TYM-3-98, a novel selective inhibitor of PI3Kδ, demonstrates promising preclinical antitumor activity in B-cell lymphomas.

AIMS: PI3Kδ is expressed predominately in leukocytes and is commonly found to be aberrantly activated in human B-cell lymphomas. Although PI3Kδ has been intensively targeted for discovering anti-lymphoma drugs, the application of currently approved PI3Kδ inhibitors has been limited due to unwanted systemic toxicities, thus warranting the development of novel PI3Kδ inhibitors with new scaffolds. MAIN METHODS: We designed TYM-3-98, an indazole derivative, and evaluated its selectivity for all four PI3K isoforms, as well as its efficacy against various B-cell lymphomas both in vitro and in vivo. KEY FINDINGS: We identified TYM-3-98 as a highly selective PI3Kδ inhibitor over other PI3K isoforms at both molecular and cellular levels. It showed superior antiproliferative activity in several B-lymphoma cell lines compared with the approved first-generation PI3Kδ inhibitor idelalisib. TYM-3-98 demonstrated a concentration-dependent PI3K/AKT/mTOR signaling blockage followed by apoptosis induction. In vivo, TYM-3-98 showed good pharmaceutical properties and remarkably reduced tumor growth in a human lymphoma xenograft model and a mouse lymphoma model. SIGNIFICANCE: Our findings establish TYM-3-98 as a promising PI3Kδ inhibitor for the treatment of B-cell lymphoma.

Discovery of Highly Selective and Orally Bioavailable PI3Kδ Inhibitors with Anti-Inflammatory Activity for Treatment of Acute Lung Injury.

PI3Kδ is a promising target for the treatment of inflammatory disease; however, the application of PI3Kδ inhibitors in acute respiratory inflammatory diseases is rarely investigated. In this study, through scaffold hopping design, we report a new series of 1H-pyrazolo[3,4-d]pyrimidin-4-amine-tethered 3-methyl-1-aryl-1H-indazoles as highly selective and potent PI3Kδ inhibitors with significant anti-inflammatory activities for treatment of acute lung injury (ALI). There were 29 compounds designed, prepared, and subjected to PI3Kδ inhibitory activity evaluation and anti-inflammatory activity evaluation in macrophages. (S)-29 was identified as a candidate with high PI3Kδ inhibitory activity, isoform selectivity, and high oral bioavailability. The in vivo administration of (S)-29 at 10 mg/kg dosage could significantly ameliorate histopathological changes and attenuate lung inflammation in lung tissues of LPS-challenged mice. Molecular docking demonstrated the success of scaffold hopping design. Overall, (S)-29 is a potent PI3Kδ inhibitor which might be a promising candidate for the treatment of ALI.

A novel sesquiterpene lactone fraction from Eupatorium chinense L. suppresses hepatocellular carcinoma growth by triggering ferritinophagy and mitochondrial damage.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive tumor with limited treatment options, and it is the third leading cause of cancer-related deaths. Hence, novel therapeutic strategies are required to treat HCC. Eupatorium chinense L. is a traditional Chinese medicine (TCM) that can effectively neutralize heat and smoothen the flow of "Qi" through the liver. However, the anti-HCC effects of Eupatorium chinense L. remain unknown. PURPOSE: The present study investigated the anti-HCC effects and the underlying mechanisms of the electrophilic sesquiterpenes isolated from E. chinense L. (EChLESs) in the regulation of ferroptosis and apoptosis in HCC cells. STUDY DESIGN/METHODS: Cell viability was assessed by the MTT assay. Cell apoptosis was confirmed by flow cytometry and western blotting assay. Ferroptosis was assessed by flow cytometry, transmission electron microscopy, and western blotting assay. Ferritinophagy was detected by acridine orange staining and western blotting assay. Small interfering RNA of nuclear receptor coactivator 4 (NCOA4) was used to confirm the role of ferritinophagy in the therapeutic effect of EChLESs on HCC cells. A mouse xenograft model was constructed to determine the inhibitory effect of EChLESs on HCC in vivo. RESULTS: EChLESs induced apoptosis by disrupting mitochondrial membrane potential depolarization and mitochondrial reactive oxygen species. EChLESs induced ferroptosis as noted by a significant increase in mitochondrial disruption, lipid peroxidation, and intracellular iron level and decreased glutathione level. The apoptosis inhibitor Z-VAD-FMK and lipid reactive oxygen species scavenger ferrostatin 1 attenuated EChLESs-induced cell death. NCOA4-mediated ferritinophagy through autophagic flux was the crucial pathway for ferroptosis induced by EChLESs. NCOA4 knockdown alleviated EChLESs-induced cell death. EChLESs controlled the expression of NCOA4 at the transcriptional and post-transcriptional levels. In the in vivo experiment, EChLESs suppressed HCC growth in the xenograft tumor mouse model. CONCLUSION: EChLESs enhances cell apoptosis through mitochondrial dysfunction and ferroptosis through NCOA4-mediated ferritinophagy. Thus, Eupatorium chinense L. could be a potential TCM for treating HCC.

Hydnocarpin D attenuates lipopolysaccharide-induced acute lung injury via MAPK/NF-κB and Keap1/Nrf2/HO-1 pathway.

BACKGROUND: Acute lung injury (ALI) is a complex pulmonary destructive disease with limited therapeutic approaches. Hydnocarpin D (HD) is a flavonolignan isolated from Hydnocarpus wightiana which possesses antioxidant and anti-inflammatory properties. However, whether HD has beneficial effects on ALI as well as its underlying mechanism remains to be elucidated. PURPOSE: This study evaluated the protective effect of HD in ALI and the underlying molecular mechanisms. METHODS: In vivo, the role of HD on lipopolysaccharide (LPS)-induced ALI in mice was tested by determination of neutrophil infiltration, levels of inflammatory cytokines, lung histology and edema, vascular and alveolar barrier disruption. In vitro, murine macrophage RAW 264.7 cells were used to investigate the molecular mechanisms RESULTS: Administration of HD protected mice against LPS-induced ALI, including ameliorating the histological alterations in the lung tissues, and decreasing lung edema, protein content of bronchoalveolar lavage fluid, infiltration of inflammatory cell and secretion of cytokines. Moreover, HD blocked the phosphorylation of TLR-4, NF-κB, and ERK in LPS-induced lung injury. In vitro, HD inhibited LPS-induced oxidative stress and inflammation in RAW 264.7 cells, which largely depend upon the upregulation of antioxidant defensive Nrf2 pathway, thereby suppressing LPS-activated proinflammatory mediator secretion, NLRP3 inflammasome, and MAPK/NF-κB signaling pathway. CONCLUSION: HD attenuates oxidative stress and inflammation against LPS-induced ALI via MAPK/NF-κB and Keap1/Nrf2/HO-1 pathway, and is a promising novel therapeutic candidate for ALI.

Inhibitory effect of hydnocarpin D on T-cell acute lymphoblastic leukemia via induction of autophagy-dependent ferroptosis.

Hydnocarpin D (HD) is a bioactive flavonolignan compound that possesses promising anti-tumor activity, although the mechanism is not fully understood. Using T cell acute lymphoblastic leukemia (T-ALL) cell lines Jurkat and Molt-4 as model system, we found that HD suppressed T-ALL proliferation in vitro, via induction of cell cycle arrest and subsequent apoptosis. Furthermore, HD increased the LC3-II levels and the formation of autophagolysosome vacuoles, both of which are markers for autophagy. The inhibition of autophagy by either knockdown of ATG5/7 or pre-treatment of 3-MA partially rescued HD-induced apoptosis, thus suggesting that autophagy enhanced the efficacy of HD. Interestingly, this cytotoxic autophagy triggered ferroptosis, as evidenced by the accumulation of lipid ROS and decrease of GSH and GPX4, while inhibition of autophagy impeded ferroptotic cell death. Our study suggests that HD triggers multiple cell death processes and is an interesting compound that should be evaluated in future preclinical studies.

Inhibition of retinoic acid receptor α phosphorylation represses the progression of triple-negative breast cancer via transactivating miR-3074-5p to target DHRS3.

BACKGROUND: Retinoids are promising agents in the treatment of different types of neoplasia including estrogen receptor-positive breast cancers, whereas refractoriness/low sensitivity is observed in triple-negative breast cancer (TNBC) subtype. However, the reason for these diverse retinoid-sensitivity remains elusive. METHODS: Determinants of retinoid sensitivity were investigated using immunohistochemistry of primary patient samples, and identified retinoic acid receptor α (RARα) as a putative factor. The anti-tumor activity of hypo-phosphorylated RARα was investigated in TNBC cell models and a xenograft mouse model. Next, miRNA sequencing analysis was performed to identify the target miRNA of RARα, and luciferase reporter was used to confirm the direct target gene of miR-3074-5p. RESULTS: We discovered that serine-77 residue of RARα was constantly phosphorylated, which correlated with TNBC's resistance to retinoids. Overexpression of a phosphorylation-defective mutant RARαS77A mimicked activated RARα and repressed TNBC cell progression both in vitro and in vivo, via activating cell cycle arrest, apoptosis, and cytotoxic autophagy, independent of RARα agonists. We further revealed that the anti-tumor action of RARαS77A was, at least in part, mediated by the up-regulation of miR-3074-5p, which directly targeted DHRS3, a reductase negatively associated with TNBC patient survival. Our results suggest that the inhibition of RARαS77 phosphorylation by either expressing RARαS77A or inhibiting RARα's phosphokinase CDK7, can bypass RA stimuli to transactivate tumor-suppressive miR-3074-5p and reduce oncogenic DHRS3, thus overcoming the RA-resistance of TNBC. CONCLUSION: The novel regulatory network, involving RARαS77 phosphorylation, miR-3074-5p, and DHRS3, emerges as a new target for TNBC treatment.

Autophagy Inhibition Potentiates the Anticancer Effects of a Bendamustine Derivative NL-101 in Acute T Lymphocytic Leukemia.

Acute T lymphocytic leukemia (T-ALL) is an aggressive hematologic resulting from the malignant transformation of T-cell progenitors. Drug resistance and relapse are major difficulties in the treatment of T-ALL. Here, we report the antitumor potency of NL-101, a compound that combines the nitrogen mustard group of bendamustine with the hydroxamic acid group of vorinostat. We found NL-101 exhibited efficient antiproliferative activity in T-ALL cell lines (IC50 1.59-1.89 µM), accompanied by cell cycle arrest and apoptosis, as evidenced by the increased expression of Cyclin E1, CDK2, and CDK4 proteins and cleavage of PARP. In addition, this bendamustine-derived drug showed both a HDACi effect as demonstrated by histone hyperacetylation and p21 transcription and a DNA-damaging effect as shown by an increase in γ-H2AX. Intriguingly, we found that NL-101-induced autophagy in T-ALL cells through inhibiting Akt-mTOR signaling pathway, as indicated by an increase in LC3-I to LC3-II conversion and decrease of p62. Furthermore, inhibition of autophagy by 3-methyladenine increased apoptotic cell death by NL-101, suggesting a prosurvival role of autophagy. In summary, our finding provides rationale for investigation of NL-101 as a DNA/HDAC dual targeting drug in T-ALL, either as a single agent or in combination with autophagy inhibitors.

Valtrate from Valeriana jatamansi Jones induces apoptosis and inhibits migration of human breast cancer cells in vitro.

Valtrate is a principle compound isolated from Valeriana jatamansi Jones, a traditional Chinese folk medicine originally used to treat various nervous disorders. Here, we found that valtrate exhibited significant anti-cancer activity in vitro, especially in human breast cancer cells, while displayed relatively low cytotoxicity to normal human breast epithelial cells (MCF 10A). Valtrate induced cell cycle arrest at G2/M stage and apoptosis in MDA-MB-231 and MCF-7 cells, with reduced expression of p-Akt (Ser 473), cyclin B1 and caspase 8, and increased expression of p21, p-cdc2, cleaved-caspase 3, cleaved-caspase 7 and poly (ADP-ribose) polymerase (PARP). In addition, valtrate inhibited cell migration through down-regulation of MMP-9 and MMP-2 expression. These results demonstrate that valtrate possesses anti-breast cancer activities via cell cycle arrest, apoptosis, and inhibition of cell migration, thus supporting valtrate as a potential antitumor agent.

Jatamanvaltrate P induces cell cycle arrest, apoptosis and autophagy in human breast cancer cells in vitro and in vivo.

Jatamanvaltrate P is a novel iridoid ester isolated from Valeriana jatamansi Jones, a traditional medicine used to treat nervous disorders. In this study, we found that Jatamanvaltrate P possessed notable antitumor properties and therefore evaluated its anticancer effects against human breast cancer cells in vitro and in vivo. Jatamanvaltrate P inhibited the growth and proliferation of MCF-7 and triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-453 and MDA-MB-468) in a concentration-dependent manner, while displayed relatively low cytotoxicity to human breast epithelial cells (MCF-10A). Treatment with Jatamanvaltrate P induced G2/M-phase arrest in TNBC and G0/G1-phase arrest in MCF-7 cells. Further study of the molecular mechanisms of this cytotoxic compound demonstrated that Jatamanvaltrate P enhanced cleavage of PARP and caspases, while decreased the expression levels of cell cycle-related Cyclin B1, Cyclin D1 and Cdc-2. It also activated autophagy, as indicated by the triggered autophagosome formation and increased LC3-II levels. Autophagy inhibition by 3-MA co-treatment undermined Jatamanvaltrate P-induced cell death. Finally, Jatamanvaltrate P exhibited a potential antitumor effect in MDA-MB-231 xenografts without apparent toxicity. These results suggest that Jatamanvaltrate P is a potential therapeutic agent for breast cancer, providing a basis for development of the compound as a novel chemotherapeutic agent.

Am80-GCSF synergizes myeloid expansion and differentiation to generate functional neutrophils that reduce neutropenia-associated infection and mortality.

Neutrophils generated by granulocyte colony-stimulating factor (GCSF) are functionally immature and, consequently, cannot effectively reduce infection and infection-related mortality in cancer chemotherapy-induced neutropenia (CCIN). Am80, a retinoic acid (RA) agonist that enhances granulocytic differentiation by selectively activating transcription factor RA receptor alpha (RARα), alternatively promotes RA-target gene expression. We found that in normal and malignant primary human hematopoietic specimens, Am80-GCSF combination coordinated proliferation with differentiation to develop complement receptor-3 (CR3)-dependent neutrophil innate immunity, through altering transcription of RA-target genes RARß2, C/EBPε, CD66, CD11b, and CD18 This led to generation of functional neutrophils capable of fighting infection, whereas neutralizing neutrophil innate immunity with anti-CD18 antibody abolished neutrophil bactericidal activities induced by Am80-GCSF Further, Am80-GCSF synergy was evaluated using six different dose-schedule-infection mouse CCIN models. The data demonstrated that during "emergency" granulopoiesis in CCIN mice undergoing transient systemic intravenous bacterial infection, Am80 effect on differentiating granulocytic precursors synergized with GCSF-dependent myeloid expansion, resulting in large amounts of functional neutrophils that reduced infection. Importantly, extensive survival tests covering a full cycle of mouse CCIN with perpetual systemic intravenous bacterial infection proved that without causing myeloid overexpansion, Am80-GCSF generated sufficient numbers of functional neutrophils that significantly reduced infection-related mortality in CCIN mice. These findings reveal a differential mechanism for generating functional neutrophils to reduce CCIN-associated infection and mortality, providing a rationale for future therapeutic approaches.

Sequential treatment with aurora B inhibitors enhances cisplatin-mediated apoptosis via c-Myc.

UNLABELLED: Platinum compound such as cisplatin is the first-line chemotherapy of choice in most patients with ovarian carcinoma. However, patients with inherent or acquired cisplatin resistance often experience relapse. Therefore, novel therapies are urgently required to treat drug-resistant ovarian carcinoma. Here, we showed that compared to the non-functional traditional simultaneous treatment, sequential combination of Aurora B inhibitors followed by cisplatin synergistically enhanced apoptotic response in cisplatin-resistant OVCAR-8 cells. This effect was accompanied by the induction of polyploidy in a c-Myc-dependent manner, as c-Myc knockdown reduced the efficacy of the combination by suppressing the expression of Aurora B and impairing cellular response to Aurora B inhibitor, as indicated by the decreased polyploidy and hyperphosphorylation of histone H1. In c-Myc-deficient SKOV3 cells, c-Myc overexpression restored Aurora B expression, induced polyploidy after inhibition of Aurora B, and sensitized cells to this combination therapy. Thus, our report reveals for the first time that sequential treatment of Aurora B inhibitors and cisplatin is essential to inhibit ovarian carcinoma by inducing polyploidy and downregulating c-Myc and that c-Myc is identified as a predictive biomarker to select cells responsive to chemotherapeutical combinations targeting Aurora B. Collectively, these studies provide novel approaches to overcoming cisplatin chemotherapy resistance in ovarian cancer. KEY MESSAGE: Pretreatment of Aurora B inhibitors augment apoptotic effects of cisplatin. The synergy of Aurora B inhibitor with cisplatin is dependent on c-Myc expression. c-Myc-dependent induction of polyploidy sensitizes cells to cisplatin.

DJ-1 as a human oncogene and potential therapeutic target.

DJ-1 is a cancer- and Parkinson's disease-associated protein that participates in different intracellular signaling pathways to protect cells from toxic stresses. DJ-1 expression, oxidation, localization, and phosphorylation are often altered in human tumors, and DJ-1 has been implicated in various aspects of transformation, including uncontrolled proliferation, invasion, metastasis, and resistance to chemotherapy and apoptosis. Despite the strong relationship between DJ-1 and cancer, which made it a particularly attractive therapeutic target for cancer treatment, the detailed mechanisms of how this oncogene coordinates altered signaling with cell survival remains elusive. In this commentary, we discuss the role of DJ-1 in transformation, highlight some of the significant aspects of and prospects for therapeutically targeting the DJ-1 signaling in cancer, and describe what the future may hold.

The lost intrinsic fragmentation of MAT1 protein during granulopoiesis promotes the growth and metastasis of leukemic myeloblasts.

MAT1, an assembly factor and targeting subunit of both cyclin-dependent kinase-activating kinase (CAK) and general transcription factor IIH (TFIIH) kinase, regulates cell cycle and transcription. Previous studies show that expression of intact MAT1 protein is associated with expansion of human hematopoietic stem cells (HSC), whereas intrinsically programmed or retinoic acid (RA)-induced MAT1 fragmentation accompanies granulocytic differentiation of HSC or leukemic myeloblasts. Here we determined that, in humanized mouse microenvironment, MAT1 overexpression resisted intrinsic MAT1 fragmentation to sustain hematopoietic CD34+ cell expansion while preventing granulopoiesis. Conversely, we mimicked MAT1 fragmentation in vitro and in a mouse model by overexpressing a fragmented 81-aa MAT1 polypeptide (pM9) that retains the domain for assembling CAK but cannot affix CAK to TFIIH-core. Our results showed that pM9 formed ΔCAK by competing with MAT1 for CAK assembly to mimic MAT1 fragmentation-depletion of CAK. This resulting ΔCAK acted as a dominant negative to inhibit the growth and metastasis of different leukemic myeloblasts, with or without RA resistance, by concurrently suppressing CAK and TFIIH kinase activities to inhibit cell cycle and gene transcription. These findings suggest that the intrinsically programmed MAT1 expression and fragmentation regulate granulopoiesis by inversely coordinating CAK and TFIIH activities, whereas pM9 shares a mechanistic resemblance with MAT1 fragmentation in suppressing myeloid leukemogenesis.

Efficacy of all-trans retinoid acid in preventing nickel induced cardiotoxicity in myocardial cells of rats.

Nickel, a metal commonly found in battery plants and welding factories, has potential cardiotoxicity, while all-trans retinoid acid (atRA) can promote cardiovascular repair and myocardial recovery. The purpose of this study was to investigate whether atRA could prevent cardiotoxicity induced by nickel both in vitro and in vivo. In the study, a rat myocardial cell line (H9c2) exposed to different concentrations of nickel chloride (NiCl(2)) displayed apoptotic features accompanied by reactive oxygen species generation. In addition, NiCl(2) also caused obvious apoptosis and systolic dysfunction in primary myocardial cells. Treatment with atRA efficiently attenuated the cytotoxicities triggered by NiCl(2) as it significantly mitigated ROS generation and decreased MAP kinases activity in NiCl(2)-treated cardiomyocytes. Additionally, NiCl(2) exposure caused obvious arrhythmia in Sprague-Dawley rats with the maximum tolerance dose of NiCl(2) between 2 and 3mg/kg. A combinational intragastric administration of 40mg/kg atRA can partially reverse NiCl(2)-induced arrhythmia in rats. Our results suggested that atRA might have therapeutic potential in alleviating the adverse effects of nickel on the cardiovascular system.