Mechanism and Function of Angiogenin in Apoptosis Regulation.

Anti-apoptotic Bcl-2 (B-cell lymphoma 2) proteins such as Bcl-2 itself, Bcl-xL and Mcl-1, prevent mitochondrial activation and thereby the induction of the intrinsic apoptotic signaling pathway. Since the avoidance of cell death is a prerequisite for malignant transformation, anti-apoptotic proteins are frequently overexpressed in tumor cells. Additionally, there is growing evidence that Bcl-2, Bcl-xL and Mcl-1 also play a role in other cellular processes, such as cell cycle regulation, DNA repair and autophagy induction, which can also be important for the onset and progression of cancer. One of the most frequently diagnosed malignancies worldwide is colorectal cancer. Even though improvements in treatment and population screenings have led to a decreased mortality in many countries, especially patients with metastasized colorectal cancer still face a poor prognosis. Until now, it has been an open question whether and to which extend anti-apoptotic Bcl-2 proteins influence colorectal cancer initiation, progression and metastasation, with a few studies showing contradictory results. Hence, the purpose of this work was to shed more light on the role Bcl-2, Bcl-xL and Mcl-1 play for the maintenance of intestinal tissue homeostasis and for colorectal cancer development and outgrowth. First, their expression levels in the human intestinal mucosa as well as in adenoma and adenocarcinoma tissue were determined. This revealed a significant increase of Bcl-xL in the malignant state and an unaltered expression of Bcl-2. By contrast, Mcl-1 has been found to be significantly downregulated in colorectal cancer specimens. Results obtained in subsequent in vitro experiments clearly showed that Mcl-1 has an anti-proliferative effect which cancer cells preclude by downregulation of the protein. Neither in intestinal epithelial cells nor in colorectal cancer cells has a cell cycle inhibiting mode of action been described so far for Mcl-1. Additionally, further in vitro experiments have shown for the first time that the siRNA mediated silencing of anti-apoptotic Bcl-2 proteins significantly decreased the migratory capacity and invasiveness of human colorectal cancer cells. In a second step, protein functions were studied in further detail in vivo. Since previous publications showed that the constitutive deletion of both Bcl-xL and Mcl-1 induces embryonic lethality, two intestine-specific knockout mouse models were generated during this work. For the first time, they allowed to study functioning of Bcl-xL and Mcl-1 in the murine intestine. The mouse models revealed a strong discrepancy between Bcl-xL and Mcl-1, regarding their influence on tissue maintenance and tumorigenesis. While Bcl-xL turned out to be dispensable for normal tissue homeostasis, it has been found to be a crucial factor for colorectal cancer cell survival in a chemically induced tumor model. This confers Bcl-xL tumor-promoting properties and explains its overexpression in human adenomas and adenocarcinomas. The loss of Mcl-1, by contrast, caused a severe intestinal phenotype, comprising high levels of cell death, an accompanying increase of proliferation and chronic inflammation. From an age of about six months, spontaneous tumorigenesis was observed in intestine-specific Mcl-1 knockout mice which was promoted by the loss of the anti-proliferative effect Mcl-1 exerts on intestinal epithelial cells and the inflammatory environment. Therefore, it has been proven that Mcl-1 possesses tumor-suppressing properties in the intestine, what explains its downregulation in human colorectal cancer specimens. The presented results highly recommend the utilization of Mcl-1 sparing inhibitors in the context of colorectal cancer treatment. A first step towards clinical application was done in this work by treating viable human colorectal cancer tissue ex vivo with the Bcl-xL/Bcl-2-specific inhibitor ABT-737. Subsequent analyses revealed a significantly decreased viability of human colorectal cells in presence of the inhibitor. Since proliferation turned out to be unaltered under ABT-737 treatment, inhibition of Bcl-xL in combination with classical chemotherapy could be an interesting approach for further studies with a focus on clinical applicability.

Angiogenin, as a member of the ribonuclease superfamily, is an angiogenic protein. The angiogenic ability of angiogenin plays an important role in many physical and pathological processes. Angiogenin can induce endothelial cell migration, proliferation, tubulation, as well as inhibition of cellular apoptosis. Angiogenin can be used to modulate the angiogenetic process of tissue engineered constructions via local delivery. Furthermore, angiogenin can also be regarded as a biomarker for diagnostic evaluation of malignancy, or as a target for cancer therapy. This paper presents a comprehensive overview of the angiogenic mechanisms of angiogenin, as well as its potential application in the process of wound healing and treatment of ischemic diseases and malignancy.

Anticancer drug development is now an emerging field of research all over the world. In this study we attempt to synthesize a novel, simple, inexpensive and safe chemical agents Benzoin thiosemicarbazone (BTSC) and studied against Ehrlich Ascites Carcinoma (EAC) cells bearing Swiss Albino mice by monitoring in vivo tumor cell growth inhibition, survival time of tumor bearing swiss albino mice. MTT colorimetric assay was done to assess the in vitro effect of the test compound. The intrinsic apoptotic pathway induced by BTSC was evidenced by p53 or tumor protein, B-cell lymphoma 2 (BCL-2), B-cell lymphoma extra-large (BCL-xL), BCL-2 associated X protein (BAX), cleavage of caspase-9 and caspase-3 and poly-ADP ribose polymerase (PARP-1). Reactive oxygen species (ROS) generation after BTSC treatment was determined by 2´, 7´- dicholorodihydrofluorescein diacetate (DCFH-DA) staining. The compound was found to possess pronounced anticancer effect. Maximum cell growth inhibition, enhancement of life span was found 73.53% and 52.17% at the dose of 8 mg/kg (i.p) respectively. The induction of apoptosis by BTSC occurred through an ROS-dependent mitochondria-mediated intrinsic pathway rather than an extrinsic pathway, and was regulated by the BCL-2 protein family. The compound exerted a potent antitumor effect toward Ehrlich ascites carcinoma cells through the induction of apoptosis via an intrinsic pathway. Thus, this study provides evidence to carry out further researches in a way to formulate novel anticancer drugs.

Flavonoids are a group of polyphenol compounds that exist extensively in dietary foods of vegetables and fruits, and the biological effects of flavonoids are anti-oxidant, anti-microbial, and anti-inflammatory activities. Recently, more and more studies have showed that flavonoids have the anti-tumor and anti-carcinogenesis effects. Therefore, the anti-tumor effects of flavanone （flavanone、2'-OH flavanone、3'-OH flavanone、4'-OH flavanone、5'-OH flavanone、6'-OH flavanone、7'-OH flavanone） in the osteosarcoma cell line （U-2 OS、143 B、MG-63、SaOS2） were analyzed. The result of MTT assay indicated that effect of flavanone、2'-OH flavanone and 3'-OH flavanone showed the most potent cytotoxic effect in the osteosarcoma cell line, and we also found that 2'-OH flavanoneare was the most potent cytotoxic compound. The result of cell’s live and death number calculation showed that 2'-OH flavanone induced the death number increasing of osteosarcoma cells. The further way, we found the phenomenon of chromatin condensation、mitochondria membrane potential change and diploid cell （sub-G1 phase） appearance after treatment to 143 B cell. Then, we analyzed the expression of apoptotic protein molecule by western blotting. The result showed the activation of caspase-3、caspase-8 and caspase-9. Therefore, the activation of caspase evidenced the occurrence of apoptosis. The result showed that 2'-OH flavanone increased the expression of TRAIL（TNF-released apoptosis-inducing ligand） and DR5 （Death receptor 5）. On the other hand, the result also showed that 2'-OH flavanone induced cytochrome c was released from mitochondria. We also found the anti-apoptotic proteins （Bcl-2、Bcl-xL）were inhibited, and the pro-apoptotic proteins（Bid、Bax、Bcl-xS）were activated. Therefore, the 2'-OH flavanone induced the 143 B cell apoptosis through TRAIL-DR5 pathway.Finally, we conducted the in vivo experiment（143 B-BALB/c nude mice）. The result showed that tumor growth were suppressed by 2'-OH flavanone. These results indicated that 2'-OH flavanone activated the extrinsic pathway and the intrinsic pathway through TRAIL-DR5 pathway, and it had potent effect to tumor growth in in vivo model. Therefore, 2'-hydroxyflavanone is a potent biological activity compound for the development of cancer chemotherapy.

Objective To explore the changes and related mechanism of the apoptosis of pancreatic islet cells in neonatal mice. Methods Mice islets at 1-,3-,8-week after birth were isolated by collagenase. The apoptotic rate was detected with Annexin V-FITC/PI double staining by flow cytometry. The morphological changes of apoptotic cells were detected by electron microscope. The apoptotic rate of islet β-cell was detected by immunofluorescence with Tunel/Insulin double staining. The expression of apoptosis-related genes was detected by real-time quantitative PCR. The expression of the pro-apoptotic Fas, FasL and Cleaved Caspase-3 and anti-apoptotic Bcl-2 protein were detected by Western blotting. One-way analysis of variance was used for data analysis. Results (1)The apoptotic rate of islet cells increased significantly at 3 w, which was markedly higher than that at 1 w and 8 w ((10.53±2.61)% vs (1.80±0.69)%, (3.26±0.94)%, F=32.09, P<0.01). Early ultrastructural changes of apoptosis appeared at 3 w. Tunel+/Insulin+ cells increased markedly at 3 w compared with that of 1 w and 8 w.(2)The mRNA expression of the apoptosis-related genes was dynamically changed. Among them, Fas and FasL were higher than that at 1 w and 8 w (1.53±0.21 vs 1.00±0.00, 0.46±0.24, F=24.85, P<0.01; 2.63±0.56 vs 1.00±0.00, 0.52±0.14, F= 32.77, P<0.01), while Bcl-2 was lower than that at 1 w and 8 w (0.30±0.23 vs 1.00±0.00, 1.71±0.00, F= 78.06, P<0.01).(3)Compared with 1 w and 8 w, the protein level of Fas, FasL and cleaved caspase-3 were significantly increased at 3 w (2.05±0.16 vs 1.00±0.00, 0.59±0.24, F=61.47, P<0.01; 3.54±0.86 vs 1.00±0.00, 0.72±0.26, F=27.04, P<0.01; 5.74±0.59 vs 1.00±0.00, 3.11±0.20, F=128.79, P<0.01), while Bcl-2 protein was markedly decreased at 3 w (0.62±0.13 vs 1.00±0.00, 1.90±0.10, F=151.08, P<0.01). Conclusion The Fas/FasL and Bcl-2 mediated apoptosis signaling pathways may be involved in the regulation of apoptosis in neonatal mice islet cells. Key words: Pancreatic islets; Apoptosis; Gene; Neonatal mice

Redox-Dependent Translocation of p53 to Mitochondria or Nucleus in Human Melanocytes after UVA- and UVB-Induced Apoptosis

Peyronie’s disease (PD) is a connective tissue disorder where formation of fibrous plaques in tunica albuginea (TA) and erectile tissue can result in penile deformity, pain, and erectile dysfunction. Fibrosis, its major pathological manifestation, arises from fibroblast proliferation and accumulation of extracellular matrix; PD progresses with formation of plaques or even ectopic calcification having the appearance of scar tissue, which prevent TA expansion during erections. The mechanisms underpinning PD are unclear, and relatively little is known about the disease itself. To date corrective surgery is the sole effective treatment. A greater understanding of PD pathophysiology at the molecular level has the potential to help develop novel medical therapeutic approaches. The aim of this study was to investigate the activation of the apoptotic intrinsic apoptotic pathway in plaques from PD patients. Tunica albuginea from either PD and control patients were assessed for the expression of bax, bcl-2, caspase 9 and 3 using immunohistochemistry, and by measurement of apoptotic cells using TUNEL assay. Bax overexpression was observed in metaplasic bone tissue, in fibroblasts and in myofibroblast of plaques from PD patients. Little or no bcl-2 immunostaining was detected in samples from either patients or controls. Caspase 3 immunostaining was very strong in fibrous tissue, in metaplasic bone osteocytes and in primary ossification center osteoblasts. Moderate caspase 9 immunostaining was seen in fibrous cells plaques and in osteocytes and osteoblasts of primary ossification centers from PD patients. Control samples were negative for caspase 9 immunostaining. In PD patients the TUNEL immunoassay showed intense immunostaining of fibroblasts and myofibroblasts, the absence of apoptotic cells in metaplasic bone tissue and on the border between fibrous and metaplasic bone tissue. Apoptotic cell death occurs in stabilized PD plaques and is partly induced by the intrinsic mitochondrial pathway. The present findings can have clinical implications and may help devise improved treatment strategies. A therapeutic approach aimed at enhancing apoptosis-inducing molecules would at least help delay the progression of PD. Identification of target molecules for gene construct or biological or chemical reagent delivery to target sites could contribute to induce PD plaque stabilization.

Prostate cancer (PCa) is a malignant tumor for which there are no effective treatment strategies. In this study, we developed a targeted strategy for prostate-specific membrane-antigen (PSMA)-positive PCa in vitro based on 2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (ACUPA) modified polyethylene glycol (PEG)-Cholesterol micelles containing wogonin (WOG), which was named ACUPA-M-WOG. ACUPA-M-WOG was conventionally prepared using a self-assembling method, which produced stable particle size and ζ potential. Moreover, ACUPA-M-WOG showed good drug encapsulating capacity and drug release profiles. Fluorescence activated cell sorting (FACS) results suggested that ACUPA modified PEG-Cholesterol micelles could effectively enhance the drug uptake on PSMA(+) PCa cells, and the cytotoxicity of ACUPA-M-WOG was stronger than other controls according to in vitro cellular proliferation and apoptosis assays, separately through methyl thiazolyl tetrazolium (MTT) and Annexin V/Propidium Iodide (PI) staining. Finally, the molecular mechanisms of ACUPA-M-WOG’s effects on human PSMA(+) PCa were investigated, and were mainly the intrinsic or extrinsic apoptosis signaling pathways. The Western blot results suggested that ACUPA-M-WOG could enhance the WOG-induced apoptosis, which was mainly via the intrinsic signaling pathway rather than the extrinsic signaling pathway. In conclusion, ACUPA-M-WOG was successfully developed for WOG-selective delivery to PSMA(+) PCa cells and had stronger inhibition than free drugs, which might make it an effective strategy for PSMA(+) PCa.

Squamous cell carcinoma of the lung is one of the most aggressive cancers, and its aggressiveness is in part due to its intrinsic high rate of metastasis. Moreover, the process of epithelial-mesenchymal transition(EMT) appears to be involved in these neoplastic processes. Furthermore, EMT-type cells share many biological characteristics with the function of angiogenin(ANG) in squamous cell lung carcinoma. We conducted immunohistochemical analysis to detect the expression of ANG, E-cadherin, vimentin, N-cadherin, β-catenin and TGF-β1 in 60cases of squamous cell lung carcinoma tissues. Western blot analysis was adopted to detect the protein expression levels of ANG and EMT markers. The effects of ANG on proliferation, migration and invasion of squamous cell lung carcinoma cells was analyzed by Cell Counting Kit-8, scratch assay and Transwell invasion chamber in order to reveal the role of ANG in the process of EMT in squamous cell lung carcinoma. The results revealed that ANG was aberrantly expressed in the squamous cell lung carcinoma specimens and was closely correlated with the differentiation of the cell lines. The expression of ANG was also significantly associated with metastasis and the stage of the squamous cell lung carcinoma cases. In addition, we validated that ANG influenced the expression of vimentin, E-cadherin, N-cadherin, β-catenin and TGF-β1 in SK-MES-1 cells. Most importantly, overexpression of ANG enhanced the migration and invasion of SK-MES-1 cells, while knockdown resulted in opposite effects. In the present study, we found that ANG plays an important role in EMT in squamous cell lung carcinoma and may be a valuable therapeutic target for squamous cell lung carcinoma.

Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells

Alkylphosphocholines (APCs) represent a new class of anti-tumour agents. In contrast to most of the conventional anti-cancer drugs, these compounds do not act on the cellular DNA but operate on the level of the plasma membrane. APCs are synthetic phospholipids, which show only marginal substrate features to phospholipid metabolising enzymes. Due to small chemical alterations they are stable compounds and exhibit a strongly increased resistance to phospholipase degradation. As a result APCs accumulate in the plasma membrane of the target cells and lead to dramatic alterations, which finally result in death. One of the most significant mechanisms of APC induced tumour cell death is via the initiation of apoptosis (programmed cell death). In the present study, the focus was set on the investigation of the apoptosis-inducing potency of one special APC-compound, the S-NC-2. The induction of apoptosis in neoplastic cells was clearly demonstrated by the use of different microscopic and biochemical methods, the investigation of initiator- and effector-caspases-activation as well as the cleavage of specific death substrates that were shown to be specific markers of programmed cell death. One important regulatory element of S-NC-2 induced apoptosis could be identified as the CD95 death receptor. Immunohistochemical studies demonstrated a clustering of the death receptor molecules upon S-NC-2 treatment as well as a translocation of the receptors to specific microdomains of the plasma membrane - the so called lipid rafts. The use of genetically modified cell lines, which were deficient in important molecules of the CD95 signal transduction pathway, or even in the death receptor itself, demonstrated a dramatic reduction of the apoptosis inducing potency of S-NC-2. Furthermore, the participation of the mitochondrial and the lysosomal pathway could be identified in the signalling pathway. The loss of the mitochondrial/lysosomal membrane integrity as well as the release of pro-apoptotic molecules like cytochrome c and cathepsin B, were obvious markers for the participation of these organelles in S-NC-2 induced cell death. Moreover, depending on the cell type, apoptosis could be blocked by the overexpression of the anti-apoptotic protein Bcl-2. An additional particular feature of APCs is their selectivity towards tumour cells that could also be demonstrated for the compound S-NC-2. The comparison of primary peripheral blood lymphocytes and leukaemia cells on their sensitivity to S-NC-2 treatment showed a strong cytotoxic effect in neoplastic cells, whereas normal cells were spared. As the majority of anti-cancer agents damage besides tumour cells also fast growing healthy tissue, it is of great importance to find new strategies of chemotherapeutic application. Promising candidates are the APCs and the knowledge about the mode of action of one special APC, the S-NC-2, which was achieved in this study, can be accounted as an interesting and helpful tool for the development of new targeted anti-cancer drugs.

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.

Objective To partially purify the toxic factor secreted by A. corymbifera and to analyze the mechanism of A. corymbifera-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Methods Glycoprotein secreted by A. corymbifera was purified by Con A Lectin chromatography. The influence of different protein fractions on HUVEC apoptosis was determined by flow eytometer. Both denaturing and nondenaturing deglycosylation of purified glycoprotein was performed and the ability of the protein moiety and carbohydrate moiety to induce HUVEC apoptosis was evaluated respectively. Activation of related caspases during A. corymbifera-induced apoptosis was analyzed by Western blot. The role of caspase-8 and -9 in HUVEC apoptosis was investigated using caspase inhibitors. Caspase inhibitors were used to stop the suppression of HUVEC viability by XTT assay. Results Flow cytometric analysis shows the total protein as well as the glycoprotein fraction of A. corymbifera may induce HUVEC apoptosis in a dose dependent manner. In contrast, similar activity was not observed in the non-glycoprotein fraction. Neither deglycosylated protein nor carbohydrate moiety is able to induce HUVEC apoptosis alone. In the apoptotic signaling pathway, caspase9, caspase-3 and cytochrome C were activated significantly, except caspase-8. Moreover, caspase-9 inhibitor, instead of caspase-8 inhibitor, completely abrogates A. corymbifera-induced HUVEC apoptosis. Caspase9 and caspase-3 inhibitors completely waived the suppression of HUVEC viability by A. corymbifera. Conclusion Glycoprotein secreted by A. corymbifera is associated with HUVEC apoptosis. Intact glycoprotein is essential for the apoptotic progress. Intrinsic apoptotic signaling pathway mediates A. corymbifera-induced HUVEC apoptosis. Key words: Mucorales; Absidia corymbifera; Glycoprotein; Human umbilical vein endothelial cell; Apoptosis

The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TNFSF12, CD255) (TWEAK) can stimulate apoptosis in certain cancer cells. Previous studies suggest that TWEAK activates cell death indirectly, by inducing TNFα-mediated autocrine signals. However, the underlying death-signaling mechanism has not been directly defined. Consistent with earlier work, TWEAK assembled a proximal signaling complex containing its cognate receptor FN14, the adaptor TRAF2, and cellular inhibitor of apoptosis protein 1 (cIAP1). Neither the death domain adaptor Fas-associated death domain nor the apoptosis-initiating protease caspase-8 associated with this primary complex. Rather, TWEAK induced TNFα secretion and TNF receptor 1-dependent assembly of a death-signaling complex containing receptor-interacting protein 1 (RIP1), FADD, and caspase-8. Knockdown of RIP1 by siRNA prevented TWEAK-induced association of FADD with caspase-8 but not formation of the FN14-TRAF2-cIAP1 complex and inhibited apoptosis activation. Depletion of the RIP1 E3 ubiquitin ligase cIAP1 enhanced assembly of the RIP1-FADD-caspase-8 complex and augmented cell death. Conversely, knockdown of the RIP1 deubiquitinase CYLD inhibited these functions. Depletion of FADD, caspase-8, BID, or BAX and BAK but not RIP3 attenuated TWEAK-induced cell death. Pharmacologic inhibition of the NF-κB pathway or siRNA knockdown of RelA attenuated TWEAK induction of TNFα and association of RIP1 with FADD and caspase-8. These results suggest that TWEAK triggers apoptosis by promoting assembly of a RIP1-FADD-caspse-8 complex via autocrine TNFα-TNFR1 signaling. The proapoptotic activity of TWEAK is modulated by cIAP1 and CYLD and engages both the extrinsic and intrinsic signaling pathways.

Cyclin A1 (Ccna1), a member of the mammalian A type cyclins, is most abundantly expressed in spermatocytes and is essential for spermatogenesis in the mouse. Ccna1- deficient spermatocytes arrest at late meiotic prophase and undergo apoptosis. To further delineate the mechanisms and key factors involved in this process, we have examined changes in expression of genes involved in both intrinsic and extrinsic signaling pathways that trigger apoptosis in the mutant spermatocytes. Our results show that both pathways are involved, and that the factors involved in the intrinsic pathway were expressed earlier than those involved in the extrinsic pathway. We have also begun to identify in vivo Ccna1-interacting proteins, using an unbiased biochemical approach, and identified 14-3-3, a key regulator of apoptosis, as a Ccna1-interacting protein. Expression levels of 14-3-3 proteins remain unchanged between wild type and mutant testes but there were differences in the subcellular distribution. In wild type control, 14-3-3 is detected in both cytosolic and nuclear fractions whereas it is restricted to the cytoplasm in mutant testes. This differential distribution of 14-3-3 may contribute to the induction of apoptosis in Ccna1-deficient spermatocytes. These results provide insight into the apoptotic mechanisms and pathways that are triggered when progression through the meiotic cell cycle is defective in male gametogenesis.