Apoptogenic Effects Research Articles

A new 1,2,3-triazole-indirubin hybrid suppresses tumor growth and pulmonary metastasis by mitigating the HGF/c-MET axis in hepatocellular carcinoma

IntroductionHepatocellular carcinoma (HCC) is a fatal cancer that is often diagnosed at the advanced stages which limits the available therapeutic options. The interaction of HGF with c-MET (a receptor tyrosine kinase) results in the activation of c-MET which subsequently triggers the PI3K/Akt/mTOR axis. Overexpression of c-MET in HCC tissues has been demonstrated to contribute to tumor progression and metastasis. ObjectivesWe aimed to synthesize triazole-indirubin conjugates, examine their growth suppressor efficacy in cell-based assays, and investigate the antitumor as well as antimetastatic activity of lead cytotoxic agent in the orthotopic mice model. MethodsA series of triazole-indirubin hybrids were synthesized and cytotoxicity, apoptogenic, and antimigratory effect of the lead compound (CRI9) was evaluated using MTT assay, cell cycle analysis, annexin-V/PI assay, TUNEL assay, and wound healing assay. The effect of CRI9 on the operation of the HGF/c-MET/PI3K/Akt/mTOR axis was examined using western blotting and transfection experiments. Acute toxicity, antitumor, and antimetastatic activity of CRI9 were examined in NCr nude mice. The expression of c-MET/PI3K/Akt/mTOR, CD31, and Ki-67 was examined using immunohistochemistry and western blotting. ResultsAmong the new compounds, CRI9 consistently displayed potent cytotoxicity against HGF-induced HCC cells. CRI9 induced apoptosis as evidenced by increased sub G1 cells, annexin-V+/PI+ cells, TUNEL+ cells, and cleavage of procaspase-3 and PARP. CRI9 inhibited HGF-induced phosphorylation of c-METY1234/1235 and subsequently suppressed the PI3K/Akt/mTOR axis. Also, depletion of c-MET or inhibition of c-MET by CRI9 resulted in suppression of the PI3K/Akt/mTOR axis. CRI9 showed no toxic effects in NCr nude mice and displayed a potent antitumor and antimetastatic effect in the orthotopic HCC mice model. CRI9 also reduced the levels of phospho-c-MET, CD31, and Ki-67 and suppressed the activation of the PI3K/Akt/mTOR axis in tumor tissues. ConclusionCRI9 has been identified as a new inhibitor of the c-MET/PI3K/Akt/mTOR axis in HCC preclinical models.

Cinnamaldehyde potentiates cytotoxic and apoptogenic effects of doxorubicin in prostate cancer cell line.

Nowadays, herbal medicine has been utilized to treat various diseases such as cancer, which showed successful therapeutic efficacy in previous studies. This study for the first time evaluated the cytotoxic potential of cinnamaldehyde (CIN) alone and in combination with doxorubicin (DOX), a well-known potent anti-tumor agent, on the proliferation of prostatic cancer cell line (PC3). The cytotoxicity and apoptotic activities of CIN and DOX, either separately or together, were determined on PC3 cells by the MTT test and Annexin V/PI assay, respectively. To further investigate which apoptotic pathway participated in cell death a collection of prominent markers of apoptosis induction including caspase-3/7 activations, mitochondrial membrane potential (MMP), and phosphatidyl serine translocation were detected. The different concentrations of CIN and DOX significantly inhibited the proliferation of PC3 cells in a concentration-dependent way within a 24-h treatment. In addition, the induction of apoptosis by CIN was accompanied by an increase in the activation of caspase-3/7 in PC3 cells with IC50 concentrations of 12.5 and 10 μg/mL for CIN and DOX, respectively. Moreover, the morphological observations obtained from flow cytometry MMP and caspase-3/7 activity assays, altogether, revealed the potential effect of CIN on apoptosis induced in PC3 cells by DOX. Taken together, the current study concluded that the combination of CIN and DOX could lead to the production of a potential therapeutic agent for prostate cancer. However, further in vivo and clinical studies are still needed to validate this combination in prostate cancer therapy.

Stylosin Exerts Cytotoxic and Apoptogenic Effects in Human Malignant Glioblastoma Line and Hepatocellular Carcinoma Cells: The Role of Reactive Oxygen Species

Stylosin Exerts Cytotoxic and Apoptogenic Effects in Human Malignant Glioblastoma Line and Hepatocellular Carcinoma Cells: The Role of Reactive Oxygen Species

Review Focusing on Molecular Mechanisms of Induction of Apoptosis in Different Cancers by a Flavone: Luteolin

Luteolin (3’,4’,5,7-tetrahydroxyflavone), an emerging anticancer flavone, has been reported to have other activities like antioxidant, antidiabetic, antimicrobial, anti-inflammatory, neuroprotective, cardioprotective and anti-allergic activities. In recent times, more attention has been paid to search the mechanism of apoptogenic effects of luteolin. Reports about the anticancer mechanism of luteolin published in Google Scholar, Scopus, Web of Science and PubMed are summarized in this study. Luteolin has shown potential against different cancers like breast, gastric, lung, hepatocellular, prostate etc. Mechanism of apoptosis in breast cancer cells is reported as inhibition of epidermal growth factor receptor-mediated pathway in MDA-MB 231 ER negative breast cancer cells by luteolin. Induction of apoptosis was linked with the inactivation of the critical kinases ERK and AKT in addition to P70S6K and mTOR in prostate cancer cells. Our literature survey shows that luteolin is a lead compound for anticancer chemotherapy. Further molecular mechanisms of induction of apoptosis should be explored in respect to luteolin in different cancer treatments.

Tumor integrin targeted theranostic iron oxide nanoparticles for delivery of caffeic acid phenethyl ester: preparation, characterization, and anti-myeloma activities.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells preferentially in the bone marrow. Currently, emerging chemotherapy drugs with improved biosafety profiles, such as immunomodulatory agents and protease inhibitors, have been used in clinics to treat MM in both initial therapy or maintenance therapy post autologous hematopoietic stem cell transplantation (ASCT). We previously discovered that caffeic acid phenethyl ester (CAPE), a water-insoluble natural compound, inhibited the growth of MM cells by inducing oxidative stress. As part of our continuous effort to pursue a less toxic yet more effective therapeutic approach for MM, the objective of this study is to investigate the potential of CAPE for in vivo applications by using magnetic resonance imaging (MRI)-capable superparamagnetic iron oxide nanoparticles (IONP) as carriers. Cyclo (Arg-Gly-Asp-D-Phe-Cys) (RGD) is conjugated to IONP (RGD-IONP/CAPE) to target the overexpressed αvβ3 integrin on MMcells for receptor-mediated internalization and intracellular delivery of CAPE. A stable loading of CAPE on IONP can be achieved with a loading efficiency of 48.7% ± 3.3% (wt%). The drug-release studies indicate RGD-IONP/CAPE is stable at physiological (pH 7.4) and basic pH (pH 9.5) and subject to release of CAPE at acidic pH (pH 5.5) mimicking the tumor and lysosomal condition. RGD-IONP/CAPE causes cytotoxicity specific to human MM RPMI8226, U266, and NCI-H929 cells, but not to normal peripheral blood mononuclear cells (PBMCs), with IC50s of 7.97 ± 1.39, 16.75 ± 1.62, and 24.38 ± 1.71μM after 72-h treatment, respectively. Apoptosis assays indicate RGD-IONP/CAPE induces apoptosis of RPMI8226 cells through a caspase-9 mediated intrinsic pathway, the same as applying CAPE alone. The apoptogenic effect of RGD-IONP/CAPE was also confirmed on the RPMI8226 cells co-cultured with human bone marrow stromal cells HS-5 in a Transwell model to mimic the MM microenvironment in the bone marrow. In conclusion, we demonstrate that water-insoluble CAPE can be loaded to RGD-IONP to greatly improve the biocompatibility and significantly inhibit the growth of MMcells in vitro through the induction of apoptosis. This study paves the way for investigating the MRI-trackable delivery of CAPE for MM treatment in animal models in the future.

Combretastatin A-4 suppresses the invasive and metastatic behavior of glioma cells and induces apoptosis in them: in-vitro study.

The most common primary brainmalignancy, glioblastoma multiforme, is tremendously resistant to conventionaltreatmentsdue to its potency for metastasis to surrounding brain tissue. Temozolomide is a chemotherapeutic agent that currently is administrated during the treatment procedure. Studies have attempted to investigate new agents with higher effectiveness and fewer side effects. Combretastatin A-4 (CA-4), a natural compound derived from Combretum caffrum, has been recently considered for its potent antitumor activities in a wide variety of preclinical solid tumor models. Our findings have shown that CA-4 exerts potent anti-proliferative and apoptotic effects on glioma cells, and ROS generation may be involved in these cellular events. CA-4 has imposed G2 arrest in U-87 cells. We also observed that CA-4 significantly reduced the migration and invasion capability of U-87 cells. Furthermore, the gene expression and enzyme activity of MMP-2 and MMP-9 were significantly inhibited in the presence of CA-4. We also observed a considerable decrease in PI3K and Akt protein expression following treatment with CA-4. In conclusion, our findings showed significant apoptogenic and anti-metastatic effects of CA-4 on glioma cells and also suggested that the PI3K/Akt/MMP-2/-9 and also ROS pathway might play roles in these cellular events.

Development, Characterization and Pharmacological Evaluation of Cannabidiol-Loaded Long Circulating Niosomes.

Cannabidiol (CBD) is a promising drug candidate with pleiotropic pharmacological activity, whose low aqueous solubility and unfavorable pharmacokinetics have presented obstacles to its full clinical implementation. The rational design of nanocarriers, including niosomes for CBD encapsulation, can provide a plausible approach to overcoming these limitations. The present study is focused on exploring the feasibility of copolymer-modified niosomes as platforms for systemic delivery of CBD. To confer steric stabilization, the niosomal membranes were grafted with newly synthesized amphiphilic linear or star-shaped 3- and 4-arm star-shaped copolymers based on polyglycidol (PG) and poly(ε-caprolactone) (PCL) blocks. The niosomes were prepared by film hydration method and were characterized by DLS, cryo-TEM, encapsulation efficacy, and in vitro release. Free and formulated cannabidiol were further investigated for cytotoxicity and pro-apoptotic and anti-inflammatory activities in vitro in three human tumor cell lines. The optimal formulation, based on Tween 60:Span60:Chol (3.5:3.5:3 molar ration) modified with 2.5 mol% star-shaped 3-arm copolymer, is characterized by a size of 235 nm, high encapsulation of CBD (94%), and controlled release properties. Niosomal cannabidiol retained the antineoplastic activity of the free agent, but noteworthy superior apoptogenic and inflammatory biomarker-modulating effects were established at equieffective exposure vs. the free drug. Specific alterations in key signaling molecules, implicated in programmed cell death, cancer cell biology, and inflammation, were recorded with the niosomal formulations.

Cytotoxic Activity of Phytoconstituents Isolated from Monotheca buxifolia against Hepatocellular Carcinoma Cell Line HepG2: In Vitro and Molecular Docking Studies.

Natural products and conventional chemotherapeutic drugs are believed to enhance anticancer treatment efficacy while lowering toxicity. The current study investigates the cytotoxic and apoptogenic effects of Monotheca buxifolia bioactive compounds on HepG2 cell lines. MTT assay was used to assess the effect on the viability of HepG2 cells. Morphological changes were investigated. Annexin-V-FITC/PI was used to demonstrate apoptotic activity. A molecular dynamics simulation study was carried out to investigate the compound binding pattern in the active site of the PPRAδ protein. MTT and annexin V-FITC/PI assays revealed that the isolated compounds lauric acid, oleanolic acid, and bis(2-ethylhexyl) phthalate inhibited the growth of hepatocellular cancer cells. The IC50 value for lauric acid was 56.46 ± 1.20 μg/mL, 31.94 ± 1.03 μg/mL for oleanolic acid, and 83.80 ± 2.18 μg/mL for bis(2-ethylhexyl) phthalate. Apoptosis was observed in 29.5, 52.1 and 22.4% of HepG2 cells treated with lauric acid, oleanolic acid, and bis(2-ethylhexyl) phthalate, respectively, after 24 h of treatment. Morphological assays and Hoechst staining microscopy revealed that the treatment caused morphological changes in the cell membrane and nuclear condensation. The high fluctuation indicates that various interactions were highly potent and widely adopted, and vice versa. Oleanolic acid displayed high residue fluctuation, remaining stable in the active site of the PPRAδ protein and involved in various interactions while remaining locally fluctuating in the binding sites of the other two compounds. These findings concluded that lauric acid, oleanolic acid, and bis(2-ethylhexyl) phthalate have a significant apoptogenic effect against HepG2 cells in inducing apoptosis. Our findings suggest that these bioactive compounds could be used as adjuvant therapies.

Cell cycle arrest and apoptotic studies of Terminalia chebula against MCF-7 breast cancer cell line: an in vitro and in silico approach.

Breast cancer is a leading cause of mortality in women, and alternative therapies with fewer side effects are actively being explored. Breast cancer is a significant global health concern, and conventional treatments like radiotherapy and chemotherapy often have side effects. Medicinal plant extracts offer a promising avenue for the development of effective and safe anticancer therapies. Terminalia chebula, a plant known for its medicinal properties, was selected for investigation in this study. We aimed to assess the antiproliferative effects of TCF extract on breast cancer cells and explore the potential role of saccharopine, a phytochemical found in TCF, as an anticancer agent. MCF7 breast cancer cell lines were exposed to TCF extract, and cell viability and apoptosis assays were performed to evaluate the antiproliferative and apoptogenic effects. Molecular docking studies were conducted to assess the binding affinity of saccharopine with EGFRs. Molecular dynamics simulations and binding energy calculations were employed to analyze the stability of the EGFR-saccharopine complex. The TCF extract exhibited significant antiproliferative effects on MCF7 breast cancer cells and induced apoptosis in a dose-dependent manner. Molecular docking analysis revealed that saccharopine demonstrated a higher binding affinity with EGFR compared to the reference compound (17b-estradiol). The subsequent MDS simulations indicated stable binding patterns and conformation of the EGFR-saccharopine complex, suggesting a potential role in inhibiting EGFR-mediated signaling pathways. The investigation of Terminalia chebula fruit extract and its phytochemical saccharopine has revealed promising antiproliferative effects and a strong binding affinity with EGFR. These findings provide a foundation for future research aimed at isolating saccharopine and conducting in vivo studies to evaluate its potential as a targeted therapy for breast cancer. The development of novel anticancer agents from plant sources holds great promise in advancing the field of oncology and improving treatment outcomes for breast cancer patients.

Assessment of the In Vitro and In Vivo Antitumor Activity of Hemocyanins from Helix aspersa, Helix lucorum, and Rapana venosa in a Graffi Myeloid Tumor Model.

Hemocyanins are oxygen-transporting glycoproteins in the hemolymph of some invertebrate species that attracted scientific interest as potential anticancer agents. The present study aims to assess the in vitro and in vivo anticancer activity of hemocyanins isolated from Helix aspersa, Helix lucorum, and Rapana venosa in the Graffi myeloid tumor model. The in vitro antitumor activity of the hemocyanins was determined by a MTT test and cytomorphological analysis by fluorescent and transmission electron microscopy. The in vivo effects of the hemocyanins were examined in hamsters transplanted with Graffi tumor. The serum antibody titers against the tested hemocyanins and tumor antigen were determined by ELISA. Histopathological assessment of the morphological features related to antitumor effect, immune system response, and toxicity in some internal organs was performed. The results of in vitro studies indicated that the tested hemocyanins induced significant antiproliferative and apoptogenic effects. The in vivo investigations demonstrated a protective antitumor effect, expressed in reduced transplantability, suppression of tumor growth and metastasis, reduced mortality, prolonged survival time, and absence of toxic side effects. The present study indicated that the antitumor activity of the studied hemocyanins was due to both immune stimulation and direct effects on the tumor cells, and they displayed their potential as therapeutic agents against hematological malignances.

Abstract 6306: Pharmacological deuteration of SCID mice using the water-isotopologue deuterium oxide (D2O) inhibits tumor growth in bioluminescent models of human malignant melanoma and pancreatic ductal adenocarcinoma

Abstract Since its initial discovery as a natural isotopologue of dihydrogen oxide (1H2O), extensive research has focused on the biophysical, biochemical, and pharmacological effects of deuterated water [2H2O (D2O, also referred to as ‘heavy water’)]. Here, using diverse panels of cultured human malignant melanoma and pancreatic ductal adenocarcinoma (PDAC) cells we have profiled (i) D2O-induced phenotypic anti-proliferative and apoptogenic effects, (ii) redox- and proteotoxicity-directed stress response gene expression, and (iii) phosphoprotein-signaling related to endoplasmic reticulum (ER) and MAP-kinase stress response pathways. Differential RT-qPCR array analysis revealed early modulation of stress response gene expression elicited by D2O (90%; ≤6 h) confirmed by independent RT-qPCR analysis. Immunoblot-analysis revealed rapid (< 6 h) onset of D2O-induced MAP-kinase signaling (p-JNK) together with ER stress response upregulation (p-eIF2α, ATF4, XBP1s, DDIT3/CHOP) attributable to deuteration-induced alteration of hydrogen bonding triggering proteotoxic stress. Next, we tested the chemotherapeutic efficacy of D2O-based drinking water supplementation in bioluminescent murine models of malignancy (A375-luc melanoma and BxPC-3-luc orthotopic PDAC xenografts in SCID mice). Time course of systemic deuteration (30% D2O in drinking water fed continuously for up to 21 days) was established using time-resolved whole-body proton magnetic resonance imaging (MRI) and isotope-ratio mass spectrometry (IR-MS)-based plasma (D/H)-analysis. In both models, D2O supplementation significantly suppressed tumor growth and metastasis with downregulated expression of PCNA/Ki67 while increasing tumor levels of DDIT3/CHOP, HO-1, and p-eIF2α. Taken together, these data demonstrate for the first time that pharmacological induction of systemic deuteration by oral administration of D2O, associated with induction of cellular ER stress, reduces tumor burden and metastasis in murine models of human malignant melanoma and PDAC. Citation Format: Jana Jandova, Jean-Philippe Galons, David L. Dettman, Georg T. Wondrak. Pharmacological deuteration of SCID mice using the water-isotopologue deuterium oxide (D2O) inhibits tumor growth in bioluminescent models of human malignant melanoma and pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6306.

A Study of Ungernia trisphaera Bunge and Thymus transcaspicus Cytotoxicity in Cancer Cell Lines

Objective: Thymus transcaspicus (Lamiaceae) is an Iranian species of Thymus, and Ungernia trisphaera Bunge belongs to the family Amaryllidaceae. Cytotoxic properties of total ethanolic extract of T. transcaspicus and U. trisphaera were investigated against different cell lines including B16F10, PC3, U87, and MCF-7. Methods: The proliferation, cell cycle, and migration of the cells were determined by thiazolyl blue tetrazolium bromide (MTT) test, propidium iodide (PI) staining, and scratch assay respectively. The effects of U. trisphaera extract (UTE) on the activity of matrix metalloproteinases (MMPs) and angiogenesis were evaluated by the gelatin zymography method and chicken chorioallantoic membrane model, respectively. The GSH, SOD, and MDA were evaluated by colorimetric method. Results: The results showed that UTE could inhibit the development of malignant cells in a concentration- dependent manner, while the inhibitory effect of T. transcaspicus extract (TTE) was not significant compared to the control group. The UTE-induced sub-G1 peak apoptosis compared to the control group indicated that apoptotic cell death is involved in UTE-induced cytotoxicity. MMPs activity was significantly decreased 48 hours after treatment. Moreover, GSH level and SOD activity were significantly decreased while MDA and ROS levels were significantly increased after 24 hours treatment. In addition, combination of UTE (1.5-25 μg/mL) with doxorubicin (6 μg/mL) showed an additive cell growth inhibitory effect. Conclusion: UTE demonstrated cytotoxic and apoptogenic effects in different cancer cell lines, and it was found that apoptosis plays a crucial role in the cytotoxicity of UTE. Thus, U. trisphaera can be considered a potential medicinal herb in cancer treatment after comprehensive pharmacological and toxicological studies.

Systemic deuteration of SCID mice using the water-isotopologue deuterium oxide (D2 O) inhibits tumor growth in an orthotopic bioluminescent model of human pancreatic ductal adenocarcinoma.

Since its initial discovery as a natural isotopologue of dihydrogen oxide (1 H2 O), extensive research has focused on the biophysical, biochemical, and pharmacological effects of deuterated water (2 H2 O [D2 O, also referred to as "heavy water"]). Using a panel of cultured human pancreatic ductal adenocarcinoma (PDAC) cells we have profiled (i) D2 O-induced phenotypic antiproliferative and apoptogenic effects, (ii) redox- and proteotoxicity-directed stress response gene expression, and (iii) phosphoprotein-signaling related to endoplasmic reticulum (ER) and MAP-kinase stress response pathways. Differential array analysis revealed early modulation of stress response gene expression in both BxPC-3 and PANC-1 PDAC cells elicited by D2 O (90%; ≤6 h; upregulated: HMOX1, NOS2, CYP2E1, CRYAB, DDIT3, NFKBIA, PTGS1, SOD2, PTGS2; downregulated: RUNX1, MYC, HSPA8, HSPA1A) confirmed by independent RT-qPCR analysis. Immunoblot-analysis revealed rapid (≤6 h) onset of D2 O-induced MAP-kinase signaling (p-JNK, p-p38) together with ER stress response upregulation (p-eIF2α, ATF4, XBP1s, DDIT3/CHOP). Next, we tested the chemotherapeutic efficacy of D2 O-based drinking water supplementation in an orthotopic PDAC model employing firefly luciferase-expressing BxPC-3-FLuc cells in SCID mice. First, feasibility and time course of systemic deuteration (30% D2 O in drinking water; 21 days) were established using time-resolved whole-body proton magnetic resonance imaging and isotope-ratio mass spectrometry-based plasma (D/H)-analysis. D2 O-supplementation suppressed tumor growth by almost 80% with downregulated expression of PCNA, MYC, RUNX1, and HSP70 while increasing tumor levels of DDIT3/CHOP, HO-1, and p-eIF2α. Taken together, these data demonstrate for the first time that pharmacological induction of systemic deuteration significantly reduces orthotopic tumor burden in a murine PDAC xenograft model.

Combination of RNase Binase and AKT1/2 Kinase Inhibitor Blocks Two Alternative Survival Pathways in Kasumi-1 Cells

Treatment of malignant neoplasms often requires the use of combinations of chemotherapeutic agents. However, in order to select combinations that are effective against specific tumor cells, it is necessary to understand the mechanisms of action of the drugs that make up the combination. Bacillus pumilus ribonuclease (binase) is considered as an adjuvant antitumor agent, and the sensitivity of malignant cells to the apoptogenic effect of binase depends on the presence of certain oncogenes. In the acute myelogenous leukemia cell line Kasumi-1, binase blocks the proliferation pathway mediated by the mutant tyrosine kinase KIT, which, as shown in our work, activates an alternative proliferation pathway through AKT kinase. In Kasumi-1 cells, binase in combination with an Akt1/2 inhibitor induces apoptosis, and their toxic effects add up: the Akt1/2 inhibitor blocks the binase-induced pathway after suppression of the KIT-dependent pathway. Thus, a combination of binase and AKT kinase inhibitors can effectively block various pathways of tumor cell proliferation and be used for their elimination.

Pyrimidine-2,4-dione targets STAT3 signaling pathway to induce cytotoxicity in hepatocellular carcinoma cells

Signal transducer and activator of transcription 3 (STAT3) is a tumorigenic transcription factor that is persistently activated in various human cancers including hepatocellular carcinoma (HCC). Therefore, STAT3 is considered as a prominent target to counteract the uncontrolled proliferation of cancer cells. In the present report, pyrimidine-2,4-diones (N-methyluracil derivatives) (MNK1-MNK14) were synthesized in an ionic liquid (BMIm PF6) medium employing a ligand-free Suzuki-Miyaura cross-coupling process. Among the 14 derivatives, compound MNK8 showed good cytotoxicity towards both the tested cell lines and did not display a toxic effect against normal hepatocytes (LO2). MNK8 significantly increased the Sub-G1 cell count in both cell lines and the cytotoxic effect of MNK8 was found to be mediated through the suppression of constitutive phosphorylation of STAT3Y705. It also decreased the DNA interaction ability of nuclear STAT3 in HCC cells. MNK8 downregulated the levels of apoptosis-related proteins (such as Bcl-2, cyclin D1, survivin) and increased cleaved caspase-3 inferring the apoptogenic effect of MNK8. It also reduced the CXCL12-triggered cell migration and invasion in in vitro assay systems. Overall, MNK8 has been demonstrated as a new inhibitor of STAT3 signaling cascade in HCC cells.

Synthesis, molecular structure, DFT studies, in silico docking and molecular dynamics simulations of 2,6 dimethoxychalcone derivatives as BRD4 inhibitors

Methoxyphenyl chalcone derivatives (MAHB1 and MAHB3) have been synthesized and characterised by NMR (1H&13C) spectra and single crystal X-ray diffraction techniques. The crystal structures revealed the crystallization of the both compounds in the monoclinic systems and P2(1)/c space groups. Supramolecular architecture unveiled the stability of compounds and build of crystal packings upon short contacts and weak interactions. Density functional theory (DFT) with B3LYP functional under 6–311++G(d,p) basis set have been adopted to calculate the geometry and investigate intramolecular charge transfer (ICT) using natural bond orbital analysis (NBO) as well as the frontier molecular orbitals (FMOs), hardness, softness, energy gap and other chemical reactivity parameters. Hirshfeld surface and energy frameworks were adopted to evaluate the different intermolecular interaction and energies based on the anisotropy of the topology. The inhibitory activity of the compounds against the first bromodomain of human BRD4 protein have been assessed by using in silico molecular docking and molecular dynamics simulations against 5H21 protein (homo sapiens), which revealed prominent interactions of both the ligands at the active site of BRD4 protein showing with a reasonable conformity as similar with the co-crystal ligand interactions. Furthermore, apo and holo states in the molecular dynamic simulations confirmed the stability and structural constancy of the protein-ligand complexes with minimal deviation based on RMSD and RMSF plots. Both the simulation results confirmed the hydrogen bonds of the compounds with the vital amino acid ASN140 which is necessary for the inhibition of BRD4 as well as exhibiting similar hydrophobic residues interactions. Furthermore, the newly synthesized compounds were screened against human leukemia MV4–11 cells based on MTT and apoptosis assays system. These compounds exhibited anti-proliferation activity and induced the apoptotic effect. This study indicated that the MAHB1 is a promising compound against leukaemia with IC50 value of 79.10 μM and exhibited better apoptogenic effect. Furthermore, the expression of BRD4 in MV4–11 cell line was measured by western blotting, while the protein thermal shift assay was conducted to determine the impact of MAHB1 compound against the thermal stability of BRD4.

The Novel Imipridone ONC212 Induces Pronounced Anti-Leukemia Effects in Vitro and In Vivo and Is Highly Synergistic with the BCL-2 Inhibitor ABT-199

Imipridones constitute a novel class of molecules that includes the first-in-class anti-cancer compound ONC201. ONC201 has shown promising clinical activity against hematological and solid malignancies (Allen et al., Oncotarget, 2016). ONC212 is a second-generation imipridone with higher potency than ONC201. We first confirmed the overall in vitro anti-tumor effects of ONC212 using the collection of 1,088 human cancer cell lines available from the Genomic of Drug Sensitivity in Cancer Project. Ranking the sensitivity of these cancer cell lines to ONC212 by tumor type revealed that leukemias and lymphomas were especially sensitive. In fact, ONC212 exerted prominent apoptogenic effects in AML cell lines with > 20-fold potency compared to ONC201 (ED50s at 72 hr; 6.4 μM vs 258.7 nM, respectively for OCI-AML3 cells). Similar results were observed in primary AML cells, but there was no activity against normal bone marrow (BM) cells. We previously demonstrated that ONC201 induces apoptosis via an atypical integrated stress response (ISR, Ishizawa et al., Sci Signal, 2016). Similarly, ONC212 showed ISR activation accompanying its apoptogenic effects in AML cells which was agnostic to the mutation status of p53. However, because of the higher sensitivity of wild-type p53 cells to ONC212, we speculated that this effect could also reflect a p53-dependent apoptosis mechanism. Indeed, p53 knockdown reduced the ONC212-induced apoptosis in MV4;11 and MOLM13 cells, suggesting both, p53-dependent and -independent, mechanisms.We next investigated the effects of ONC212 in vivo in a systemic AML xenograft model using OCI-AML3 cells expressing luciferase. Biweekly oral administration of 50 mg/kg ONC212 markedly inhibited AML expansion and prolonged overall survival (p = 0.0003). Median survival increased from 43 d in controls to 49 d in the ONC212-treated group (+14%). For in vivo functional assessment of ONC212's anti-tumor effects against leukemia stem and progenitor cells, we utilized our patient-derived xenograft (PDX) mouse model. We treated primary PDX cells with ONC212 (250 nM, 36 hr) ex vivo, and then injected identical numbers of viable cells (treated or untreated) into recipient NSG mice. After one month, the percentages of human CD45+ cells in the peripheral blood, spleen, and BM were significantly decreased in the ONC212-treated group. The median survival in this group was remarkably different: 82 d compared to 36 d for controls (+128%, p<0.0001). These results indicated that ONC212 targets AML stem cells with potential to eradicate the disease.Because BCL-2 is generally considered protective against ISR-mediated apoptosis and ONC212 induced p53-mediated apoptosis as demonstrated above, we hypothesized that the BCL-2 inhibitor ABT-199 or the p53 activator nutlin-3a could further sensitize AML cells to ONC212. Indeed, the in vitro combination of ONC212 plus ABT-199 or nutlin-3a synergistically induced apoptosis in AML cells. Furthermore, the combination of ONC212 and ABT-199 showed highly significant synergistic anti-leukemia effects in vivo in a MOLM-13 xenograft mouse model. The combinatorial treatment prolonged overall median survival from 20 d for untreated controls to 21 d for each agent as monotherapy to 30 d for the combination.Since the G-protein-coupled receptor (GPCR) dopamine receptor D2 is the putative target of first-generation imipridone ONC201, we performed a PathHunter β-arrestin screening to determine if ONC212 also bound GPCRs. ONC212 did not engage DRD2 but instead specifically engaged the orphan GPCR GPR132, which is a putative tumor suppressor when activated. Interestingly, expression of GPR132 mRNA was highly correlated with sensitivity to ONC212. Furthermore, GPR132 overexpression induced cell death and ONC212 treatment induced GPR132 mRNA expression in AML cell lines, suggesting that GPR132 could be a potential therapeutic target in hematological malignancies.Taken together, ONC212, and its putative target GPR132, represent a promising novel anti-cancer strategy for AML therapy that warrants further development. DisclosuresPrabhu:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. Oster:Oncoceutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Stogniew:Oncoceutics: Employment. Andreeff:Daiichi Sankyo: Consultancy.

Inhibition of Translation Initiation and Heat Shock Factor 1 (HSF1): A Novel Strategy Targeting FLT3 Double Mutant and Non-FLT3 Mutant AML Progenitor/Stem Cells

The transcription factor Heat shock factor 1 (HSF1) is a key sensor of proteotoxic stress and plays a major role in cancer biology. In various tumors, downregulation of HSF1 inhibits growth and induces cell death, and its activation was reported to be an adverse prognostic factor in breast and lung cancers. We have reported that inhibition of translation represses DNA-binding of HSF1 in cancers; large scale drug screening identified the eIF4a antagonist rohinitib (RHT) as a potent inhibitor of the HSF1 activation state (Santagata et al, Science 2013). Review of public databases provided us with additional rationale for targeting HSF1 and eIF4a in acute myeloid leukemia (AML): 1) mRNA levels of HSPA8, a primary HSF1 target, are correlated with poor prognosis in AML; 2) eIF4a mRNA levels are highest in AML among 12 cancer types; and 3) Gene set enrichment analysis using microarray dataset of functionally-defined leukemia stem cells (LSCs) and non-LSCs (Nature, Ng et al), based on intra-patient comparisons, reveals that a gene set for translation initiation is highly enriched in LSCs (NES 2.73, FDR = 0.000, p= 0.000).RHT induced marked apoptogenic effects in seven human AML cell lines in culture at low nanomolar concentrations (LD50s; 9.5 to 99.5 nM, IC50s; 4.7 to 8.8 nM, based on AnnexinV/PI-positivity at 72hr). RHT inhibited HSF1 transcriptional activity, as shown by 70% reduction in HSPA8 mRNA levels, and 50% reduction in a luciferase reporter driven by the HSPA1A or HSPA6 promoters. We then tested primary samples from 17 AML patients and bone marrow (BM) samples from eight healthy donors. RHT potently induced apoptosis in AML cells, while relatively sparing normal BM cells (%apoptosis: 65.4% vs 6.67%, P < 0.0001). Importantly, a similarly significant difference in sensitivity was also observed between AML and normal stem/progenitor cell populations (CD45+CD34+CD38-). We further found that the activity of RHT was significantly higher in FLT3-ITD than in FLT3-wt AMLs. Comparing isogenic versions of the cell line Ba/F3, RHT more potently killed FLT3-ITD cells than wild-type (wt) cells (LD50s; 65.3 vs 20.1 nM). Immunoblot analysis showed higher activation-associated phospho-HSF1 (Serine 326) in FLT3-ITD Ba/F3 than in FLT3-wt cells, and growth inhibition by HSF1 knockdown was FLT3-ITD-dependent in those lines, indicating greater dependence of FLT3-ITD cells on HSF1. In an AML xenograft model with GFP-luciferase labeled MOLM-13 cells, RHT significantly reduced tumor burden as measured by bioluminescence imaging and reduction in GFP+ leukemic cells peripheral blood (PB) and BM by day 16. Median survival of the treatment group was significantly longer (24 vs 18 days, p < 0.0001).We also found that RHT was synergistic when combined with FLT3 inhibitors of Type I (sorafenib, quizartinib, midostaurin) or Type II (gilteritinib, and E-6201). Immunoblot analysis showed that oncogenic FLT3 activation is associated with increased phosphorylation of HSF1 (S326), suggesting that decreased phosphorylation of the S326 site associated with FLT3 inhibition and diminished DNA-binding by HSF1 driven by eIF4a inhibition combine to promote synergistic lethality. In support of this, HSF1 knockdown sensitized MOLM-13 and FLT3-ITD+ Ba/F3 cells to FLT3 inhibitors. Importantly, similar combinatorial synergistic effects with type II FLT3 inhibitors were observed in cells with dual mutations of FLT3-ITD and FLT3-D835Y/H. This observation has significant clinical relevance because the development of secondary point mutations is a major resistance mechanism against FLT3 inhibitors.Finally, to test the hypothesis that eIF4a inhibition impairs LSC functions, we treated primary patient-derived xenograft AML PDX cells with RHT ex vivo, then injected identical numbers of live cells into recipient NSG mice. Subsequent leukemia engraftment was delayed by one week vs. controls, and human CD45+ cells in PB and BM at 4 weeks post transplantation were significantly reduced (n = 9, 3.30% vs 0.26% in PB, P < 0.0001). This reduced engraftment capacity of RHT-treated PDX AML-LSCs indicates a toxic effect of eIF4a inhibition on AML-LSCs.Collectively, these preclinical findings strongly support the further development of eIF4a inhibitors for the treatment of AML, especially for high-risk FLT3-mutated AML, with additional potential as LSC-targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

SARS-Cov-2 spike protein fragment 674–685 protects mitochondria from releasing cytochrome c in response to apoptogenic influence

In spite of numerous studies, many details of SARS-Cov-2 interaction with human cells are still poorly understood. The 674–685 fragment of SARS-Cov-2 spike protein is homologous to the fragment of α-cobratoxin underlying its interaction with α7 nicotinic acetylcholine receptors (nAChRs). The interaction of 674–685 peptide with α7 nAChR has been predicted in silico. In the present paper we confirm this prediction experimentally and investigate the effect of SARS-Cov-2 spike protein peptide on mitochondria, which express α7 nAChRs to regulate apoptosis-related events. We demonstrate that SARS-Cov-2 spike protein peptide 674–685 competes with the antibody against 179–190 fragment of α7 nAChR subunit for the binding to α7-expressing cells and mitochondria and prevents the release of cytochrome c from isolated mitochondria in response to 0.5 mM H2O2 but does not protect intact U373 cells against apoptogenic effect of H2O2. Our data suggest that the α7 nAChR-binding portion of SARS-Cov-2 spike protein prevents mitochondria-driven apoptosis when the virus is uncoated inside the cell and, therefore, supports the infected cell viability before the virus replication cycle is complete.

Preparation of caffeic acid phenethyl ester‐incorporated nanoparticles and their inhibitory effects on multiple myeloma cell growth

Multiple myeloma (MM) is a plasma cell cancer characterized by accumulation of malignant cells, preferentially in the bone marrow, that leads to severe bone disease in humans. This disease resulted in approximately 13,000 deaths last year in the United States. Caffeic acid phenethyl ester (CAPE), a phenolic compound found in bee glue, is reported to have an array of bioactive properties including anticancer effect. We previously reported that CAPE inhibits MM cell growth through induction of apoptosis and oxidative stress. It is established that iron oxide nanoparticles (IONPs) enhance drug delivery to tissues/cells and conjugation of a small peptide RGD with drug loaded IONPs improves cancer cell targeting. The purpose of this study is to improve the biocompatibility of CAPE through encapsulation onto IONPs (IONP/CAPE) and evaluate the cytotoxic and apoptogenic effects of IONP/CAPE with RGD conjugated as a targeting ligand for human MM cells. We hypothesize that RGD-IONP/CAPE may enhance the intracellular delivery of CAPE to MM cells thus demonstrating more potent inhibition on the growth of MM cells than CAPE alone. We prepared RGD-IONP/CAPE and characterized these NPs through determination of the hydrodynamic sizes and zeta potentials. We optimized the stability of these nanomaterial complexes with various stabilizers at different pH and evaluated the inhibitory effect of the most stable formulation on MM cell growth. Our results show that the developed RGD-IONP/CAPE were stable at neutral and basic conditions, and subject to release of CAPE at acidic pH (5.5) in vitro. The RGD-IONP/CAPE caused a decreased MM cell viability in a time (24, 48, 72 hours) and dose (1, 5, 10, 20 and 30 μM) -dependent manner. Interestingly, the growth-inhibitory effects of RGD-IONP/CAPE at 10 μM was equivalent to the parent drug at 30 μM, indicating a 3-fold enhancement in CAPE delivery efficiency under the experimental condition. Apoptogenic effect of RGD-IONP/CAPE was assessed using western blotting. Protein expression of apoptosis-executioner caspase 3 was examined, and the results indicated that caspase 3 was activated. In conclusion, we constructed a novel CAPE-loaded IONPs with RGD conjugation which showed more potent antimyeloma effect than parental CAPE.