8-ethoxy-2-(4-fluorophenyl)-3-nitro-2h-chromene and Multiple-Myeloma

S14161 is a pan-Class I PI3K inhibitor that induces blood cancer cell death, but its mechanism is largely unknown. In the present study, we evaluated the role of S14161 in autophagy, an emerging event in cell destination. Multiple myeloma cell lines RPMI-8226, OPM2, KMS11 and leukemia cell line K562 were treated with S14161. The results showed that S14161 induced autophagy as demonstrated by increased LC3-II and decreased p62, which were prevented by autophagy inhibitors including 3-methyladenine and bafilomycin A1. Mechanistic studies showed that S14161 had no effects on Vps34 expression, but increased Beclin 1 and decreased Bcl-2, two major regulators of autophagy. Furthermore, S14161 dissociated the Beclin 1/Bcl-2 complex and enhanced the formation of Beclin 1/Vps34 complex. Moreover, S14161 inhibited the mTORC1 signaling transduction. S14161 downregulated activation of mTOR and its two critical targets 4E-BP1 and p70S6K, suggesting S14161 inhibited protein synthesis. Taken together, these results demonstrated that Class I PI3K regulates autophagy by modulating protein synthesis and the Beclin 1 signaling pathway. This finding helps understanding the roles of PI3K in autophagy and cancer treatment.

Topics: Autophagy; Beclin-1; Benzopyrans; Cell Line, Tumor; Class III Phosphatidylinositol 3-Kinases; Gene Expression; Hematologic Neoplasms; Humans; K562 Cells; Leukemia; Mechanistic Target of Rapamycin Complex 1; Microtubule-Associated Proteins; Multiple Myeloma; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; RNA-Binding Proteins; Signal Transduction

Side population (SP) cells are an enriched source of cancer-initiating cells with stemness characteristics, generated by increased ABC transporter activity, which has served as a unique hallmark for multiple myeloma (MM) stem cell studies. Here we isolated and identified MM SP cells via Hoechst 33342 staining. Furthermore, we demonstrate that SP cells possess abnormal cell cycle, clonogenicity, and high drug efflux characteristics-all of which are features commonly seen in stem cells. Interestingly, we found that bortezomib, As2O3, and melphalan all affected apoptosis and clonogenicity in SP cells. We followed by characterizing the miRNA signature of MM SP cells and validated the specific miR-451 target tuberous sclerosis 1 (TSC1) gene to reveal that it activates the PI3K/Akt/mTOR signaling in MM SP cells. Inhibition of miR-451 enhanced anti-myeloma novel agents' effectiveness, through increasing cells apoptosis, decreasing clonogenicity, and reducing MDR1 mRNA expression. Moreover, the novel specific PI3K/Akt/mTOR signaling inhibitor S14161 displayed its prowess as a potential therapeutic agent by targeting MM SP cells. Our findings offer insights into the mechanisms regulating MM SP cells and provide a novel strategy to overcome resistance to existing therapies against myeloma.

Topics: Animals; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Benzopyrans; Blotting, Western; Bortezomib; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Melphalan; Mice, Inbred NOD; Mice, SCID; MicroRNAs; Multiple Myeloma; Neoplastic Stem Cells; Oxides; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; Side-Population Cells; Signal Transduction; TOR Serine-Threonine Kinases; Transplantation, Heterologous; Tuberous Sclerosis

We previously reported a PI3K inhibitor S14161 which displays a promising preclinical activity against multiple myeloma (MM) and leukemia, but the chiral structure and poor solubility prevent its further application.. Six S14161 analogs were designed based on the structure-activity relationship; activity of the compounds in terms of cell death and inhibition of PI3K were analyzed by flow cytometry and Western blotting, respectively; anti-myeloma activity in vivo was performed on two independent xenograft models.. Among the six analogs, BENC-511 was one of the most potent compounds which significantly inhibited PI3K activity and induced MM cell apoptosis. BENC-511 was able to inactivate PI3K and its downstream signals AKT, mTOR, p70S6K, and 4E-BP1 at 1 μM but had no effects on their total protein expression. Consistent with its effects on PI3K activity, BENC-511 induced MM cell apoptosis which was evidenced by the cleavage of Caspase-3 and PARP. Notably, addition of insulin-like growth factor 1 and interleukin-6, two important triggers for PI3K activation in MM cells, partly blocked BENC-511-induced MM cell death, which further demonstrated that PI3K signaling pathway was critical for the anti-myeloma activity of BENC-511. Moreover, BENC-511 also showed potent oral activity against myeloma in vivo. Oral administration of BENC-511 decreased tumor growth up to 80% within 3 weeks in two independent MM xenograft models at a dose of 50 mg/kg body weight, but presented minimal toxicity. Suppression of BENC-511 on MM tumor growth was associated with decreased PI3K/AKT activity and increased cell apoptosis.. Because of its potent anti-MM activity, low toxicity (LD50 oral >1.5 g/kg), and easy synthesis, BENC-511 could be developed as a promising agent for the treatment of MM via suppressing the PI3K/AKT signaling pathway.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzopyrans; Caspase 3; Cell Line, Tumor; Female; Flow Cytometry; Humans; Immunoblotting; Insulin-Like Growth Factor I; Interleukin-6; Mice; Mice, Nude; Molecular Structure; Multiple Myeloma; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Signal Transduction; Structure-Activity Relationship; Tumor Burden; Xenograft Model Antitumor Assays

D-cyclins are universally dysregulated in multiple myeloma and frequently overexpressed in leukemia. To better understand the role and impact of dysregulated D-cyclins in hematologic malignancies, we conducted a high-throughput screen for inhibitors of cyclin D2 transactivation and identified 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161), which inhibited the expression of cyclins D1, D2, and D3 and arrested cells at the G(0)/G(1) phase. After D-cyclin suppression, S14161 induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of leukemia, S14161 inhibited tumor growth without evidence of weight loss or gross organ toxicity. Mechanistically, S14161 inhibited the activity of phosphoinositide 3-kinase in intact cells and the activity of the phosphoinositide 3-kinases α, β, δ, and γ in a cell-free enzymatic assay. In contrast, it did not inhibit the enzymatic activities of other related kinases, including the mammalian target of rapamycin, the DNA-dependent protein kinase catalytic subunit, and phosphoinositide-dependent kinase-1. Thus, we identified a novel chemical compound that inhibits D-cyclin transactivation via the phosphoinositide 3-kinase/protein kinase B signaling pathway. Given its potent antileukemia and antimyeloma activity and minimal toxicity, S14161 could be developed as a novel agent for blood cancer therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Base Sequence; Benzopyrans; Biological Transport, Active; Cell Line, Tumor; Cell Membrane; Cyclin D; DNA Primers; Drug Evaluation, Preclinical; G1 Phase; Humans; K562 Cells; Leukemia; Mice; Mice, SCID; Molecular Structure; Multiple Myeloma; NIH 3T3 Cells; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcriptional Activation; Xenograft Model Antitumor Assays