GNAI1 and GNAI3 Reduce Colitis-Associated Tumorigenesis in Mice by Blocking IL6 Signaling and Down-regulating Expression of GNAI2.

BACKGROUND & AIMS: Interleukin 6 (IL6) and tumor necrosis factor contribute to the development of colitis-associated cancer (CAC). We investigated these signaling pathways and the involvement of G protein subunit alpha i1 (GNAI1), GNAI2, and GNAI3 in the development of CAC in mice and humans. METHODS: B6;129 wild-type (control) or mice with disruption of Gnai1, Gnai2, and/or Gnai3 or conditional disruption of Gnai2 in CD11c+ or epithelial cells were given dextran sulfate sodium (DSS) to induce colitis followed by azoxymethane (AOM) to induce carcinogenesis; some mice were given an antibody against IL6. Feces were collected from mice, and the compositions of microbiomes were analyzed by polymerase chain reactions. Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) isolated from spleen and colon tissues were analyzed by flow cytometry. We performed immunoprecipitation and immunoblot analyses of colon tumor tissues, MDSCs, and mouse embryonic fibroblasts to study the expression levels of GNAI1, GNAI2, and GNAI3 and the interactions of GNAI1 and GNAI3 with proteins in the IL6 signaling pathway. We analyzed the expression of Gnai2 messenger RNA by CD11c+ cells in the colonic lamina propria by PrimeFlow, expression of IL6 in DCs by flow cytometry, and secretion of cytokines in sera and colon tissues by enzyme-linked immunosorbent assay. We obtained colon tumor and matched nontumor tissues from 83 patients with colorectal cancer having surgery in China and 35 patients with CAC in the United States. Mouse and human colon tissues were analyzed by histology, immunoblot, immunohistochemistry, and/or RNA-sequencing analyses. RESULTS: GNAI1 and GNAI3 (GNAI1;3) double-knockout (DKO) mice developed more severe colitis after administration of DSS and significantly more colonic tumors than control mice after administration of AOM plus DSS. Development of increased tumors in DKO mice was not associated with changes in fecal microbiomes but was associated with activation of nuclear factor (NF) κB and signal transducer and activator of transcription (STAT) 3; increased levels of GNAI2, nitric oxide synthase 2, and IL6; increased numbers of CD4+ DCs and MDSCs; and decreased numbers of CD8+ DCs. IL6 was mainly produced by CD4+/CD11b+, but not CD8+, DCs in DKO mice. Injection of DKO mice with a blocking antibody against IL6 reduced the expansion of MDSCs and the number of tumors that developed after CAC induction. Incubation of MDSCs or mouse embryonic fibroblasts with IL6 induced activation of either NF-κB by a JAK2-TRAF6-TAK1-CHUK/IKKB signaling pathway or STAT3 by JAK2. This activation resulted in expression of GNAI2, IL6 signal transducer (IL6ST, also called GP130) and nitric oxide synthase 2, and expansion of MDSCs; the expression levels of these proteins and expansion of MDSCs were further increased by the absence of GNAI1;3 in cells and mice. Conditional disruption of Gnai2 in CD11c+ cells of DKO mice prevented activation of NF-κB and STAT3 and changes in numbers of DCs and MDSCs. Colon tumor tissues from patients with CAC had reduced levels of GNAI1 and GNAI3 and increased levels of GNAI2 compared with normal tissues. Further analysis of a public human colorectal tumor DNA microarray database (GSE39582) showed that low Gani1 and Gnai3 messenger RNA expression and high Gnai2 messenger RNA expression were significantly associated with decreased relapse-free survival. CONCLUSIONS: GNAI1;3 suppresses DSS-plus-AOM-induced colon tumor development in mice, whereas expression of GNAI2 in CD11c+ cells and IL6 in CD4+/CD11b+ DCs appears to promote these effects. Strategies to induce GNAI1;3, or block GNAI2 and IL6, might be developed for the prevention or therapy of CAC in patients.

Cytotoxic Sesquiterpenoid Quinones and Quinols, and an 11-Membered Heterocycle, Kauamide, from the Hawaiian Marine Sponge Dactylospongia elegans.

Several known sesquiterpenoid quinones and quinols (1-9), and kauamide (10), a new polyketide-peptide containing an 11-membered heterocycle, were isolated from the extracts of the Hawaiian marine sponge Dactylospongia elegans. The planar structure of 10 was determined from spectroscopic analyses, and its relative and absolute configurations were established from density functional theory (DFT) calculations of the GIAO NMR shielding tensors, and advanced Marfey's analysis of the N-MeLeu residue, respectively. Compounds 1 and 3 showed moderate inhibition of ß-secretase 1 (BACE1), whereas 1-9 exhibited moderate to potent inhibition of growth of human glioma (U251) cells. Compounds 1-2 and 4-7 were also active against human pancreatic carcinoma (Panc-1) cells.

An Unusual Benzoisoquinoline-9-one Derivative and Other Related Compounds with Antiproliferative Activity from Hawaiian Endophytic Fungus Peyronellaea sp. FT431.

A new polyketide containing the benzoisoquinoline-9-one moiety, peyronetide A (1), and three other new derivatives peyronetides B⁻D (2⁻4), as well as one known compound (5) were purified from the cultured broth of the endophytic fungus Peyronellaea sp. FT431, which was isolated from the Hawaiian indigenous plant, Verbena sp. The structures of the new compounds were determined through the analysis of HRMS and NMR spectroscopic data. Compounds 1, 2, and 5 showed cytotoxic activities against TK-10 (human kidney adenocarcinoma cells), cisplatin sensitive A2780S (human ovarian carcinoma cells), and cisplatin resistant A2780CisR cell lines, with IC50 values between 6.7 to 29.2 µM.

Factorial Design Based Multivariate Modeling and Optimization of Tunable Bioresponsive Arginine Grafted Poly(cystaminebis(acrylamide)-diaminohexane) Polymeric Matrix Based Nanocarriers.

Desired characteristics of nanocarriers are crucial to explore its therapeutic potential. This investigation aimed to develop tunable bioresponsive newly synthesized unique arginine grafted poly(cystaminebis(acrylamide)-diaminohexane) [ABP] polymeric matrix based nanocarriers by using L9 Taguchi factorial design, desirability function, and multivariate method. The selected formulation and process parameters were ABP concentration, acetone concentration, the volume ratio of acetone to ABP solution, and drug concentration. The measured nanocarrier characteristics were particle size, polydispersity index, zeta potential, and percentage drug loading. Experimental validation of nanocarrier characteristics computed from initially developed predictive model showed nonsignificant differences (p > 0.05). The multivariate modeling based optimized cationic nanocarrier formulation of <100 nm loaded with hydrophilic acetaminophen was readapted for a hydrophobic etoposide loading without significant changes (p > 0.05) except for improved loading percentage. This is the first study focusing on ABP polymeric matrix based nanocarrier development. Nanocarrier particle size was stable in PBS 7.4 for 48 h. The increase of zeta potential at lower pH 6.4, compared to the physiological pH, showed possible endosomal escape capability. The glutathione triggered release at the physiological conditions indicated the competence of cytosolic targeting delivery of the loaded drug from bioresponsive nanocarriers. In conclusion, this unique systematic approach provides rational evaluation and prediction of a tunable bioresponsive ABP based matrix nanocarrier, which was built on selected limited number of smart experimentation.

Bioactive polyprenylated benzophenone derivatives from the fruits extracts of Garcinia xanthochymus.

Two new polycyclic prenylated xanthones (1 and 2) and a new phenylpropanoid glycoside (3), along with seven known compounds (4-10) were isolated from the fruits of Garcinia xanthochymus. The structures were elucidated by 1D- and 2D-NMR, and HRMS experiments. The isolates were evaluated for their inhibitory effects against the viability of U251MG glioblastoma and MDA-MB-231 breast cancer cells that harbor an aberrantly active signal transducer and exhibit activation of transcription 3 (STAT3), and compared to normal NIH3T3 mouse fibroblasts. Among the isolates, compounds 1, 2, 5, and 6-9 inhibited the viability of glioma cancer cells with IC50 values in the range of 1.6-6.5µM. Furthermore, treatment of U251MG with 6 and 7 inhibited intracellular STAT3 tyrosine phosphorylation and glioma cell migration in vitro, respectively.

Heliotropiumides A and B, new phenolamides with N-carbamoyl putrescine moiety from Heliotropium foertherianum collected in Hawaii and their biological activities.

Two new compounds heliotropiumides A (1) and B (2), phenolamides each with an uncommon carbamoyl putrescine moiety, were isolated from the seeds of a naturalized Hawaiian higher plant, Heliotropium foertherianum Diane & Hilger in the borage family, which is widely used for the treatment of ciguatera fish poisoning. The structures of compounds 1 and 2 were characterized based on MS spectroscopic and NMR analysis, and DP4+ calculations. The absolute configuration (AC) of compound 1 was determined by comparison of its optical rotation with those reported in literature. Compound 2 showed inhibition against NF-κB with an IC50 value of 36µM.

Verbenanone, an octahydro-5H-chromen-5-one from a Hawaiian-Plant Associated Fungus FT431.

A new secondary metabolite verbenanone (1) with a unique (4aS,8aS)-octahydro-5H-chromen-5-one moiety has been obtained from the endophytic fungus FT431, which was isolated from the native Hawaiian plant Verbena sp. The structure of compound 1 was characterized based on NMR and MS spectroscopic analysis. The absolute configuration (AC) of compound 1 was determined by Mosher acids. Compound 1 was tested against A2780 and A2780cisR, but it was inactive.

A New N-methoxypyridone from the Co-Cultivation of Hawaiian Endophytic Fungi Camporesia sambuci FT1061 and Epicoccum sorghinum FT1062.

A new N-methoxypyridone analog (1), together with four known compounds, was isolated from the co-culture of Hawaiian endophytic fungi Camporesia sambuci FT1061 and Epicoccum sorghinum FT1062. The structure of the new compound was elucidated as 11S-hydroxy-1-methoxyfusaricide (1) by extensive spectroscopic analysis and comparison with the literature. The absolute configuration of 1 was determined by comparison with the experimental and calculated ECD spectra. The absolute configuration of compound 3 was investigated and renamed as (+)-epipyridone by comparison of the optical rotation and CD spectrum with those of 1. The other known compounds were identified as epicoccarine B (2), D8646-2-6 (4), and iso-D8646-2-6 (5). Compounds 4 and 5 showed modest inhibitory activity towards pathogenic fungi. Epicoccarine B (2) inhibited A2780 and TK-10 with an IC50 value of 22 µM.

Bromotyrosine-derived metabolites from an Indonesian marine sponge in the family Aplysinellidae (Order Verongiida).

Seven new bromotyrosine-derived metabolites, purpuramine M-N (1-2), araplysillin VII-XI (3-7) and six known compounds (8-13) were isolated from an Indonesian sponge belonging to the family Aplysinellidae (Order Verongiida). The structures of the new compounds were determined by extensive NMR experiments and mass spectrometric measurements. These compounds were screened against BACE1 and five cancer cell lines.

A Resveratrol Analogue Promotes ERKMAPK-Dependent Stat3 Serine and Tyrosine Phosphorylation Alterations and Antitumor Effects In Vitro against Human Tumor Cells.

(E)-4-(3,5-dimethoxystyryl)phenyl acetate (Cmpd1) is a resveratrol analog that preferentially inhibits glioma, breast, and pancreatic cancer cell growth, with IC50 values of 6-19 µM. Notably, the human U251MG glioblastoma tumor line is the most sensitive, with an IC50 of 6.7 µM, compared with normal fibroblasts, which have an IC50 > 20 µM. Treatment of U251MG cells that harbor aberrantly active signal transducer and activator of transcription (Stat) 3 with Cmpd1 suppresses Stat3 tyrosine705 phosphorylation in a dose-dependent manner in parallel with the induction of pserine727 Stat3 and extracellular signal-regulated kinase/mitogen-activated protein kinase 1/2 (pErk1/2(MAPK)). Inhibition of pErk1/2(MAPK) induction by the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] blocked both the pserine727 Stat3 induction and ptyrosine705 Stat3 suppression by Cmpd1, indicating dependency on the mitogen-activated protein/extracellular signal-regulated kinase kinase-Erk1/2(MAPK) pathway for Cmpd1-induced modulation of Stat3 signaling. Cmpd1 also blocked epidermal growth factor-stimulated pStat1 induction, whereas upregulating pSrc, pAkt, p-p38, pHeat shock protein 27, and pmammalian target of rapamycin levels. However, pJanus kinase 2 and pEpidermal growth factor receptor levels were not significantly altered. Treatment of U251MG cells with Cmpd1 reduced in vitro colony formation, induced cell cycle arrest in the G2/M phase and cleavage of caspases 3, 8, and 9 and poly(ADP ribose) polymerase, and suppressed survivin, myeloid cell leukemia 1, Bcl-xL, cyclin D1, and cyclin B1 expression. Taken together, these data identify a novel mechanism for the inhibition of Stat3 signaling by a resveratrol analog and suggest that the preferential growth inhibitory effects of Cmp1 occur in part by Erk1/2(MAPK)-dependent modulation of constitutively active Stat3.

Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts.

Computer-aided lead optimization derives a unique, orally bioavailable inhibitor of the signal transducer and activator of transcription (Stat)3 Src homology 2 domain. BP-1-102 binds Stat3 with an affinity (K(D)) of 504 nM, blocks Stat3-phospho-tyrosine (pTyr) peptide interactions and Stat3 activation at 4-6.8 µM, and selectively inhibits growth, survival, migration, and invasion of Stat3-dependent tumor cells. BP-1-102-mediated inhibition of aberrantly active Stat3 in tumor cells suppresses the expression of c-Myc, Cyclin D1, Bcl-xL, Survivin, VEGF, and Krüppel-like factor 8, which is identified as a Stat3 target gene that promotes Stat3-mediated breast tumor cell migration and invasion. Treatment of breast cancer cells with BP-1-102 further blocks Stat3-NF-κB cross-talk, the release of granulocyte colony-stimulating factor, soluble intercellular adhesion molecule 1, macrophage migration-inhibitory factor/glycosylation-inhibiting factor, interleukin 1 receptor antagonist, and serine protease inhibitor protein 1, and the phosphorylation of focal adhesion kinase and paxillin, while enhancing E-cadherin expression. Intravenous or oral gavage delivery of BP-1-102 furnishes micromolar or microgram levels in tumor tissues and inhibits growth of human breast and lung tumor xenografts.

Sensitization of pancreatic cancer cells to radiation by cerium oxide nanoparticle-induced ROS production.

Side effect of radiation therapy (RT) remains the most challenging issue for pancreatic cancer treatment. In this report we determined whether and how cerium oxide nanoparticles (CONPs) sensitize pancreatic cancer cells to RT. CONP pretreatment enhanced radiation-induced reactive oxygen species (ROS) production preferentially in acidic cell-free solutions as well as acidic human pancreatic cancer cells. In acidic environments, CONPs favor the scavenging of superoxide radical over the hydroxyl peroxide resulting in accumulation of the latter whereas in neutral pH CONPs scavenge both. CONP treatment prior to RT markedly potentiated the cancer cell apoptosis both in culture and in tumors and the inhibition of the pancreatic tumor growth without harming the normal tissues or host mice. Taken together, these results identify CONPs as a potentially novel RT-sensitizer as well as protectant for improving pancreatic cancer treatment. FROM THE CLINICAL EDITOR: Pancreatic tumors remain some of the most notoriously treatment-unresponsive malignancies. Cerium oxide nanoparticles may be capable of sensitizing such cells to radiotherapy, as demonstrated in this study.

Retraction of "Identification of Purine-Scaffold Small-Molecule Inhibitors of Stat3 Activation by QSAR Studies".

[This retracts the article DOI: 10.1021/ml100224d.].

The R(h)oads to Stat3: Stat3 activation by the Rho GTPases.

The signal transducer and activator of transcription-3 (Stat3) is a member of the STAT family of cytoplasmic transcription factors. Overactivation of Stat3 is detected with high frequency in human cancer and is considered a molecular abnormality that supports the tumor phenotype. Despite concerted investigative efforts, the molecular mechanisms leading to the aberrant Stat3 activation and Stat3-mediated transformation and tumorigenesis are still not clearly defined. Recent evidence reveals a crosstalk close relationship between Stat3 signaling and members of the Rho family of small GTPases, including Rac1, Cdc42 and RhoA. Specifically, Rac1, acting in a complex with the MgcRacGAP (male germ cell RacGAP), promotes tyrosine phosphorylation of Stat3 by the IL6-receptor family/Jak kinase complex, as well as its translocation to the nucleus. Studies have further revealed that the mutational activation of Rac1 and Cdc42 results in Stat3 activation, which occurs in part through the upregulation of IL6 family cytokines that in turn stimulates Stat3 through the Jak kinases. Interestingly, evidence also shows that the engagement of cadherins, cell to cell adhesion molecules, specifically induces a striking increase in Rac1 and Cdc42 protein levels and activity, which in turn results in Stat3 activation. In this review we integrate recent findings clarifying the role of the Rho family GTPases in Stat3 activation in the context of malignant progression.

SS-4 is a highly selective small molecule inhibitor of STAT3 tyrosine phosphorylation that potently inhibits GBM tumorigenesis in vitro and in vivo.

Glioblastoma (GBM) is a highly aggressive cancer with a dismal prognosis. Constitutively active STAT3 has a causal role in GBM progression and is associated with poor patient survival. We rationally designed a novel small molecule, SS-4, by computational modeling to specifically interact with STAT3. SS-4 strongly and selectively inhibited STAT3 tyrosine (Y)-705 phosphorylation in MT330 and LN229 GBM cells and inhibited their proliferation and induced apoptosis with an IC50 of ∼100 nM. The antiproliferative and apoptotic actions of SS-4 were Y-705 phosphorylation dependent, as evidenced by its lack of effects on STAT3 knockout (STAT3KO) cells or STAT3KO cells that overexpressed a phospho-Y705 deficient (STAT3Y705F) mutant, and the recovery of effects when wild-type STAT3 or a phospho-serine (S)727 deficient mutant was expressed in STAT3KO cells. SS-4 increased the expression of STAT3 repressed genes, while decreasing the expression of STAT3 promoted genes. Importantly, SS-4 markedly reduced the growth of GBM intracranial tumor xenografts. These data together identify SS-4 as a potent STAT3 inhibitor that selectively blocks Y705-phosphorylation, induces apoptosis, and inhibits growth of human GBM models in vitro and in vivo.

Natural product preferentially targets redox and metabolic adaptations and aberrantly active STAT3 to inhibit breast tumor growth in vivo.

Dysregulated gene expression programs and redox and metabolic adaptations allow cancer cells to survive under high oxidative burden. These mechanisms also represent therapeutic vulnerabilities. Using triple-negative breast cancer (TNBC) as a model, we show that compared to normal human breast epithelial cells, the TNBC cells, MDA-MB-231 and MDA-MB-468 that harbor constitutively active STAT3 also express higher glucose-6-phosphate dehydrogenase (G6PD), thioredoxin reductase (TrxR)1, NADPH, and GSH levels for survival. Present studies discover that the natural product, R001, targets these adaptation mechanisms. Treatment of TNBC cells with R001 inhibited constitutively active STAT3, STAT3-regulated gene expression, and the functions of G6PD and TrxR1. Consequently, in the TNBC, but not normal cells, R001 suppressed GSH levels, but raised NADPH levels, reflective of a loss of mitochondrial respiration and which led to reactive oxygen species (ROS) induction, all of which led to loss of viable cells and inhibition of anchorage-dependent and independent growth. R001 treatment further led to early pyroptosis and late DNA damage, cell cycle arrest, and apoptosis only in the TNBC cells. Oral administration of 5 mg/kg R001 inhibited MDA-MB-468 xenografts growth in mice, with reduced pY705-STAT3, G6PD, TrxR1, and GSH levels. R001 serves as a therapeutic entity that targets the vulnerabilities of TNBC cells to inhibit tumor growth in vivo.

Inhibition of STAT3-ferroptosis negative regulatory axis suppresses tumor growth and alleviates chemoresistance in gastric cancer.

Chemotherapy is still one of the principal treatments for gastric cancer, but the clinical application of 5-FU is limited by drug resistance. Here, we demonstrate that ferroptosis triggered by STAT3 inhibition may provide a novel opportunity to explore a new effective therapeutic strategy for gastric cancer and chemotherapy resistance. We find that ferroptosis negative regulation (FNR) signatures are closely correlated with the progression and chemoresistance of gastric cancer. FNR associated genes (GPX4, SLC7A11, and FTH1) and STAT3 are upregulated in 5-FU resistant cells and xenografts. Further evidence demonstrates that STAT3 binds to consensus DNA response elements in the promoters of the FNR associated genes (GPX4, SLC7A11, and FTH1) and regulates their expression, thereby establishing a negative STAT3-ferroptosis regulatory axis in gastric cancer. Genetic inhibition of STAT3 activity triggers ferroptosis through lipid peroxidation and Fe2+ accumulation in gastric cancer cells. We further develop a potent and selective STAT3 inhibitor, W1131, which demonstrates significant anti-tumor effects in gastric cancer cell xenograft model, organoids model, and patient-derived xenografts (PDX) model partly by inducing ferroptosis, thus providing a new candidate compound for advanced gastric cancer. Moreover, targeting the STAT3-ferroptosis circuit promotes ferroptosis and restores sensitivity to chemotherapy. Our finding reveals that STAT3 acts as a key negative regulator of ferroptosis in gastric cancer through a multi-pronged mechanism and provides a new therapeutic strategy for advanced gastric cancer and chemotherapy resistance.