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Dynamics of Bcl-xL in Water and Membrane: Molecular Simulations  [PDF]
Atanu Maity, Seema Yadav, Chandra S. Verma, Shubhra Ghosh Dastidar
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0076837
Abstract: The Bcl2 family of proteins is capable of switching the apoptotic machinery by directly controlling the release of apoptotic factors from the mitochondrial outer membrane. They have ‘pro’ and ‘anti’-apoptotic subgroups of proteins which antagonize each other’s function; however a detailed atomistic understanding of their mechanisms based on the dynamical events, particularly in the membrane, is lacking. Using molecular dynamics simulations totaling 1.6μs we outline the major differences between the conformational dynamics in water and in membrane. Using implicit models of solvent and membrane, the simulated results reveal a picture that is in agreement with the ‘hit-and run’ concept which states that BH3-only peptides displace the tail (which acts as a pseudo substrate of the protein itself) from its binding pocket; this helps the membrane association of the protein after which the BH3 peptide becomes free. From simulations, Bcl-xL appears to be auto-inhibited by its C-terminal tail that embeds into and covers the hydrophobic binding pocket. However the tail is unable to energetically compete with BH3-peptides in water. In contrast, in the membrane, neither the tail nor the BH3-peptides are stable in the binding pocket and appear to be easily dissociated off as the pocket expands in response to the hydrophobic environment. This renders the binding pocket large and open, thus receptive to interactions with other protein partners. Principal components of the motions are dramatically different in the aqueous and in the membrane environments and provide clues regarding the conformational transitions that Bcl-xL undergoes in the membrane, in agreement with the biochemical data.
Quercetin Potentiates Doxorubicin Mediated Antitumor Effects against Liver Cancer through p53/Bcl-xl  [PDF]
Guanyu Wang, Jiawei Zhang, Luying Liu, Sherven Sharma, Qinghua Dong
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0051764
Abstract: Background The dose-dependent toxicities of doxorubicin (DOX) limit its clinical applications, particularly in drug-resistant cancers, such as liver cancer. In this study, we investigated the role of quercetin on the antitumor effects of DOX on liver cancer cells and its ability to provide protection against DOX-mediated liver damage in mice. Methodology and Results The MTT and Annexin V/PI staining assay demonstrated that quercetin selectively sensitized DOX-induced cytotoxicity against liver cancer cells while protecting normal liver cells. The increase in DOX-mediated apoptosis in hepatoma cells by quercetin was p53-dependent and occurred by downregulating Bcl-xl expression. Z-VAD-fmk (caspase inhibitor), pifithrin-α (p53 inhibitor), or overexpressed Bcl-xl decreased the effects of quercetin on DOX-mediated apoptosis. The combined treatment of quercetin and DOX significantly reduced the growth of liver cancer xenografts in mice. Moreover, quercetin decreased the serum levels of alanine aminotransferase and aspartate aminotransferase that were increased in DOX-treated mice. Quercetin also reversed the DOX-induced pathological changes in mice livers. Conclusion and Significance These results indicate that quercetin potentiated the antitumor effects of DOX on liver cancer cells while protecting normal liver cells. Therefore, the development of quercetin may be beneficial in a combined treatment with DOX for increased therapeutic efficacy against liver cancer.
Adenovirus-mediated siRNA targeting Bcl-xL inhibits proliferation, reduces invasion and enhances radiosensitivity of human colorectal cancer cells
Jinsong Yang, Ming Sun, Aiping Zhang, Chengyu Lv, Wei De, Zhaoxia Wang
World Journal of Surgical Oncology , 2011, DOI: 10.1186/1477-7819-9-117
Abstract: RT-PCR and Western blot assays were performed to determine the expression of Bcl-xL mRNA and protein in CRC cells and normal human intestinal epithelial cell line. Then, adenovirus-mediated RNA interference technique was employed to inhibit the expression of Bcl-xL gene in CRC cells. The proliferation of CRC cells was analyzed by MTT and colony formation assay. The migration and invasion of CRC cells was determined by wound-healing and tranwell invasion assays. Additionally, the in vitro and in vivo radiosensitivity of CRC cells was determined by clonogenic cell survival assay and murine xnograft model, respectively.The levels of Bcl-xL mRNA and protein expression were significantly higher in human CRC cells than in normal human intestinal epithelial cell line. Ad/shBcl-xL could significantly reduce the expression of Bcl-xL protein in CRC cells. Also, we showed that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit proliferation and colony formation of CRC cells. Ad/shBcl-xL could significantly suppress migration and invasion of CRC cells. Moreover, Ad/shBcl-xL could enhance in vitro and in vivo radiosensitivity of CRC cells by increasing caspase-dependent apoptosis.Targeting Bcl-xL will be a promising strategy to inhibit the metastatic potential and reverse the radioresistance of human CRC.Colorectal cancer, one of the most prevalent cancers in the world, is the second most common malignancy and the second leading cause of cancer related mortality in developed countries [1]. In spite of much progress made in diagnostic and therapeutic methods, the prognosis of CRC patients with distant metastasis still remains poor. Therefore, it is necessary to understand the molecular signaling mechanisms of CRC development so as to provide important insights into more effective therapeutic strategies.Bcl-xL, an anti-apoptotic member, plays important roles in tumor progression and development [2]. Bcl-xL molecule may inhibit apoptosis by maintaining the perme
Functions of BCL-XL at the Interface between Cell Death and Metabolism  [PDF]
Judith Michels,Oliver Kepp,Laura Senovilla,Delphine Lissa,Maria Castedo,Guido Kroemer,Lorenzo Galluzzi
International Journal of Cell Biology , 2013, DOI: 10.1155/2013/705294
Abstract: The BCL-2 homolog BCL-XL, one of the two protein products of BCL2L1, has originally been characterized for its prominent prosurvival functions. Similar to BCL-2, BCL-XL binds to its multidomain proapoptotic counterparts BAX and BAK, hence preventing the formation of lethal pores in the mitochondrial outer membrane, as well as to multiple BH3-only proteins, thus interrupting apical proapoptotic signals. In addition, BCL-XL has been suggested to exert cytoprotective functions by sequestering a cytosolic pool of the pro-apoptotic transcription factor p53 and by binding to the voltage-dependent anion channel 1 (VDAC1), thereby inhibiting the so-called mitochondrial permeability transition (MPT). Thus, BCL-XL appears to play a prominent role in the regulation of multiple distinct types of cell death, including apoptosis and regulated necrosis. More recently, great attention has been given to the cell death-unrelated functions of BCL-2-like proteins. In particular, BCL-XL has been shown to modulate a number of pathophysiological processes, including—but not limited to—mitochondrial ATP synthesis, protein acetylation, autophagy and mitosis. In this short review article, we will discuss the functions of BCL-XL at the interface between cell death and metabolism. 1. Introduction According to current models, cell death most often proceeds via either of two relatively independent subroutines, apoptosis, and necrosis [1, 2]. For a long time, apoptotic and necrotic instances of cell death have exclusively been identified based on morphological criteria [2]. In addition, while apoptosis was believed to constitute the sole regulated (i.e., genetically encoded, and hence susceptible to pharmacological modulation) modality of cell death, necrosis was viewed as a purely accidental process [3]. Recently, a functional classification of cell death mechanisms, based on measurable biochemical features, has been proposed [1], and the concept of regulated necrosis has gained large consensus [4]. In this scenario, the true relevance of additional processes that were previously catalogued as bona fide cell death subroutines is being reevaluated. In particular, while macroautophagy (hereafter referred to as autophagy) turned out to constitute a prominent homeostatic and cytoprotective mechanism [5, 6], autophagic cell death (a lethal subroutine that is mediated, rather than merely accompanied, by autophagy) has been shown to occur in a limited number of, mostly developmental, scenarios [1, 7]. Along similar lines, mitotic catastrophe, a signaling cascade elicited in
Cyclin B1/Cdk1 Phosphorylation of Mitochondrial p53 Induces Anti-Apoptotic Response  [PDF]
Danupon Nantajit,Ming Fan,Nadire Duru,Yunfei Wen,John C. Reed,Jian Jian Li
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0012341
Abstract: The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53+/+ status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53?/? cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53?/? cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53.
Significance of Bcl-xL in human colon carcinoma  [cached]
You-Li Zhang, Li-Qun Pang, Ying Wu, Xiao-Yan Wang, Chong-Qiang Wang, Yu Fan
World Journal of Gastroenterology , 2008,
Abstract: AIM: To investigate the clinical significance of Bcl-xL gene in the pathogenesis of human colon carcinoma.METHODS: Fifty-six pair tissue samples from patients with colon cancer were collected, and protein level of the Bcl-xL gene was measured by immunohistochemistry method. The correlation of Bcl-xL expression with clinical index was evaluated. After human colon cancer cell line HT29 was transfected with Bcl-xL small interfering RNA (siRNA), the anchorage-independent growth of cancer cells was detected by colony formation in soft agar and invasion ability of cancer cells was determined by a transwell model.RESULTS: The Bcl-xL expression was higher in cancerous tissue samples than in normal tissue samples (38.78 ±11.36 vs 0.89 ± 0.35, P < 0.001), and was associated with the pathological grade, lymphnode metastasis and Duke’s stage of colorectal carcinoma. Transfection with Bcl-xL siRNA inhibited the colony formation and invasion ability of human colon cancer cell line HT29 in vitro.CONCLUSION: Bcl-xL gene plays an important role in carcinogenesis of human colorectal carcinoma and is associated with malignant biological behaviors of human colorectal carcinoma.
The Anti-Apoptotic Bcl-xL Protein, a New Piece in the Puzzle of Cytochrome C Interactome  [PDF]
Ivano Bertini,Soizic Chevance,Rebecca Del Conte,Daniela Lalli,Paola Turano
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0018329
Abstract: A structural model of the adduct between human cytochrome c and the human anti-apoptotic protein Bcl-xL, which defines the protein-protein interaction surface, was obtained from solution NMR chemical shift perturbation data. The atomic level information reveals key intermolecular contacts identifying new potentially druggable areas on cytochrome c and Bcl-xL. Involvement of residues on cytochrome c other than those in its complexes with electron transfer partners is apparent. Key differences in the contact area also exist between the Bcl-xL adduct with the Bak peptide and that with cytochrome c. The present model provides insights to the mechanism by which cytochrome c translocated to cytosol can be intercepted, so that the apoptosome is not assembled.
BcL-xL Conformational Changes upon Fragment Binding Revealed by NMR  [PDF]
Clémentine Aguirre, Tim ten Brink, Olivier Walker, Florence Guillière, Dany Davesne, Isabelle Krimm
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0064400
Abstract: Protein-protein interactions represent difficult but increasingly important targets for the design of therapeutic compounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approaches for the elaboration of protein-protein surface inhibitors from fragment-like molecules. One major challenge in targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are therefore required at the early steps of the drug design process. In this report we present a fast NMR method able to probe subtle conformational changes upon fragment binding. The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding. We illustrate the method by the retrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 4′-fluoro-[1,1′-biphenyl]-4-carboxylic acid that was previously shown to bind one of the Bcl-xL hot spots. The CSP-based approach shows that the protein undergoes a subtle conformational rearrangement upon interaction, for residues located in helices 2, 3 and the very beginning of 5. Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound forms of the Bcl-xL protein. These NMR-based results are in total agreement with previous molecular dynamic calculations that evidenced a high flexibility of Bcl-xL around the binding site. Here we show that CSP of protein amine protons are useful and reliable structural probes. Therefore, we propose to use CSP simulation to assess protein conformational changes upon ligand binding in the fragment-based drug design approach.
Curcumin induces human HT-29 colon adenocarcinoma cell apoptosis by activating p53 and regulating apoptosis-related protein expression
Song, G.;Mao, Y.B.;Cai, Q.F.;Yao, L.M.;Ouyang, G.L.;Bao, S.D.;
Brazilian Journal of Medical and Biological Research , 2005, DOI: 10.1590/S0100-879X2005001200007
Abstract: curcumin, a major yellow pigment and active component of turmeric, has multiple anti-cancer properties. however, its molecular targets and mechanisms of action on human colon adenocarcinoma cells are unknown. in the present study, we examined the effects of curcumin on the proliferation of human colon adenocarcinoma ht-29 cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method and confirmed the curcumin-induced apoptosis by morphology and dna ladder formation. at the same time, p53, phospho-p53 (ser15), and other apoptosis-related proteins such as bax, bcl-2, bcl-xl, pro-caspase-3, and pro-caspase-9 were determined by western blot analysis. the colon adenocarcinoma cells were treated with curcumin (0-75 μm) for 0-24 h. we observed that p53 was highly expressed in ht-29 cells and curcumin could up-regulate the serine phosphorylation of p53 in a time- and concentration-dependent manner. an increase in expression of the pro-apoptotic factor bax and a decrease in expression of the anti-apoptotic factor bcl-2 were also observed in a time-dependent manner after exposure of 50 μm curcumin, while the expression of the anti-apoptotic factor bcl-xl was unchanged. curcumin could also down-regulate the expression of pro-caspase-3 and pro-caspase-9 in a time-dependent manner. these data suggest a possible underlying molecular mechanism whereby curcumin could induce the apoptosis signaling pathway in human ht-29 colon adenocarcinoma cells by p53 activation and by the regulation of apoptosis-related proteins. this property of curcumin suggests that it could have a possible therapeutic potential in colon adenocarcinoma patients.
Curcumin induces human HT-29 colon adenocarcinoma cell apoptosis by activating p53 and regulating apoptosis-related protein expression  [cached]
Song G.,Mao Y.B.,Cai Q.F.,Yao L.M.
Brazilian Journal of Medical and Biological Research , 2005,
Abstract: Curcumin, a major yellow pigment and active component of turmeric, has multiple anti-cancer properties. However, its molecular targets and mechanisms of action on human colon adenocarcinoma cells are unknown. In the present study, we examined the effects of curcumin on the proliferation of human colon adenocarcinoma HT-29 cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method and confirmed the curcumin-induced apoptosis by morphology and DNA ladder formation. At the same time, p53, phospho-p53 (Ser15), and other apoptosis-related proteins such as Bax, Bcl-2, Bcl-xL, pro-caspase-3, and pro-caspase-9 were determined by Western blot analysis. The colon adenocarcinoma cells were treated with curcumin (0-75 μM) for 0-24 h. We observed that p53 was highly expressed in HT-29 cells and curcumin could up-regulate the serine phosphorylation of p53 in a time- and concentration-dependent manner. An increase in expression of the pro-apoptotic factor Bax and a decrease in expression of the anti-apoptotic factor Bcl-2 were also observed in a time-dependent manner after exposure of 50 μM curcumin, while the expression of the anti-apoptotic factor Bcl-xL was unchanged. Curcumin could also down-regulate the expression of pro-caspase-3 and pro-caspase-9 in a time-dependent manner. These data suggest a possible underlying molecular mechanism whereby curcumin could induce the apoptosis signaling pathway in human HT-29 colon adenocarcinoma cells by p53 activation and by the regulation of apoptosis-related proteins. This property of curcumin suggests that it could have a possible therapeutic potential in colon adenocarcinoma patients.
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