Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218

The integrity of lysosomes is of vital importance to survival of tumor cells. We demonstrated that LW-218, a synthetic flavonoid, induced rapid lysosomal enlargement accompanied with lysosomal membrane permeabilization in hematological malignancy. LW-218-induced lysosomal damage and lysosome-dependent cell death were mediated by cathepsin D, as the lysosomal damage and cell apoptosis could be suppressed by depletion of cathepsin D or lysosome alkalization agents, which can alter the activity of cathepsins. Lysophagy, was initiated for cell self-rescue after LW-218 treatment and correlated with calcium release and nuclei translocation of transcription factor EB. LW-218 treatment enhanced the expression of autophagy-related genes which could be inhibited by intracellular calcium chelator. Sustained exposure to LW-218 exhausted the lysosomal capacity so as to repress the normal autophagy. LW-218-induced enlargement and damage of lysosomes were triggered by abnormal cholesterol deposition on lysosome membrane which caused by interaction between LW-218 and NPC intracellular cholesterol transporter 1. Moreover, LW-218 inhibited the leukemia cell growth

Targeted therapy with a selective BCL-2 inhibitor in older patients with acute myeloid leukemia

ABSTRACT Background: Older patients with acute myeloid leukemia are particularly difficult to treat, as they have a high risk of comorbidities, poor performance status and less tolerability to chemotherapy, as well as a more aggressive disease biology, responsible for the resistance to treatment. There is a need to explore novel therapeutic agents that are more effective and tolerable. Venetoclax, a BCL-2 inhibitor is a promising agent, as BCL-2 overexpression is present in 84% of acute myeloid leukemia patients at diagnosis and 95% of patients at relapse and has been associated with leukemia cell survival, chemotherapy resistance and poor prognosis. Objective: To review the available data about venetoclax in acute myeloid leukemia and how it can influence the treatment in older patients. Methods: Using the Pubmed database, we selected 29 articles published within the last 15 years, considering preclinical and clinical trials and review studies that combined venetoclax with acute myeloid leukemia. Results: Venetoclax has demonstrated promising results in preclinical and clinical trials, especially in patients with poor prognosis and the IDH mutation, with an excellent side-effect profile. However, resistance seems to develop rapidly with venetoclax monotherapy, because of antiapoptotic escape mechanisms. Conclusions: While the results with the use of venetoclax seem encouraging, it is not likely that targeting a single pathway will result in long-term disease control. The solution includes the use of combined therapy to block resistance mechanisms and enhance apoptosis, by reducing MCL-1, increasing BIM or inhibiting the complex IV in the mitochondria.

Abt-737 enhances apoptosis of gastric cancer cells induced by MCL-1 small-molecule inhibitor UMI-77 / 中国药理学通报

Aim: To investigate the promoting effect of Bcl-2/Bcl-xL inhibitor ABT-737 on apoptosis of gastric cancer cells induced by small molecule Mcl-1 inhibitor UMI-77, and to explore its possible mechanism. Methods: The response of gastric cancer MGC-803 and HGC-27 cells to different concentrations of UMI-77 was detected by MTS assay. In the UMI-77-resistant cell lines, the effect of treatment with UMI-77/ABT-737 alone or in combination on cell viability was detected by MTS assay. The apoptotic rate and the changes of the mitochondrial membrane potential were analyzed by flow cytometry. The cleavage of caspase-9, caspase-3 and PARP-1, as well as the expression level of Bcl-2 family members and IAP proteins, were determined by Western blot. Results: Compared with MGC-803 cells, HGC-27 cells were resistant to UMI-77. Treatment with ABT-737 alone in HGC-27 cells also induced minimal level of cell death. While treatment with both agents induced much greater decreased cell viability. All the dead cells were positive for Annexin V and mitochondrial membrane potential collapsed. Caspase-9, caspase-3 and its substrate PARP-1 were cleaved. All of these proved that the sensitization effect was achieved by activating the mitochondrial apoptotic pathway. Protein levels of XIAP, cIAP1 and cIAP2 decreased after treatment with UMI-77 plus ABT-737. It also resulted in the increase of NOXA and Bcl-2 along with the decline of PUMA and Mcl-1. Conclusions: The combination of UMI-77 and ABT-737 could significantly increase the sensitivity of gastric cancer cells to the Mcl-1 small molecule inhibitor UMI-77.

Discovery of natural BH3 mimetics and research on related mechanism / 中国药理学与毒理学杂志

In the past two decades,with the increase of smoking population,more and more people are suffering from small cell lung cancer(SCLC).Besides,it is difficult to find an effective way to cure SCLC,since patience can easily develop drug resistance.On the other hand,with the development of science and technology,people began to study the anti-cancer strategy to increase apoptosis,such as inhibiting the overexpression of survival factors.In these survival factors,BCL-2 family has attracted a lot of attention.BH3-only protein is a member of BCL-2 family and it can directly inhibit the expression of BCL-2 protein,thereby prompting apoptosis.Since the BH3-only protein itself is difficult to become a clinical drug, to find alternatives BH3-only protein-BH3 mimetics is particularly important. Plus, more and more researchers have paid attention on the natural BH3 mimetic since it has less side-effect than artificial BH3 mimetics.To find possible BH3 mimetics,we made a primary screening with this pharma-cophore on a small molecular compounds library via Discovery Studio software. And then MTS assay were introduced to verify the activity of compounds. After that, we use Western Blot and Co-IP meth-ods to test the effect of BH3 mimetics.And finally use CDOCKER to predict the further mechanism on autophagy and apoptosis.In our studies, we found 3 possible BH3 mimetics compounds from 170,000 natural small molecular compounds via pharmacophore-based virtual screening.Furthermore,we dem-onstrated AD23,one of the 3 possible natural BH3 mimetics,induced autophagy and apoptosis simulta-neously in dose-time dependence in SCLC cell line. Finally, we use Molecular Docking to predict the further mechanism on autophagy and apoptosis. We believe our works would provide evidences and clues for the structural optimizing and further study of new drugs in the future.

Research progress on MCL-1 and its inhibitors in hematological malignancies / 中国肿瘤临床

Myeloid cell leukemia-1 (MCL-1) protein is one of the key antiapoptotic protein members of the B-cell lymphoma-2 protein family. Overexpression of MCL-1 is closely related to not only tumor progression but also resistance to targeted therapy and traditional chemotherapeutic drug. MCL-1 and its inhibitors have been studied in recent years. The mimetics of MCL-1 endogenous ligand BH3 have resulted in significant breakthroughs. In this study, the research progress on MCL-1 and its inhibitors in hematological malignan-cies is reviewed.

Mechanism of BH3 mimetics promoting cell apoptosis and its latest clinical research progress / 中国药理学与毒理学杂志

Targeting cell apoptosis is currently the most promising therapy for cancer treatment. The BH3-only protein, which is a member of Bcl-2 family, can bind to the pro-survival members of the family and neutralize their functional activities to induce apoptosis (ie, to isolate pro-apoptotic members of the Bcl-2 family). BH3 mimetics, a kind of small molecule compounds, has the ability to mimic the BH3-only protein to induce apoptosis. The prototype of BH3 mimetics is ABT-737, who can selectively targets on BCL-XL, BCL-2 and BCL-W (but not MCL-1 and A1). ABT-263, a derivative of ABT-737, has a better performance of inducing apoptosis and inhibiting the growth of tumor in clinical trials. At this stage, some presumably BH3 mimetics has entered the clinical stage, while a large part of them is still being characterized and tested. Basing on the mechanism of BH3-only protein, this review summarize a variety of BH3 mimetics which have been widely recognized, and show the latest developments of newly diagnosed BH3 mimetics in the field.

Crucial Residues Modulating Interface of hBcl-B - hBaxBH3 Heterodimer as Probed by Computational Methods

ABSTRACT Cancerous cells develop resistance to cell death by over expression of anti-apoptotic proteins, which are specific to interact with pro-apoptotic and BH3-only proteins of Bcl-2 family. Delineating crucial residues mediating the heterodimer complexes (anti-apoptotic proteins - pro-apoptotic/BH3-only proteins) is indispensable to develop specific antagonists to anti-apoptotic proteins. In these backgrounds, we have herein reported crucial residues of hBaxBH3 and hBcl-B (an anti-apoptotic protein specifically interacts with human Bax but does not interact with human Bak) for hetero dimerization of the polypeptides and as well validated the structural determinants of the polypeptides through variety of virtual 'alanine mutants' and 'switch mutants' by using an array of computational methods. Residues such as D53, S60, E61, K64, E69 and D71 of hBaxBH3 and R45, H50, F53, F54, Y57, M71, S74, V75, R86, V88, T89, F93 and F159 of hBcl-B were found to be crucial residues of the polypeptides for intermolecular interaction leading hetero dimerization. Moreover, 'pharmacophoric residues' for the hBaxBH3 and hBcl-B have also been figured out and rationalized.

Granzyme B, B-cell lymphoma/leukemia-2 gene and BH3-interacting domain death agonist in cell apoptais / 国际外科学杂志

Granzyme B is an important effect factor of cytotoxic T lymphocytes in the immune killer function,can quickly induce cell apoptosis of the target cell.It is complicated for the reasons and the ways of the occurrence of the cell apoptosis,many genes are involved in gene regulation of cell apoptosis,and the B-cell lymphoma/leukemia-2 gene family members play a crucial role in the process of cell apoptosis.This paper mainly reviews the correlation of GrB and Bcl-2,Bid with cell apoptosis.

Therapeutic Modulation of Apoptosis: Targeting the BCL-2 Family at the Interface of the Mitochondrial Membrane

A vast portion of human disease results when the process of apoptosis is defective. Disorders resulting from inappropriate cell death range from autoimmune and neurodegenerative conditions to heart disease. Conversely, prevention of apoptosis is the hallmark of cancer and confounds the efficacy of cancer therapeutics. In the search for optimal targets that would enable the control of apoptosis, members of the BCL-2 family of anti- and pro-apoptotic factors have figured prominently. Development of BCL-2 antisense approaches, small molecules, and BH3 peptidomimetics has met with both success and failure. Success-because BCL-2 proteins play essential roles in apoptosis. Failure-because single targets for drug development have limited scope. By examining the activity of the BCL-2 proteins in relation to the mitochondrial landscape and drawing attention to the significant mitochondrial membrane alterations that ensue during apoptosis, we demonstrate the need for a broader based multi-disciplinary approach for the design of novel apoptosis-modulating compounds in the treatment of human disease.

Analysis of the NF-kappaB and Apoptosis Inducing Genes in Bladder Tumor / 대한비뇨기과학회지

PURPOSE: A multi-subunit transcription factor NF-kappaB mediates the antiapoptotic signals in several cancer cell lines and it is activated in a broad range of human tumors. In this study, we investigated whether the expression levels of the NF-kappaB and the apoptosis inducing genes were related to the pathogenesis and clinical properties of human bladder tumor. MATERIALS AND METHODS: The expressions of NF-kappaB, BCL2-associated X protein (BAX), BCL2-associated death protein (BAD) and BH3-interacting domain death agonist protein (BID) were investigated by performing immunohistochemical staining on 133 archival bladder tissue paraffin blocks; these blocks included 122 transitional cell carcinomas of the urinary bladder and 11 normal bladder mucosae. RESULTS: The expression levels of NF-kappaB were significantly higher in the bladder tumors than those of the normal bladder mucosae (p=0.001). The expression levels of BAX in the superficial and low-grade (grade 1 and 2) bladder tumors were significantly enhanced more than those of the high-grade and invasive cases (p=0.042 and p=0.045, respectively), while the expression levels of BAD in the tumor tissues and low-grade tumors were significantly elevated compared with those of the normal mucosae and high grade tumor (p=0.007 and p=0.048, respectively). But the expressions of BID were not correlated with any pathologic and clinical properties. CONCLUSIONS: The expressions of the NF-kappaB and apoptosis inducing genes such as BAX and BAD are strongly associated with the pathogenesis and clinical properties of bladder tumor. (Korean J Urol 2007;48:483-488)

The expression of BH3-only Bcl-2 family members in mouse ovary and influence of gonadotropin on Nix expression in granulosa cells / 대한산부인과학회지

OBJECTIVE: In the ovary apoptosis eliminates granulosa cells (GC) during follicle atresia. While we identified many cell death regulatory molecules not yet characterized in the ovary, of particular interest were members of the Bcl-2 family which contain only the Bcl-2 homology (BH)-3 domain. The objectives of this study was to investigate and compare the expression patterns of BH3-only Bcl-2 family members in various organs and evaluate their function in ovarian granulosa cells. METHODS: Total RNA was extracted from GC, ovaries, uteri, hearts (low rate of cell turnover) and livers (high rate of cell turnover) of prepubertal female mice. BH3-only Bcl-2 family members were cloned to make riboprobe. The expression patterns in the tissues evaluated by Northern blot analysis. Nix mRNA expression in ovarian granulosa cells after gonadotropin treatment also compared by Northern blot analysis. RESULTS: Bad, Bid, Bim, Bmf, Map-1 and Nix were expressed in granulosa cells. Nix was most abundantly expressed in GC. In contrast, Blk was expressed in the ovary, liver, heart and uterus, but not in GC. Bmf, a sensor or microfilament disassembly, was expressed in GC, ovary and uterus, with limited to no expression in non-reproductive tissues. Nix mRNA expression was not regulated by gonadotropin after 42 hour. CONCLUSION: These studies will help to complete a molecular blueprint of the regulatory network that controls GC death during follicular atresia. In addition, these data, which show a tissue/cell-selective profile of BH3-only expression, may also explain the known variation in the in vivo apoptotic response of different tissues/cells to generic stimuli that should be globally lethal.

Role for The Bcl-2 Family Proteins and BH3 Domain in Apoptosis / 生物化学与生物物理进展

Apoptosis action is primarily exerted at the level of mitochondira, in which Bcl-2 family of proteins play an important role in its regulation. Bcl-2 family consists of anti-apoptotic and pro-apoptotic members. The anti-apoptotic members usually exist in the outer mitochondrial membrane and inhibit cell death via interaction with pro-apoptotic counterparts BH3 domain. Pro-apoptotic members are commonly localize in the cytoplasm. A series of events occured, such as typical Bax conformational change, BAD and Bik phosphorylation as well as Bid and Bim proteolysis in response to several death stimuli. As a result, these pro-apoptotic proteins directly integrate to the outer mitochondrial membranes. Finally, mitochondrial permeability transition pore is opened, by followed the release of apoptogenic factors from the mitochondrial intermembrane space, including cytochrome c, apoptosis inducing factor(AIF) and Smac, then the activation of downstream caspases and execution of cell death.

Expression,Purification of PUMA-BH3 Death Domain Peptide in E.coli and Identification of Its Pro-apoptotic Activity / 中国生物工程杂志

The Bcl-2 family of proteins play a central role in the control of apoptosis, a fundamental process for both human health and disease, by mitochondrial pathway. PUMA(p53 up-regulated modulator of apoptosis protein) is one of BH3-only members of Bcl-2 family , its function is to promote cell apoptosis. To obtain BH3 death domain peptide of PUMA and detect its biological activity, the synthesized double-stranded oligomeric nucleotide encoding PUMA-BH3 peptide was cloned into expression vector pTYB2,thus generating a construct of pTYB2-PUMA-BH3 which expressed PUMA-BH3-intein-chitin binding domain fusion protein. Then the recombinant plasmid was transformed into E.coli BL-21 (DE3) and fusion protein was expressed under induction by IPTG. The soluble PUMA-BH3 peptide was purified from chitin affinity chromatography by DTT reduction. Through measuring mitochondria viability(MTT),mitochondria permeability transition(MPT) and the translocation of cytochrome c(Cyt c ) assayed by western blotting, the biological pro-apoptotic activity of PUMA-BH3 peptide was studied. The PUMA-BH3 peptide has the effects on decreasing the mitochondria viability remarkably , inducing mitochondrial swelling and promoting Cyt c releasing from isolated mitochodria . Mitochondrial swelling and the release of Cyt c induced by PUMA-BH3 peptide concerned with the opening of MPT,which can be improved by cyclosporine A(CsA).These results indicated that recombinant PUMA-BH3 peptide might possess pro-apoptosis activity and paved a reasonable way for the study of new apoptosis regulators.