderate or stronge manner respectively.2) RB stimulated mitochondria damage which induced apoptosis and mitophagy in prostate cancer cells, RB-mediated mitophagy played a protective role and delayed cell death; MA1 mediated apoptotic, necrotic and autophagic cell death via causing oxidative stress; severe redox reaction and dramatic intracellular ATP deletion promoted autophagy process through stimulating AMPK/mTOR pathway.3) Retigeric acid B inhibited cell proliferation and migration through suppression of nuclear factor-κB signaling in prostate cancer cells, and exerted in vivo anti-tumor activity in the tumor tissue.4) Retigeric acid B acting as a novel topoisomerase Ⅱ inhibitor, induced DNA damage, impair DNA repair and activated ATF3, leading to by apoptosis in prostate cancer cells.2. Innovations1) This study first reported the anticancer activity of Retigeric acid B, and clarified the multi-mechanism of Retigeric acid B inhibiting cell proliferation and migration and inducing cell death, and verified its in vivo anti-tumor activity.2) This study first reported that Retigeric acid B was a novel Topoisomerase Ⅱ inhibitor, and firstly proposed a model of the mechanism by which RB promoted cell death by inducing DNA damage, impairing DNA repair and activating ATF3.3) This study first reported the effect of Malformin A1 on autophagy, and clarified the mechanism underlying Malformin A1-mediated apoptotic, necrotic and autophagic cell death via causing oxidative stress. |
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