Potent Inducer Of Apoptosis Research Articles

MycobacteriumtuberculosisProtein PE6 (Rv0335c), a Novel TLR4 Agonist, Evokes an Inflammatory Response and Modulates the Cell Death Pathways in Macrophages to Enhance Intracellular Survival.

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that exploits moonlighting functions of its proteins to interfere with host cell functions. PE/PPE proteins utilize host inflammatory signaling and cell death pathways to promote pathogenesis. We report that M. tb PE6 protein (Rv0335c) is a secretory protein effector that interacts with innate immune toll-like receptor TLR4 on the macrophage cell surface and promotes activation of the canonical NFĸB signaling pathway to stimulate secretion of proinflammatory cytokines TNF-α, IL-12, and IL-6. Using mouse macrophage TLRs knockout cell lines, we demonstrate that PE6 induced secretion of proinflammatory cytokines dependent on TLR4 and adaptor Myd88. PE6 possesses nuclear and mitochondrial targeting sequences and displayed time-dependent differential localization into nucleus/nucleolus and mitochondria, and exhibited strong Nucleolin activation. PE6 strongly induces apoptosis via increased production of pro-apoptotic molecules Bax, Cytochrome C, and pcMyc. Mechanistic details revealed that PE6 activates Caspases 3 and 9 and induces endoplasmic reticulum-associated unfolded protein response pathways to induce apoptosis through increased production of ATF6, Chop, BIP, eIF2α, IRE1α, and Calnexin. Despite being a potent inducer of apoptosis, PE6 suppresses innate immune defense strategy autophagy by inducing inhibitory phosphorylation of autophagy initiating kinase ULK1. Inversely, PE6 induces activatory phosphorylation of autophagy master regulator MtorC1, which is reflected by lower conversion of autophagy markers LC3BI to LC3BII and increased accumulation of autophagy substrate p62 which is also dependent on innate immune receptor TLR4. The use of pharmacological agents, rapamycin and bafilomycin A1, confirms the inhibitory effect of PE6 on autophagy, evidenced by the reduced conversion of LC3BI to LC3BII and increased accumulation of p62 in the presence of rapamycin and bafilomycin A1. We also observed that PE6 binds DNA, which could have significant implications in virulence. Furthermore, our analyses reveal that PE6 efficiently binds iron to likely aid in intracellular survival. Recombinant Mycobacterium smegmatis (M. smegmatis) containing pe6 displayed robust growth in iron chelated media compared to vector alone transformed cells, which suggests a role of PE6 in iron acquisition. These findings unravel novel mechanisms exploited by PE6 protein to subdue host immunity, thereby providing insights relevant to a better understanding of host–pathogen interaction during M. tb infection.

Stigmasterol isolated from the chloroform fraction of Adenopus breviflorus Benth fruit induces mitochondrial-dependent apoptosis in rat liver.

Decoction of Adenopus breviflorus fruit is used in folkloric medicine for treating dysmenorrhea and gonorrhea. Phytochemicals from A.breviflorus may be potent in inducing mitochondrial-dependent apoptosis via the opening of the mitochondrial permeability transition (MPT) pore. Therefore, this study investigated the in vitro effects of stigmasterol isolated from the chloroform fraction of A.breviflorus (CFAB) and also the increasing concentration of CFAB on the opening of rat liver mitochondrial permeability transition (MPT) pore. Fractionation of CFAB on column chromatography yielded a needle-like crystal which structure was elucidated by standard spectroscopic techniques. The effects of stigmasterol and CFAB on MPT pore opening were assayed spectrophotometrically. Also, the effect of CFAB on mitochondrial ATPase (mATPase) activity and cytochrome c (Cyt c) release were determined. Stigmasterol isolated from CFAB induced MPT pore opening significantly (p<0.05) when compared withthe control. Similarly, CFAB significantly (p<0.05) induced MPT pore opening in rat liver mitochondria in a concentration-dependent manner in the presence and absence of the triggering agent- calcium ion. Furthermore, the increasing concentration of CFAB significantly (p<0.05) stimulated mitochondrial ATPase (mATPase) activity and Cyt c release in a concentration-dependent manner. The study showed that stigmasterol isolated from the chloroform fraction of A.breviflorus is a potent inducer of mitochondrial-dependent apoptosis. Also, the study further revealed that CFAB possesses potent bioactive compounds which can induce the mitochondrial-dependent apoptosis through the opening of the mitochondrial permeability transition pore, activation of mitochondrial ATPase (mATPase) activity and cytochrome c release.

Optimization of PhysicoChemical Parameters for Production of Cytotoxic Secondary Metabolites and Apoptosis Induction Activities in the Culture Extract of a Marine Algal-Derived Endophytic Fungus Aspergillus sp.

The endophytic fungal community in the marine ecosystem has been demonstrated to be relevant source of novel and pharmacologically active secondary metabolites. The current study focused on the evaluation of cytotoxic and apoptosis induction potential in the culture extracts of endophytic fungi associated with Sargassum muticum, a marine brown alga. The cytotoxicity of the four marine endophytes, Aspergillus sp., Nigrospora sphaerica, Talaromyces purpureogenus, and Talaromyces stipitatus, was evaluated by the MTT assay on HeLa cells. Further, several physicochemical parameters, including growth curve, culture media, and organic solvents, were optimized for enhanced cytotoxic activity of the selected extract. The Aspergillus sp. ethyl acetate extract (ASE) showed maximum cytotoxicity on multiple cancer cell lines. Chemical investigation of the metabolites by gas chromatography–mass spectroscopy (GC-MS) showed the presence of several compounds, including quinoline, indole, 2,4-bis(1,1-dimethylethyl) phenol, and hexadecenoic acid, known to be cytotoxic in ASE. The ASE was then tested for cytotoxicity in vitro on a panel of six human cancer cell lines, namely, HeLa (cervical adenocarcinoma), MCF-7 (breast adenocarcinoma), Hep G2 (hepatocellular carcinoma), A-549 (lung carcinoma), A-431 (skin/epidermis carcinoma), and LN-229 (glioblastoma). HeLa cells were most vulnerable to ASE treatment with an IC50 value of 24 ± 2 μg/ml. The mechanism of cytotoxicity exhibited by the ASE was further investigated on Hela cells. The results showed that the ASE was capable of inducing apoptosis in HeLa cells through production of reactive oxygen species, depolarization of mitochondrial membrane, and activation of the caspase-3 pathway, which shows a possible activation of the intrinsic apoptosis pathway. It also arrested the HeLa cells at the G2/M phase of the cell cycle, eventually leading to apoptosis. Through this study, we add to the knowledge about the marine algae associated with fungal endophytes and report its potential for purifying specific compounds responsible for cytotoxicity.

Global Phosphoproteomics Reveal CDK Suppression as a Vulnerability to KRas Addiction in Pancreatic Cancer.

Among human cancers that harbor mutant (mt) KRas, some, but not all, are dependent on mt KRas. However, little is known about what drives KRas dependency. Global phosphoproteomics, screening of a chemical library of FDA drugs, and genome-wide CRISPR/Cas9 viability database analysis were used to identify vulnerabilities of KRas dependency. Global phosphoproteomics revealed that KRas dependency is driven by a cyclin-dependent kinase (CDK) network. CRISPR/Cas9 viability database analysis revealed that, in mt KRas-driven pancreatic cancer cells, knocking out the cell-cycle regulators CDK1 or CDK2 or the transcriptional regulators CDK7 or CDK9 was as effective as knocking out KRas. Furthermore, screening of a library of FDA drugs identified AT7519, a CDK1, 2, 7, and 9 inhibitor, as a potent inducer of apoptosis in mt KRas-dependent, but not in mt KRas-independent, human cancer cells. In vivo AT7519 inhibited the phosphorylation of CDK1, 2, 7, and 9 substrates and suppressed growth of xenografts from 5 patients with pancreatic cancer. AT7519 also abrogated mt KRas and mt p53 primary and metastatic pancreatic cancer in three-dimensional (3D) organoids from 2 patients, 3D cocultures from 8 patients, and mouse 3D organoids from pancreatic intraepithelial neoplasia, primary, and metastatic tumors. A link between CDK hyperactivation and mt KRas dependency was uncovered and pharmacologically exploited to abrogate mt KRas-driven pancreatic cancer in highly relevant models, warranting clinical investigations of AT7519 in patients with pancreatic cancer.

Neuregulin 1/ErbB4/Akt signaling attenuates cytotoxicity mediated by the APP-CT31 fragment of amyloid precursor protein

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by neuronal and synaptic loss. The cytoplasmic tail of amyloid precursor protein (APP) undergoes sequential cleavage at a specific intracellular caspase site to generate the cytoplasmic terminal 31 (CT31) fragment. The APP-CT31 fragment is a potent inducer of apoptosis. The cytotoxicity of APP-CT31 in SH-SY5Y cells was evaluated by the lactate dehydrogenase (LDH) assay. TUNEL staining was used to detect apoptotic signals in SH-SY5Y cells and primary cortical neurons. The expression of apoptosis-related proteins, such as p53, PUMA (p53 up-regulated modulator of apoptosis), and cleaved was investigated by immunofluorescence analysis and Western blotting. In this study, we investigated the neuroprotective effect of neuregulin 1 (NRG1) against cytotoxicity induced by APP-CT31. Our data showed that CT31 induced cytotoxicity and apoptosis in SH-SY5Y cells and primary cortical neurons. NRG1 attenuated the neurotoxicity induced by the expression of APP-CT31. We also showed that APP-CT31 altered the expression of p53 and cleaved caspase 3. However, treatment with NRG1 rescued the APP-CT31-induced upregulation of p53 and cleaved caspase 3 expression. The protective effect of NRG1 was abrogated by inhibition of the ErbB4 receptor and Akt. These results indicate an important role of ErbB4/Akt signaling in NRG1-mediated neuroprotection, suggesting that endogenous NRG1/ErbB4 signaling represents a valuable therapeutic target in AD.

Synthesis and antitumor potential of new arylidene ursolic acid derivatives via caspase-8 activation.

Continuing our studies on NO-donating ursolic acid-benzylidene derivatives as potential antitumor agents, we designed and synthesized a series of new arylidene derivatives containing NO-donating ursolic acid and aromatic heterocyclic units. Compounds 5c and 6c showed a significant broad-spectrum antitumor activity. Compound 5c exhibited nearly three- to nine-fold higher cytotoxicity as compared with the parent drug in A549, MCF-7, HepG-2, HT-29, and HeLa cells, and it was also found to be the most potent apoptosis inducer of MCF-7 cells. More importantly, compound 5c arrested the MCF-7 cell cycle in the G1 phase, which was associated with caspase activation and a decrease of the Bcl-2/Bax ratio. Meanwhile, compound 5c caused changes in morphological features, dissipation of the mitochondrial membrane potential, and accumulation of reactive oxygen species. A docking study revealed that the nitroxyethyl moiety of compound 5c may form hydrogen bonds with caspase-8 amino acid residues (SER256 and HIS255). Together, these data suggest that NO-donating ursolic acid-arylidene derivatives are potent apoptosis inducers in tumor cells.

Synthetic Studies on Selective, Proapoptotic Isomalabaricane Triterpenoids Aided by Computational Techniques.

The isomalabaricanes comprise a large family of marine triterpenoids with fascinating structures that have been shown to be selective and potent apoptosis inducers in certain cancer cell lines. In this article, we describe the successful total syntheses of the isomalabaricanes stelletin A, stelletin E, and rhabdastrellic acid A, as well as the development of a general strategy to access other natural products within this unique family. High-throughput experimentation and computational chemistry methods were used in this endeavor. A preliminary structure-activity relationship study of stelletin A revealed the trans-syn-trans core motif of the isomalabaricanes to be critical for their cytotoxic activity.

Toxic Prediction of Pyrrolizidine Alkaloids and Structure-Dependent Induction of Apoptosis in HepaRG Cells.

Pyrrolizidine alkaloids (PAs) are common phytotoxins and could cause liver genotoxicity/carcinogenicity following metabolic activation. However, the toxicity of different structures remains unclear due to the wide variety of PAs. In this study, the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of 40 PAs were analyzed, and their toxicity was predicted by Komputer Assisted Technology (TOPKAT) using Discovery Studio software. The in silico results showed that all PAs except retronecine had good intestinal absorption, and all PAs were predicted to have different toxicity ranges. To verify the predictive results, 4 PAs were selected to investigate cell injury and possible mechanisms of the differentiation in HepaRG cells, including retronecine type of twelve-membered cyclic diester (retrorsine), eleven-membered cyclic diester (monocrotaline), noncyclic diester (retronecine), and platynecine type (platyphylline). After 24 h exposure, retronecine-type PAs exhibited concentration-dependent cytotoxicity. The high-content screening assay showed that cell oxidative stress, mitochondrial damage, endoplasmic reticulum stress, and the concentration of calcium ions increased, and neutral lipid metabolism was changed notably in HepaRG cells. Induced apoptosis by PAs was indicated by cell cycle arrest in the G2/M phase, disrupting the mitochondrial membrane potential. Overall, our study revealed structure-dependent cytotoxicity and apoptosis after PA exposure, suggesting that the prediction results of in silico have certain reference values for compound toxicity. A 1,2-membered cyclic diester seems to be a more potent apoptosis inducer than other PAs.

Pharmacological inactivation of CDK2 inhibits MYC/BCL-XL-driven leukemia in vivo through induction of cellular senescence

ABSTRACT Deregulated expression of the MYC oncogene is a frequent event during tumorigenesis and generally correlates with aggressive disease and poor prognosis. While MYC is a potent inducer of apoptosis, it often suppresses cellular senescence, which together with apoptosis is an important barrier against tumor development. For this latter function, MYC is dependent on cyclin-dependent kinase 2 (CDK2). Here, we utilized a MYC/BCL-XL-driven mouse model of acute myeloblastic leukemia (AML) to investigate whether pharmacological inhibition of CDK2 can inhibit MYC-driven tumorigenesis through induction of senescence. Purified mouse hematopoietic stem cells transduced with MYC and BCL-XL were transplanted into lethally irradiated mice, leading to the development of massive leukemia and subsequent death 15–17 days after transplantation. Upon disease onset, mice were treated with the selective CDK2 inhibitor CVT2584 or vehicle either by daily intraperitoneal injections or continuous delivery via mini-pumps. CVT2584 treatment delayed disease onset and moderately but significantly improved survival of mice. Flow cytometry revealed a significant decrease in tumor load in the spleen, liver and bone marrow of CVT2584-treated compared to vehicle-treated mice. This was correlated with induced senescence evidenced by reduced cell proliferation, increased senescence-associated β-galactosidase activity and heterochromatin foci, expression of p19ARF and p21CIP1, and reduced phosphorylation (activation) of pRb, while very few apoptotic cells were observed. In addition, phosphorylation of MYC at Ser-62 was decreased. In summary, inhibition of CDK2 delayed MYC/BCL-XL-driven AML linked to senescence induction. Our results suggest that CDK2 is a promising target for pro-senescence cancer therapy, in particular for MYC-driven tumors, including leukemia.

Induction of apoptosis in retinoid-refractory acute myelogenous leukemia by a novel AHPN analog

AbstractAcute myelogenous leukemia (AML) is a heterogeneous disease consisting of a variety of different leukemic subtypes. While acute promyelocytic leukemia displays marked sensitivity to the differentiating effects of trans-retinoic acid (tRA), other subtypes of AML display resistance. We now describe a novel compound (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC/MM002) that induces apoptosis in the tRA-resistant leukemia cell lines M07e, KG-1, and HL-60R, and in tRA-resistant patient leukemic blasts. The 3-Cl-AHPC totally inhibits leukemia colony formation at concentrations that inhibit committed human bone marrow stem cell proliferation, that is, granulocyte/macrophage colony-forming units (CFU-GMs) by only 30%. Exposure to 3-Cl-AHPC results in caspase activation and the cleavage of poly(adenosine diphosphate) (poly(ADP)) ribose polymerase. While activation of the extracellular signal-regulated kinase (ERK) and p38 pathways is not necessary for 3-Cl-AHPC-mediated apoptosis, maximal apoptosis requires c-Jun N-terminal kinase (JNK) activation. The 3-Cl-AHPC-mediated cleavage of the antiapoptotic B-cell leukemia XL (Bcl-XL) protein to a proapoptotic 18-kDa product is found in both the M07e cell line and patient leukemic blasts. The 3-Cl-AHPC treatment of mice bearing the AML 1498 cell line results in a 3.3-log kill in the leukemic blasts. While 3-Cl-AHPC does not activate retinoic nuclear receptors, it is a potent inducer of apoptosis in AML cells and may represent a novel therapy in the treatment of this disease. (Blood. 2003; 102:3743-3752)

Chaetolactam A, an Azaphilone Derivative from the Endophytic Fungus Chaetomium sp. g1.

Chaetolactam A (1), an unprecedented azaphilone derivative bearing a unique 9-oxa-7-azabicyclo[4.2.1]octan-8-onering system, together with two new compounds, 11-epi-chaetomugilide B (2) and chaetomugilide D (3) was isolated from an endophytic fungus, Chaetomium sp. g1. Notably, extensive NMR data analyses, NMR calculations with DP4 and DP4+ analyses, ECD calculations, and the RDC method were employed to establish the structure of 1. Furthermore, 2 exhibited potent apoptosis induction activity by mediating caspase-3 activation and PARP degradation at 3 μM in HL-60.

Polychemotherapy with Curcumin and Doxorubicin via Biological Nanoplatforms: Enhancing Antitumor Activity.

Doxorubicin (DOX) is a well-known chemotherapeutic agent extensively applied in the field of cancer therapy. However, similar to other chemotherapeutic agents such as cisplatin, paclitaxel, docetaxel, etoposide and oxaliplatin, cancer cells are able to obtain chemoresistance that limits DOX efficacy. In respect to dose-dependent side effect of DOX, enhancing its dosage is not recommended for effective cancer chemotherapy. Therefore, different strategies have been considered for reversing DOX resistance and diminishing its side effects. Phytochemical are potential candidates in this case due to their great pharmacological activities. Curcumin is a potential antitumor phytochemical isolated from Curcuma longa with capacity of suppressing cancer metastasis and proliferation and affecting molecular pathways. Experiments have demonstrated the potential of curcumin for inhibiting chemoresistance by downregulating oncogene pathways such as MMP-2, TGF-β, EMT, PI3K/Akt, NF-κB and AP-1. Furthermore, coadministration of curcumin and DOX potentiates apoptosis induction in cancer cells. In light of this, nanoplatforms have been employed for codelivery of curcumin and DOX. This results in promoting the bioavailability and internalization of the aforementioned active compounds in cancer cells and, consequently, enhancing their antitumor activity. Noteworthy, curcumin has been applied for reducing adverse effects of DOX on normal cells and tissues via reducing inflammation, oxidative stress and apoptosis. The current review highlights the anticancer mechanism, side effects and codelivery of curcumin and DOX via nanovehicles.

Co-Delivery of Hispolon and Doxorubicin Liposomes Improves Efficacy Against Melanoma Cells.

Hispolon is a small molecular weight polyphenol that has antioxidant, anti-inflammatory, and anti-proliferative activities. Our recent study has demonstrated hispolon as a potent apoptosis inducer in melanoma cell lines. Doxorubicin is a broad spectrum first-line treatment for various kinds of cancers. In this study, co-delivery of doxorubicin and hispolon using a liposomal system in B16BL6 melanoma cell lines for synergistic cytotoxic effects was investigated. Liposomes were prepared using a lipid film hydration method and loaded with doxorubicin or hispolon. The formulations were characterized for particle size distribution, release profile, and encapsulation efficiency (EE). In addition, in vitro cytotoxicity, in vitro cell apoptosis, and cellular uptake were evaluated. Liposomes exhibited small particle size (mean diameter ~ 100nm) and narrow size distribution (polydispersity index (< 0.2) and high drug EE% (> 90%). The release from liposomes showed slower release compared to free drug solution as an additional time required for the release of drug from the liposome lipid bilayer. Liposome loaded with doxorubicin or hispolon exhibited significantly higher cytotoxicity against B16BL6 melanoma cells as compared to doxorubicin solution or hispolon solution. Likewise, co-delivery of hispolon and doxorubicin liposomes showed two-fold and three-fold higher cytotoxicity, as compared to hispolon liposomes or doxorubicin liposomes, respectively. In addition, co-delivery of doxorubicin and hispolon in liposomes enhanced apoptosis more than the individual drugs in the liposome formulation. In conclusion, the co-delivery of hispolon and doxorubicin could be a promising therapeutic approach to improve clinical outcomes against melanoma.

Synthesis and biological assessment of ciprofloxacin-derived 1,3,4-thiadiazoles as anticancer agents

The quinolone-3-carboxylic acid scaffold is essential structure for antibacterial activity of fluoroquinolones such as ciprofloxacin. Modification of 3-carboxylic functionality in this structure can be used for switching its activity from antibacterial to anticancer. Accordingly, a series of C-3 modified ciprofloxacin derivatives containing N-(5-(benzylthio)-1,3,4-thiadiazol-2-yl)-carboxamide moiety was synthesized as novel anticancer agents. Most of compounds showed significant activity against MCF-7, A549 and SKOV-3 cancer cells in the MTT assay. In particular, compounds 13a-e and 13g were found to be as potent as standard drug doxorubicin against MCF-7 cell line (IC50s = 3.26–3.90 µM). Furthermore, the 4-fluorobenzyl derivatives 13h and 14b with IC50 values of 3.58 and 2.79 µM exhibited the highest activity against SKOV-3 and A549 cells, being as potent as doxorubicin. Two promising compounds 13e and 13g were further tested for their apoptosis inducing activity and cell cycle arrest. Both compounds could significantly induce apoptosis in MCF-7 cells, while compound 13e was more potent apoptosis inducer resulting in an 18-fold increase in the proportion of apoptotic cells at the IC50 concentration in MCF-7 cells. The cell cycle analysis revealed that compounds 13e and 13g could increase cell portions in the sub-G1 phase, inducing oligonucleosomal DNA fragmentation and apoptosis confirmed by comet assay.

Investigation of Dextran-Coated Superparamagnetic Nanoparticles for Targeted Vinblastine Controlled Release, Delivery, Apoptosis Induction, and Gene Expression in Pancreatic Cancer Cells

In the current study, the surface of superparamagnetic iron oxide (SPION) was coated with dextran (DEX), and conjugated with folic acid (FA), to enhance the targeted delivery and uptake of vinblastine (VBL) in PANC-1 pancreatic cancer cells. Numerous analyses were performed to validate the prepared FA-DEX-VBL-SPION, such as field emission scanning transmission electron microscopy, high-resolution transmission electron microscopy, dynamic light scattering (DLS), Zeta Potential, Fourier transform infrared spectroscopy, and vibrating sample magnetometry (VSM). The delivery system capacity was evaluated by loading and release experiments. Moreover, in vitro biological studies, including a cytotoxicity study, cellular uptake assessment, apoptosis analysis, and real-time PCR, were carried out. The results revealed that the obtained nanocarrier was spherical with a suitable dispersion and without visible aggregation. Its average size, polydispersity, and zeta were 74 ± 13 nm, 0.080, and −45 mV, respectively. This dual functional nanocarrier also exhibited low cytotoxicity and a high apoptosis induction potential for successful VBL co-delivery. Real-time quantitative PCR analysis demonstrated the activation of caspase-3, NF-1, PDL-1, and H-ras inhibition, in PANC-1 cells treated with the FA-VBL-DEX-SPION nanostructure. Close inspection of the obtained data proved that the FA-VBL-DEX-SPION nanostructure possesses a noteworthy chemo-preventive effect on pancreatic cancer cells through the inhibition of cell proliferation and induction of apoptosis.

Abstract 6225: Addition of TRAIL receptor agonists after treatment with ONC201 or ONC212 converts pancreatic cancer cells from anti-proliferative to apoptotic in vitro

Abstract The 7% five-year survival rate for pancreatic cancer patients has remained unchanged for decades; the need for novel therapeutic interventions is imminent. ONC201 is the founding member of the impridone class, a class of orally bioavailable small molecules which show efficacy in multiple tumor types. ONC201 and ONC212, a fluorinated imipridone, have been shown to be potent inducers of the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. Both compounds are currently in clinical trials. In the context of pancreatic cancer, ONC201 and ONC212 induce either apoptotic or anti-proliferative effects. Additionally, both compounds activate the integrated stress response leading to DR5 upregulation in various cell types. We hypothesized that exogenous addition of TRAIL receptor agonists after pre-treatment with ONC201 or ONC212 could convert cells from anti-proliferative to apoptotic. Here, we examined both rhTRAIL and TLY012, a novel TRAIL receptor agonist with a substantially improved half-life. Pancreatic cancer cell lines were screened for synergy when treated with ONC201 or ONC212 for 72 hours followed by either rhTRAIL or TLY012 for 4 hours. Four cell lines (AsPC-1, BxPC3, Capan-1, and PANC-1) showed potent synergy with both rhTRAIL and TLY012. Growth inhibition was assessed through both Cell-Titer Glo and colony assays. Apoptotic synergy was determined using Cell-Titer Glo, western blots for PARP cleavage, and sub-G1 flow cytometric assays. Further analysis showed that ONC201 activates the integrated stress response and primes pancreatic cancer cells for TRAIL-induced apoptosis through regulation of key proteins. Upregulation of the integrated stress response and downregulation of XIAP, c-FLIP, and Bcl-XL was observed after treatment with ONC201 in BxPC3 and PANC-1 cell lines. The combination of ONC201 and TRAIL receptor agonists was non-toxic to human foreskin fibroblasts. Future experiments aim to examine G1 arrest after treatment with ONC201 and ONC212 via western blot and flow cytometry, to confirm surface expression of TRAIL and DR5 via flow cytometry, and to determine efficacy in pancreatic cancer xenografts. Taken together, these data indicate a novel therapeutic strategy for treatment of patients with ONC201 and ONC212 resistant pancreatic tumors. Citation Format: Aakash V. Jhaveri, Lanlan Zhou, Isacco Ferarrini, Young Lee, Wafik S. El-Deiry. Addition of TRAIL receptor agonists after treatment with ONC201 or ONC212 converts pancreatic cancer cells from anti-proliferative to apoptotic in vitro [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6225.

Renal Tubular Cells from Hibernating Squirrels are Protected against Cisplatin Induced Apoptosis.

Hibernating 13-lined ground squirrels are characterized by tolerance of severe hypothermia and hypoperfusion during torpor, followed by periodic warm reperfusion during IBA, conditions which are lethal to nonhibernating mammals. The aim of the present study was to determine whether protection from apoptosis was specific to torpor arousal cycles during hibernation or will also apply to cisplatin treatment on squirrel renal tubular cells (RTECs) that were procured during hibernation. Squirrel and mouse RTECs were treated with cisplatin, a potent inducer of RTEC apoptosis. Squirrel RTECs subjected to cisplatin had significantly less apoptosis, no cleaved caspase-3, and increased XIAP, pAkt, and pBAD versus mouse RTECs. To determine whether XIAP and Akt1 are necessary for RTEC protection against cisplatin induced apoptotic cell death, gene expression of Akt1 or XIAP was silenced in squirrel RTECs. Squirrel RTECs deficient in Akt1 and XIAP had increased apoptosis and cleaved caspase-3 when treated with cisplatin. Our results thus demonstrates that 13-lined ground squirrel RTECs possess intrinsic intracellular mechanisms by which they protect themselves from apoptotic cell death. Cisplatin induced acute kidney injury (AKI) may be avoided in cancer patients by employing mechanisms used by squirrel RTECs to protect against cisplatin induced tubular cell apoptosis.

Auricularia auricula increases an apoptosis in human hepatocellular carcinoma cells via a regulation of the peroxiredoxin1.

Auricularia auricula (A. auricula) has been reported to have positive health effects. Therefore, this study was conducted to explore possible mechanisms of A. auricula-induced anticancer activity in hepatocellular carcinoma (HCC) cells. First, using proliferative assay including MTT assay and real-time cell electronic sensing technique, we founded that A. auricula has an antiproliferative effect on various cancer cell lines. Among five cancer cell lines, we focused on huh-7 cell line, HCC cell line, due to that A. auricula has most dramatic antiproliferative effects on huh-7 cell line. Following experiments, we founded that its antiproliferative effects was related with apoptosis-inducing activities. For more investigation, a two-dimensional electrophoresis based-proteomic analysis (2DE-GE) was employed for identification of possible target-related proteins of A. auricula-induced apoptosis. Among seven identified proteins, we focused on peroxiredoxin1 (PRDX1), which has been known as an anti-oxidative enzyme. We confirmed downregulation of expression of PRDX1 following A. auricula treatment in mRNA and protein level. In order to obtain a more validation of the correlation of A. auricula-induced cell death and anti-oxidative enzyme. We investigated the level of anti-oxidative enzymes, total glutathione (GSSG/GSH), and superoxide dismutase (SOD) levels in treated cells and PRDX1 gene-silenced cells. GSH and SOD levels were decreased in the treated cells and PRDX1 gene-silenced cells. Our findings suggest that A. auricula is a potent inducer of apoptosis in HCC cells via PRDX1-inhibition pathways. PRACTICAL APPLICATIONS: Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related mortality. The principal treatment is surgical resection or liver transplantation. However, in most patients with HCC the diagnosis is often late, thereby excluding the patients from definitive surgical resection. Chemotherapy and radiotherapy are generally ineffective. Newer treatments are needed with several being in development. In this research article, we provide regulation mechanism of PRDX1 in HCC. PRDX1 has a proliferative effect and play a role in cancer development or progression. Overexpression of PRDX1 in cancer cells implies the role of PRDX1 in the cancer therapy. PRDX1 is currently being investigated as a new target for gene therapy in cancer. A. auricula is an apoptotic inducer of HCC cells through PRDX1 pathway. Regulation of PRDX1 in HCC may contribute to cancer treatment. Therefore, the potentials of targeting apoptosis would be a viable therapeutic strategy to improve the outcome of HCC patients.

Discovery of 1,4-pentadien-3-one derivatives containing quinoxaline scaffolds as potential apoptosis inducers.

Aim: To synthesize novel antiproliferative agents. Results &methodology: A variety of 1,4-pentadien-3-one derivatives bearing quinoxaline scaffolds was designed and synthesized and their antiproliferative activities were evaluated. Notably, compounds N3 and N4 exhibited markedly greater antiproliferative activities against SMMC-7721 cells in vitro compared with the well-known antitumor drug gemcitabine. The mechanistic investigation showed that compounds N3 and N4 induced SMMC-7721 cell apoptosis by regulating the expression levels of apoptosis-related proteins. In addition, the molecular docking model further revealed that compound N3 could be a potential peroxisome proliferator-activated receptor inhibitor. Conclusion: These compounds might serve as bioactive fragments and lead compounds for developing more potent apoptosis inducers.

Discovery of new benzhydrol biscarbonate esters as potent and selective apoptosis inducers of human melanomas bearing the activated ERK pathway: SAR studies on an ERK MAPK signaling modulator, ACA-28

The recent discovery that an ERK signaling modulator [ACA-28 (2a)] preferentially kills human melanoma cell lines by inducing ERK-dependent apoptosis has generated significant interest in the field of anti-cancer therapy. In the first SAR study on 2a, here, we successfully developed candidates (2b, 2c) both of which induce more potent and selective apoptosis towards ERK-active melanoma cells than 2a, thus revealing the structural basis for inducing the ERK-dependent apoptosis and proposing the therapeutic prospect of these candidates against ERK-dependent cancers represented by melanoma.