Inhibitors Of Apoptotic Proteins Research Articles

Enzymatically Formed Peptide Assemblies Sequestrate Proteins and Relocate Inhibitors to Selectively Kill Cancer Cells.

Herein, we show that an enzymatic reaction can generate peptide assemblies that sequestrate proteins to selectively kill cancer cells. A phosphopeptide bearing the antagonistic motif (AVPI) to the inhibitors of apoptotic proteins (IAPs) enters cancer cells and normal cells by caveolin-dependent endocytosis and macropinocytosis, respectively. The AVPI-bearing peptide assemblies sequestrates IAPs and releases bortezomib (BTZ), a proteasome inhibitor, in the cytosol of cancer cells, but rescues the normal cells (namely, HS-5 cells) by trafficking the BTZ into lysosomes. Alkaline phosphatase (ALP) acts as a context-dependent signal for trafficking the peptide/BTZ assemblies and selectively induces the death of the cancer cells. The assemblies of AVPI exhibit enhanced proteolytic resistance. This work, which utilizes the difference in endocytic uptake of enzymatically formed peptide assemblies to selectively kill cancer cells, promises a new way to develop selective cancer therapeutics.

Enzymatically Formed Peptide Assemblies Sequestrate Proteins and Relocate Inhibitors to Selectively Kill Cancer Cells

AbstractHerein, we show that an enzymatic reaction can generate peptide assemblies that sequestrate proteins to selectively kill cancer cells. A phosphopeptide bearing the antagonistic motif (AVPI) to the inhibitors of apoptotic proteins (IAPs) enters cancer cells and normal cells by caveolin‐dependent endocytosis and macropinocytosis, respectively. The AVPI‐bearing peptide assemblies sequestrates IAPs and releases bortezomib (BTZ), a proteasome inhibitor, in the cytosol of cancer cells, but rescues the normal cells (namely, HS‐5 cells) by trafficking the BTZ into lysosomes. Alkaline phosphatase (ALP) acts as a context‐dependent signal for trafficking the peptide/BTZ assemblies and selectively induces the death of the cancer cells. The assemblies of AVPI exhibit enhanced proteolytic resistance. This work, which utilizes the difference in endocytic uptake of enzymatically formed peptide assemblies to selectively kill cancer cells, promises a new way to develop selective cancer therapeutics.

Therapeutic Inducers of Apoptosis in Ovarian Cancer.

Ovarian cancers remain one of the most common causes of gynecologic cancer-related death in women worldwide. The standard treatment comprises platinum-based chemotherapy, and most tumors develop resistance to therapeutic drugs. One mechanism of developing drug resistance is alterations of molecules involved in apoptosis, ultimately assisting in the cells’ capability to evade death. Thus, there is a need to focus on identifying potential drugs that restore apoptosis in cancer cells. Here, we discuss the major inducers of apoptosis mediated through various mechanisms and their usefulness as potential future treatment options for ovarian cancer. Broadly, they can target the apoptotic pathways directly or affect apoptosis indirectly through major cancer-pathways in cells. The direct apoptotic targets include the Bcl-2 family of proteins and the inhibitor of apoptotic proteins (IAPs). However, indirect targets include processes related to homologous recombination DNA repair, micro-RNA, and p53 mutation. Besides, apoptosis inducers may also disturb major pathways converging into apoptotic signals including janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), wingless-related integration site (Wnt)/β-Catenin, mesenchymal-epithelial transition factor (MET)/hepatocyte growth factor (HGF), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)/v-AKT murine thymoma viral oncogene homologue (AKT)/mammalian target of rapamycin (mTOR) pathways. Several drugs in our review are undergoing clinical trials, for example, birinapant, DEBIO-1143, Alisertib, and other small molecules are in preclinical investigations showing promising results in combination with chemotherapy. Molecules that exhibit better efficacy in the treatment of chemo-resistant cancer cells are of interest but require more extensive preclinical and clinical evaluation.

Orientin Induces G0/G1 Cell Cycle Arrest and Mitochondria Mediated Intrinsic Apoptosis in Human Colorectal Carcinoma HT29 Cells.

Colorectal carcinoma is one of the utmost diagnosed cancer with a steep increase in mortality rate. The incidence has been increasing in developing countries like India due to a westernization life style. Flavonoids have been explored widely for its various pharmacological activity including antitumor activity. Orientin, an analogue of luteolin (citrus flavonoid) isolated from rooibos and tulsi leaves is also expected to deliver significant antitumor activity similar to that of luteolin. The present study anticipates exploring the antitumor activity of orientin against colorectal carcinoma cells (HT29). Orientin exhibited remarkable cytotoxicity and antiproliferative activity against HT29 cells, which is clearly evident from tetrazolium based cytotoxicity and lactate dehydrogenase release assays. Orientin induce G0/G1 cell cycle arrest and regulates cyclin and cyclin-dependent protein kinases in order to prevent the entry of the cell cycle to the S phase. Annexin V-FITC (V-Fluorescein Isothiocyanate) dual staining reveals the apoptotic induction ability of orientin. The Bcl-2 family proteins along with the inhibitor of apoptotic proteins were regulated and the tumor suppressor p-53 expression have been decreased. In conclusion, our results proposed that orientin could be a potent chemotherapeutic agent against colorectal cancer after ascertaining their molecular mechanisms.

Drug Combinations Co-Targeting Myeloid Cell Leukemia-1 (Mcl-1) Protein Can Overcome Microenvironmentally-Induced Multi-Drug Tolerance in Non-Hodgkin Lymphomas

Ibrutinib (IBR), an inhibitor of Bruton's Tyrosine Kinase (Btk) and venetoclax (VEN), an inhibitor of Bcl-2 have been used in Chronic Lymphocytic Leukemia (CLL), but single agent responses to these drugs are often incomplete and not durable. We previously reported synergistic cytotoxicity for the IBR+VEN combination and have initiated a trial to test that combination (NCT02419560). However, we noted extensive variability in response to IBR+VEN between patient samples ex vivo, suggesting intrinsic tolerance even to this combination. CLL cells with an “activation” phenotype (CD5+/19+/69+) that occurs from interactions with the microenvironment were less sensitive to IBR+VEN, implicating the cancer microenvironment as an inducer of drug tolerance. This was supported by our finding that CLL cells display decreased sensitivity to IBR+VEN in co-culture with agonists and cells that emulate the cancer cell microenvironment. The combination of CpG-oligodeoxynucleotides (ODN), sCD40L, and IL10 (“agonist mix”) induced near complete loss of sensitivity to IBR+VEN and upregulated Mcl-1 and Bcl-xL expression in CLL cells. However, Mcl-1 or Bcl-xL inhibitors alone were weakly effective in these cells, suggesting that exploitation of these targets requires drug combinations.Here we report a combination drug screen with an Mcl-1 inhibitor in a CLL microenvironment model ex vivo and novel drug combinations that overcome multi-drug tolerance. CLL patient PBMCs were cultured in the microenvironment model consisting of HK follicular dendritic cells, HUVEC, Jurkat T cells expressing CD40L, and CpG-ODN. Markers characteristic of activation and drug tolerance in vivo were assessed in CLL (CD5+/19+) cells using flow cytometry. We found increased expression of Ki67, CD69, and anti-apoptotic proteins Mcl-1 and Bcl-xL and decreased CXCR4 expression, as expected upon microenvironmental stimuli in vivo.Also, CLL cells from 2 IBR-treated patients were still responsive to agonist-induced drug tolerance, suggesting Btk inhibition may be less effective in blocking these microenvironmental stimuli in vivo. By comparing the ratio of anti-apoptotic proteins with cognate pro-apoptotic proteins, we noted targetable dependence of CLL cells for diverse pro-survival proteins (Mcl-1 and Bcl-xL) in our model.Flow cytometry analysis demonstrated that inhibitors of Mcl-1 (S63845), Bcl-xL (A1155643), or Bcl-2 (VEN) when used as single agents failed to induce significant apoptosis in CLL cells cultured in our model, suggesting induction of multi-drug tolerance. Drug combinations inhibiting Mcl-1 with Bcl-xL, Bcl-2, or Bcl-xL and Bcl-2 have effectively overcome multi-drug tolerance. As direct and simultaneous inhibition of multiple pro-survival proteins could be fatal to healthy cells, we carried out a Mcl-1-anchored combinatorial drug screen with inhibitors of other survival pathways in 3 CLL patient PBMCs cultured in our ex vivo model to identify drug combination(s) that induce selective toxicity in multi-drug tolerant CLL cells. Cytotoxicity was determined by analyzing cleaved PARP and dead cell staining in CLL and healthy T (CD3+/5+) cells by flow cytometry. Results showed that inhibitors of apoptotic proteins (A1155643, VEN, ABT737), IRAK4 (CA-4948, compound 26, and AS2444697), intracellular TLRs (chloroquine), Hsp90 (ganetespib), CDK (ribociclib), proteasome (bortezomib), Btk (IBR), AKT (MK2206), and HDAC (SAHA and panobinostat) or activator of PP2A (DBK1532 and NZ8061) were synergistically toxic with Mcl-1 inhibitor (S63845) in CLL cells. By comparing toxicity in CLL and healthy T cells, drug combinations targeting Mcl-1 with proteasome, IRAK4, TLRs, Btk, Hsp90, and CDK showed selective toxicity in CLL cells, indicating a potential therapeutic window for these combinations. Drug combinations targeting Mcl-1 with other apoptotic proteins were highly toxic to healthy T cells.In conclusion, CLL cells in our ex vivo model display characteristics of microenvironmentally-induced multi-drug tolerance. A similar phenotype was noted using post IBR therapy samples, suggesting IBR alone may not be effective in overcoming this multi-drug tolerance in vivo. Drug combinations targeting Mcl-1 and proteasome, IRAK4, TLRs, Btk, Hsp90, or CDK selectively overcame multi-drug tolerance in CLL cells. Thus, these combinations may be effective in patients showing intrinsic tolerance to multiple drugs. DisclosuresPortell:TG therapeutics: Research Funding; AbbVie: Research Funding; Infinity: Research Funding; Genentech/Roche: Consultancy, Research Funding; Acerta: Research Funding; BeiGene: Research Funding; Kite: Research Funding; Amgen: Consultancy. Narla:University of Michigan: Patents & Royalties: Small molecule PP2A activators. Williams:Takeda: Research Funding; TG Therapeutics: Consultancy; Seattle Genetics: Consultancy; Verastem: Consultancy; Juno: Consultancy; Sandoz: Consultancy; Celgene: Consultancy, Research Funding; Novartis: Research Funding; Astra-Zeneca: Consultancy; Abbvie: Consultancy; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Kite: Consultancy; Gilead: Consultancy, Research Funding.

Chemosensitization of Prostate Carcinoma Cells with a Receptor-directed Smac Conjugate.

Second mitochondrial activator of caspase (Smac) is a short mitochondrial peptide. When released from the mitochondria into the cytoplasm, it binds to inhibitor of apoptotic proteins (IAPs) within the cytoplasm and prevents them from inhibiting apoptosis. Delivery of external synthetic Smac peptide into the cytoplasm of malignant cells could greatly improve the efficiency of apoptosis-inducing chemotherapeutic agents. In our study different conjugates based on the seven N-terminal amino acids AVPIAQK of Smac (SmacN7) were produced to obtain a cytoplasm-directed Smac variant. SmacN7 and a point mutant (AVPKAQK) were coupled either to rhodamine alone or to both rhodamine and undecylic aldehyde, which is an antagonist of the Lily-of-the-valley fragrance receptor. The fifth conjugate consisted of rhodamine coupled only to undecylic aldehyde, without SmacN7. The uptake of these five conjugates into three different human cell lines was characterized and quantified by confocal laser scanning microscopy and flow cytometry. A caspase apoptosis assay was performed for cells incubated with the five different conjugates after induction of apoptosis. The coupling of undecylic aldehyde to SmacN7 increased the cellular uptake of the correct and mutant conjugates. Caspase 3/7 apoptosis tests after induction of apoptosis with staurosporine or UV irradiation showed that the coupling of SmacN7 with undecylic aldehyde resulted in a greatly increased adjuvant pro-apoptotic effect compared to the separate components and a mutant SmacN7 peptide sequence in the LNCaP prostate carcinoma cells compared to the benign prostate hyperplasia (BPH) cells and the human embryonal kidney (HEK) cells.

Interaction of DNA-lesions induced by sodium fluoride and radiation and its influence in apoptotic induction in cancer cell lines

Fluoride is an essential trace element but also an environmental contaminant with major sources of exposure being drinking water, food and pesticides. Previous studies showed that sodium fluoride (NaF) at 5mM or more is required to induce apoptosis and chromosome aberrations and proposed that DNA damage and apoptosis play an important role in toxicity of excessive fluoride. The aim of this study is directed to understand the nature of DNA-lesions induced by NaF by allowing its interaction with radiation induced DNA-lesions. NaF 5mM was used after observing inability to induce DNA damages and apoptosis by single exposure with 50μM or 1mM NaF. Co-exposure to NaF and radiation significantly increased the frequency of aberrant metaphases and exchange aberrations in human lymphocytes and arrested the cells in G1 stage instead of apoptotic death. Flow cytometric analysis, DNA fragmentation and PARP-cleavage analysis clearly indicated that 5mM NaF together with radiation (1Gy) induced apoptosis in both U87 and K562 cells due to down regulation of expression of anti-apoptotic proteins, like Bcl2 in U87 and inhibitors of apoptotic proteins like survivin and cIAP in K562 cells. This study herein suggested that single exposure with extremely low concentration of NaF unable to induce DNA lesions whereas higher concentration induced DNA lesions interact with the radiation-induced DNA lesions. Both are probably repaired rapidly thus showed increased interactive effect. Coexposure to NaF and radiation induces more apoptosis in cancer cell lines which could be due to increased exchange aberrations through lesions interaction and downregulating anti-apoptotic genes.

Inhibitors of Apoptotic Proteins: New Targets for Anticancer Therapy

Inhibitors of apoptotic proteins (IAPs) can play an important role in inhibiting apoptosis by exerting their negative action on caspases (apoptotic proteins). There are eight proteins in this family: NAIP/BIRC1/NLRB, cellular IAP1 (cIAP1)/human IAP2/BIRC2, cellular IAP2 (cIAP2)/human IAP1/BIRC3, X-linked IAP (XIAP)/BIRC4, survivin/BIRC5, baculoviral IAP repeat (BIR)-containing ubiquitin-conjugating enzyme/apollon/BIRC6, livin/melanoma-IAP (ML-IAP)/BIRC7/KIAP, and testis-specific IAP (Ts-IAP)/hILP-2/BIRC8. Deregulation of these inhibitors of apoptotic proteins (IAPs) may push cell toward cancer and neurodegenerative disorders. Inhibitors of apoptotic proteins (IAPs) may provide new target for anticancer therapy. Drugs may be developed that are inhibiting these IAPs to induce apoptosis in cancerous cells.

Smac/DIABLO gene expression in acute myeloid leukemia patients

Background Smac/DIABLO enhances apoptosis by antagonizing the inhibitors of apoptotic proteins. The expression of Smac/DIABLO in different cancers has been reported. The study aimed to evaluate Smac/DIABLO gene expression in patients with acute myeloid leukemia (AML) and also to determine its relation to the clinical outcome and survival of these patients. Materials and methods Smac/DIABLO gene expression was studied using real-time PCR in bone marrow samples from 70 newly diagnosed AML patients. Results The Smac/DIABLO gene was expressed in 88.5% of AML patients. A total of 32 patients (51.6%) with positive Smac/DIABLO expression responded to treatment, and the remaining 30 patients (48.4%) were treatment resistant. Smac/DIABLO expression was associated with decreased lactate dehydrogenase levels and increased disease-free survival (P = 0.01 and P < 0.001, respectively). The expression was associated with an increase in the survival rate (P < 0.05). Conclusion There was an increase of Smac/DIABLO expression in AML patients, and this was associated with the treatment response, increased disease-free survival, and better overall survival. Egypt J Haematol 38:80–83 � c 2013 The Egyptian Society of Haematology.

Abstract 3254: Novel bivalent Smac mimetics as a new class of anticancer agents

Abstract Second mitochondria-derived activator of caspase (Smac) is a protein released from mitochondria in response to apoptotic stimuli which functions as an endogenous inhibitor of several members of inhibitor of apoptotic proteins (IAPs), including XIAP, cIAP-1 and cIAP-2. Smac interacts with IAPs by binding of its N-terminal AVPI tetrapeptide to one or more BIR domains in IAPs. We designed and synthesized a series of non-peptidic, cell-permeable, bivalent small-molecules which mimic the dimeric Smac protein for targeting IAPs (bivalent Smac mimetics) and performed extensive evaluations of these bivalent Smac mimetics for their interaction with IAP proteins and their activity and mechanism of function in cancer cells. Our studies show that these Smac mimetics bind to XIAP, cIAP-1 and cIAP-2 with low nano-molar affinities. They function as extremely potent XIAP antagonists by concurrently targeting both the BIR2 and BIR3 domains of XIAP and nullify its inhibition to both caspase-9 and caspase-3/-7 and potently and effectively induce rapid degradation of these two proteins in cancer cells. The most potent bivalent Smac mimetics inhibit cell growth with IC50 values at low nanomolar and effectively induce apoptosis in a subset of cancer cell lines through TNFα dependent mechanism. Furthermore, bivalent Smac mimetics can dramatically enhance the antitumor activity of TRAIL in both TRAIL-sensitive and -resistant cancer cell lines. The combination of one such bivalent Smac mimetic, SM-164 with TRAIL induces rapid tumor regression in vivo in a breast cancer xenograft model in which either agent is ineffective. Mechanistic studies demonstrate that while RIP1 plays a minimal role in TRAIL's activity as a single agent, it is required for the synergistic interaction between TRAIL and SM-164. XIAP and cIAP1 work in concert to attenuate the activity of TRAIL and SM-164 strongly sensitizes TRAIL by concurrently targeting XIAP and cIAP1. Taken together, our data suggest that bivalent Smac mimetics may have a great therapeutic potential for the treatment of human cancer by targeting multiple IAPs, either alone or in combination with TRAIL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3254. doi:10.1158/1538-7445.AM2011-3254

Surviving apoptosis.

The concept that cells subjected to chromatin cleavage during apoptosis are destined to die is being challenged. The execution phase of apoptosis is characterized by the activation of effector caspases, such as caspase-3, that cleave key regulatory or structural proteins and in particular activate apoptotic nucleases such as the caspase activated deoxyribonuclease (CAD). It is apparent that caspases of this type may become active both through non-apoptotic processing and potentially within cells that exhibit apoptotic morphology but are subsequently able to survive. In such systems caspase suppressor molecules, the inhibitors of apoptotic proteins or IAP's, may rescue cells from apoptotic nuclease(s) attack initiated by transient caspase activation. The MLL gene is involved in leukemogenic translocations in ALL and AML and is a target of nuclease cleavage during apoptosis. Translocations initiated at the site of apoptotic nuclease attack within MLL have been identified and may offer a model, with clinical relevance, for DNA damage mediated by the apoptosis system in cells destined to survive. The specificity of apoptotic cleavage combined with the potential for recovery from the execution phase of apoptosis suggests a novel and pathogenic role for apoptosis in creating translocations with leukemogenic potential.