Pro-apoptotic Smac Research Articles

Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy

BackgroundImmunogenic cell death (ICD) is a crucial approach to turn immunosuppressive tumor microenvironment (ITM) into immune-responsive milieu and improve the response rate of immune checkpoint blockade (ICB) therapy. However, cancer cells show resistance to ICD-inducing chemotherapeutic drugs, and non-specific toxicity of those drugs against immune cells reduce the immunotherapy efficiency.MethodsHerein, we propose cancer cell-specific and pro-apoptotic liposomes (Aposomes) encapsulating second mitochondria-derived activator of caspases mimetic peptide (SMAC-P)-doxorubicin (DOX) conjugated prodrug to potentiate combinational ICB therapy with ICD. The SMAC-P (AVPIAQ) with cathepsin B-cleavable peptide (FRRG) was directly conjugated to DOX, and the resulting SMAC-P-FRRG-DOX prodrug was encapsulated into PEGylated liposomes.ResultsThe SMAC-P-FRRG-DOX encapsulated PEGylated liposomes (Aposomes) form a stable nanostructure with an average diameter of 109.1 ± 5.14 nm and promote the apoptotic cell death mainly in cathepsin B-overexpressed cancer cells. Therefore, Aposomes induce a potent ICD in targeted cancer cells in synergy of SMAC-P with DOX in cultured cells. In colon tumor models, Aposomes efficiently accumulate in targeted tumor tissues via enhanced permeability and retention (EPR) effect and release the encapsulated prodrug of SMAC-P-FRRG-DOX, which is subsequently cleaved to SMAC-P and DOX in cancer cells. Importantly, the synergistic activity of inhibitors of apoptosis proteins (IAPs)-inhibitory SMAC-P sensitizing the effects of DOX induces a potent ICD in the cancer cells to promote dendritic cell (DC) maturation and stimulate T cell proliferation and activation, turning ITM into immune-responsive milieu.ConclusionsEventually, the combination of Aposomes with anti-PD-L1 antibody results in a high rate of complete tumor regression (CR: 80%) and also prevent the tumor recurrence by immunological memory established during treatments.Graphical

Novel meriolin derivatives activate the mitochondrial apoptosis pathway in the presence of antiapoptotic Bcl-2

Meriolin derivatives represent a new class of kinase inhibitors with a pronounced cytotoxic potential. Here, we investigated a newly synthesized meriolin derivative (termed meriolin 16) that displayed a strong apoptotic potential in Jurkat leukemia and Ramos lymphoma cells. Meriolin 16 induced apoptosis in rapid kinetics (within 2–3 h) and more potently (IC50: 50 nM) than the previously described derivatives meriolin 31 and 36 [1]. Exposure of Ramos cells to meriolin 16, 31, or 36 for 5 min was sufficient to trigger severe and irreversible cytotoxicity. Apoptosis induction by all three meriolin derivatives was independent of death receptor signaling but required caspase-9 and Apaf-1 as central mediators of the mitochondrial death pathway. Meriolin-induced mitochondrial toxicity was demonstrated by disruption of the mitochondrial membrane potential (ΔΨm), mitochondrial release of proapoptotic Smac, processing of the dynamin-like GTPase OPA1, and subsequent fragmentation of mitochondria. Remarkably, all meriolin derivatives were able to activate the mitochondrial death pathway in Jurkat cells, even in the presence of the antiapoptotic Bcl-2 protein. In addition, meriolins were capable of inducing cell death in imatinib-resistant K562 and KCL22 chronic myeloid leukemia cells as well as in cisplatin-resistant J82 urothelial carcinoma and 2102EP germ cell tumor cells. Given the frequent inactivation of the mitochondrial apoptosis pathway by tumor cells, such as through overexpression of antiapoptotic Bcl-2, meriolin derivatives emerge as promising therapeutic agents for overcoming treatment resistance.

Cancer-specific drug-drug nanoparticles of pro-apoptotic and cathepsin B-cleavable peptide-conjugated doxorubicin for drug-resistant cancer therapy

Chemotherapy has shown remarkable therapeutic efficacy for various types of cancer. However, drug resistance reduces the effectiveness and sensitivity of chemotherapy, leading treatment failure and cancer relapse in many clinical indications. Herein, we propose cancer-specific drug-drug nanoparticles (DD-NPs) that improve the therapeutic efficacy of chemotherapy against drug-resistant cancer. Cancer-specific and pro-apoptotic drug-drug conjugate was prepared by conjugating the pro-apoptotic peptide drug (SMAC; Ala-Val-Pro-Ile-Ala-Gln, AVPIAQ) and cathepsin B-cleavable peptide (Phe-Arg-Arg-Gly, FRRG) to a doxorubicin (DOX), resulting in SMAC-FRRG-DOX that allows self-assembled into nanoparticles. The resulting DD-NPs were specifically cleaved to pro-apoptotic SMAC and cytotoxic DOX only in cathepsin B-overexpressing cancer cells, inducing a synergy of the pro-apoptotic activity with the chemotherapy. In MCF-7 breast tumor-bearing mice, intravenously injected DD-NPs highly accumulated at targeted tumor tissues via enhanced permeability and retention (EPR) effect, releasing SMAC and DOX, which showed a synergetic pro-apoptotic/chemotherapy. Furthermore, DD-NPs greatly suppressed tumor growth and improved overall survival in a metastatic lung cancer model. Collectively, these cancer-specific drug-drug nanoparticles may be a promising strategy to treat drug-resistant cancers with high cancer cell-specificity.

High levels of X-linked Inhibitor-of-Apoptosis Protein (XIAP) are indicative of radio chemotherapy resistance in rectal cancer

BackgroundThe mainstay of treatment in rectal cancer is neoadjuvant radio chemotherapy prior to surgery, in an attempt to downstage the tumour, allowing for more complete removal during surgery. In 40 % of cases however, this neoadjuvant radio chemotherapy fails to achieve tumour regression, partly due insufficient apoptosis signaling. X-linked Inhibitor of Apoptosis Protein (XIAP) is an anti-apoptotic protein that has been reported to contribute to disease progression and chemotherapy resistance.MethodsWe obtained rectal biopsy normal and matched tumour tissue from 29 rectal cancer patients with varying degrees of tumour regression, and using Western blot, examined anti-apoptotic XIAP and pro-apoptotic Smac protein levels in these tissues, with the aim to examine whether disturbed XIAP/Smac levels may be an indicator of neoadjuvant radio chemotherapy resistance. Expression of inhibitor of apoptosis proteins cIAP-1 and cIAP-2 was also examined.ResultsWe found that levels of XIAP increased in accordance with the degree of radio chemotherapy resistance of the tissue. Levels of this protein were also significantly higher in tumour tissue, compared to matched normal tissue in highly resistant tissue. In contrast, Smac protein levels did not increase with radio chemotherapy resistance, and the protein was similarly expressed in normal and tumour tissue, indicating a shift in the balance of these proteins. Post treatment surgical resection tissue was available for 8 patients. When we compared matched tissue pre- and post- radio chemotherapy we found that XIAP levels increased significantly during treatment in both normal and tumour tissue, while Smac levels did not change. cIAP-1 and cIAP-2 levels were not differentially expressed in varying degrees of radio chemotherapy resistance, and neoadjuvant therapy did not alter expression of these proteins.ConclusionThese data indicate that disturbance of the XIAP/Smac balance may be a driver of radio chemotherapy resistance, and hence high levels of XIAP may be a useful indicator of neoadjuvant radio chemotherapy resistance in rectal cancer. Moreover, as XIAP levels increase with radio chemotherapy it is possible that a subset of more resistant tumour cells survive this treatment and may be resistant to further adjuvant treatment. Patients with resistant tumours highly expressing XIAP may benefit from alternative treatment strategies, such as Smac mimetics post neoadjuvant radio chemotherapy.

A novel long non-coding RNA T-ALL-R-LncR1 knockdown and Par-4 cooperate to induce cellular apoptosis in T-cell acute lymphoblastic leukemia cells

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic malignancy with a poor prognosis. It has been shown that long non-coding RNA (lncRNA) plays an important role in tumorigenesis. Here, we characterized a novel lncRNA, T-ALL-R-LncR1, with whole-transcriptome deep sequencing from the Jurkat leukemic T-cell line. T-ALL-R-LncR1 was not observed in human normal tissues. However, an obvious expression was observed in some tumor tissues. T-ALL-R-LncR1 was markedly expressed in neoplastic T lymphocytes of 11 cases out of 21 children with T-ALL, indicating that T-ALL-R-LncR1 might be associated with T-ALL. T-ALL-R-LncR1 knockdown predisposed Jurkat cells to undergo pro-apoptotic factor Par-4-induced apoptosis. Further studies revealed that T-ALL-R-LncR1 knockdown facilitated the formation of a Par-4/THAP1 protein complex, resulting in the activation of caspase-3 and an increase of pro-apoptotic Smac protein in T-ALL cells. Our studies indicate a potential role of suppressing the novel long non-coding RNA T-ALL-R-LncR1 in the therapy of human T-ALL.

The role of the intrinsic FAS pathway in Titanocene Y apoptosis: The mechanism of overcoming multiple drug resistance in malignant leukemia cells

Despite the advantages in the outcome of patients with acute lymphoblastic leukemia, 25% of the affected children suffer relapses. As the response to chemotherapy is essentially determined by the development of cellular drug resistance, new drugs that are capable to overcome resistance to conventional chemotherapeutics are urgently needed. With regard to this demand, we investigated the titanium-based anticancer drug Titanocene Y.Treatment with Titanocene Y leads to inhibition of tumour cell proliferation and induces apoptosis in established cell lines of leukemia, lymphoma and melanoma.The extrinsic pathway appears to be responsible, at least in part, for the effect: cell death is partly inhibited in BJAB cells overexpressing a dominant negative Fas-associated death domain (FADD) mutant and via real time PCR we found a significant up-regulation of Fas ligand in the affected cells. Apoptosis is triggered regardless of the expression of anti-apoptotic Bcl-2 and pro-apoptotic smac and the agent is also effective on cells that are multidrug resistant due to overexpression of P-gp. In combination with vincristine impressive synergistic effects could be observed, exposing Titanocene Y as a possible component for polychemotherapy.Taken together, Titanocene Y turns out to be a promising candidate for anti-tumour therapy, especially for the treatment of multidrug resistant malignancies.

Rational design, synthesis and characterization of potent, non-peptidic Smac mimics/XIAP inhibitors as proapoptotic agents for cancer therapy

Novel proapoptotic Smac mimics/IAPs inhibitors have been designed, synthesized and characterized. Computational models and structural studies (crystallography, NMR) have elucidated the SAR of this class of inhibitors, and have permitted further optimization of their properties. In vitro characterization (XIAP BIR3 and linker-BIR2–BIR3 binding, cytotox assays, early ADMET profiling) of the compounds has been performed, identifying one lead for further in vitro and in vivo evaluation.

XIAP targeting sensitizes Hodgkin lymphoma cells for cytolytic T-cell attack

AbstractThe immunosurveillance of Hodgkin lymphoma (HL) by cytotoxic T lymphocytes (CTLs) is insufficient, and the clinical experience with adoptive transfer of CTLs is limited. We have previously reported that defects in mitochondrial apoptotic pathways and elevated XIAP expression confer resistance to different apoptotic stimuli in HL cells. Here, we aimed to develop molecular strategies to overcome the resistance of HL cells against CTL-mediated killing via granzyme B (grzB). In HL cells, grzB-induced mitochondrial release of proapoptotic Smac is blocked, which results in complete abrogation of cytotoxicity mediated by CTLs. Cytosolic expression of recombinant mature Smac enhanced caspase activity induced by grzB and restored the apoptotic response of HL cells. Similarly, down-regulation of XIAP by RNA interference markedly enhanced the susceptibility of HL cells for CTL-mediated cytotoxicity. XIAP gene knockdown sensitized HL cells for killing by antigen-specific CTLs redirected by grafting with a chimeric anti-CD30scFv-CD3zeta immunoreceptor. The results suggest that XIAP targeting by Smac agonists or XIAP-siRNA can be used as a synergistic strategy for cellular immunotherapy of Hodgkin lymphoma.

Multiple kinetics of mitochondrial cytochrome c release in drug-induced apoptosis.

We investigated cytochrome c release kinetics in response to three apoptosis-inducing agents (tumor necrosis factor-alpha, staurosporine, and valinomycin) in MCF-7/Casp-3 cells stably transfected with enhanced green fluorescent protein (EGFP)-tagged cytochrome c. All three agents induced significant caspase activation in the cultures determined by monitoring the cleavage of fluorigenic caspase substrates in extracts from drug-treated MCF-7/Casp-3 cells, albeit the valinomycin-induced activation was less pronounced. Time-lapse confocal microscopy showed that tumor necrosis factor-alpha and staurosporine caused rapid, one- or multiple-step release of cytochrome c-EGFP from mitochondria. In contrast, valinomycin-induced cytochrome c-EGFP release occurred slowly over several hours. Unlike staurosporine, the valinomycin-induced cytochrome c release was not associated with translocation of the proapoptotic Bax protein to the mitochondria, and was not accompanied by co-release of the proapoptotic Smac protein. Immunoprecipitation experiments revealed that cytochrome c was also released out of the cell into the extracellular space before loss of plasma membrane integrity. Our data indicate the existence of multiple kinetics of cytochrome c release in drug-induced apoptosis.