A novel imidazo[1,2-a]pyridine derivative and its co-administration with curcumin exert anti-inflammatory effects by modulating the STAT3/NF-κB/iNOS/COX-2 signaling pathway in breast and ovarian cancer cell lines.

Introduction: Imidazo[1,2-a]pyridine derivatives with diverse pharmacological properties and curcumin, as a potential natural anti-inflammatory compound, are promising compounds for cancer treatment. This study aimed to synthesize a novel imidazo[1,2-a]pyridine derivative, (MIA), and evaluate its anti-inflammatory activity and effects on nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) pathways, and their target genes, alone and in combination with curcumin, in MDA-MB-231 and SKOV3 cell lines. Methods: We evaluated the interaction between imidazo[1,2-a]pyridine ligand, curcumin, and NF-κB p50 protein, using molecular docking studies. MTT assay was used to investigate the impacts of compounds on cell viability. To evaluate the NF-κB DNA binding activity and the level of inflammatory cytokines in response to the compounds, ELISA-based methods were performed. In addition, quantitative polymerase chain reaction (qPCR) and western blotting were carried out to analyze the expression of genes and investigate NF-κB and STAT3 signaling pathways. Results: Molecular docking studies showed that MIA docked into the NF-κB p50 subunit, and curcumin augmented its binding. The MTT assay results indicated that MIA and its combination with curcumin reduced cell viability. According to the results of the ELISA-based methods, MIA lowered the levels of inflammatory cytokines and suppressed NF-κB activity. In addition, real-time PCR and Griess test results showed that the expression of cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) genes, and nitrite production were reduced by MIA. Furthermore, the western blotting analysis demonstrated that MIA increased the expression of inhibitory κB (IκBα) and B-cell lymphoma 2 (Bcl-2)-associated X proteins (BAX), and suppressed the STAT3 phosphorylation, and Bcl-2 expression. Our findings revealed that curcumin had a potentiating role and enhanced all the anti-inflammatory effects of MIA. Conclusion: This study indicated that the anti-inflammatory activity of MIA is exerted by suppressing the NF-κB and STAT3 signaling pathways in MDA-MB-231 and SKOV3 cancer cell lines.

Accelerating Proteomics Using Broad Specificity Proteases.

Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches. Thus, multienzyme strategies are used to maximize sequence coverage in post-translational modification profiling. We present fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin. All three enzymes are remarkably fast, producing near-complete digests in 1-5 min, and cost 200-1000× less than proteomics-grade trypsin. Using FragPipe resolved a major challenge by drastically reducing the duration of the required "unspecific" searches. In-depth analyses of proteinase K, subtilisin, and thermolysin Jurkat digests identified 7374, 8178, and 8753 unique proteins with average sequence coverages of 21, 29, and 37%, including 10,000s of amino acids not reported in PeptideAtlas' >2400 experiments. While we could not identify distinct cleavage patterns, machine learning could distinguish true protease products from random cleavages, potentially enabling the prediction of cleavage products. Finally, proteinase K, subtilisin, and thermolysin enabled label-free quantitation of 3111, 3659, and 4196 unique Jurkat proteins, which in our hands is comparable to trypsin. Our data demonstrate that broad-specificity proteases enable quantitative proteomics of uncharted areas of the proteome. Their fast kinetics may allow "on-the-fly" digestion of samples in the future.

Hepatic Steatosis Alleviated by a Novel Metformin and Quercetin Combination Activating Autophagy Through the cAMP/AMPK/SIRT1 Pathway.

Non-alcoholic fatty liver disease (NAFLD) incidence and prevalence are rapidly increasing globally. The combined effects of metformin and quercetin (Que) have yet to be investigated. However, both have demonstrated the potential to reduce triglyceride (TG) levels and treat NAFLD by promoting autophagy. The objective of the present study was to elucidate the mechanism of action and assess the role of autophagy in the lipid-lowering effects of Que, both individually and in combination with metformin, in a HepG2 cell model of hepatic steatosis. Triglyceride levels and lipogenic gene expression were reduced in HepG2 cells exposed to palmitic acid (PA) when treated with Que-metformin, as evidenced by triglyceride measurements and real-time PCR. The LDH release assay also showed that this combination induced autophagy to protect HepG2 cells from PA-induced cell death. According to the Western blot analysis outcomes, Que-metformin increased LC3-I and LC3-II protein levels while decreasing p62 expression to induce autophagy. In HepG2 cells, the co-administration of Que-metformin elevated cAMP, phosphorylated AMP-activated protein kinase (p-AMPK), and Beclin-1 levels. Additionally, the inhibition of SIRT1 reversed the autophagy induced by Que-metformin. The findings of this study demonstrated for the first time that Que-metformin reduced hepatosteatosis by stimulating autophagy through the cAMP/AMPK/SIRT1 signaling pathway and diminishing inflammatory cytokines.

Co-treatment of Naringenin and Ketoprofen-RGD Suppresses Cell Proliferation via Calmodulin/PDE/cAMP/PKA Axis Pathway in Leukemia and Ovarian Cancer Cells.

Background: Naringenin (Nar) has anti-inflammatory and anticarcinogenic properties. Arginine-glycine- aspartate (RGD) is a tripeptidic sequence used as an integrin ligand and targeting system for delivering chemotherapeutic agents to cancer cells. Objectives: In this study, the inhibitory effects of Nar and ketoprofen-RGD on leukemia and ovarian cancer cells (K562 and SKOV3) were explored for the first time, focusing on their proliferation activity and their anti-inflammatory capacity. Methods: Analyses were conducted on the calmodulin (CaM)-dependent phosphodiesterase 1 (PDE1) activation by ketoprofen-RGD, Nar, and their combination. These drugs' effects on protein kinase A (PKA) activation, intracellular cyclic adenosine monophosphate (cAMP) level, and PDE1 inhibition were identified. Later, it was also evaluated if ketoprofen-RGD alone or in combination with Nar had anti-inflammatory effects. Results: Nar improved the antagonizing consequences of ketoprofen-RGD on the CaM protein, which hinders PDE1, improving PKA activity and cAMP levels. A mixture of ketoprofen-RGD and Nar and ketoprofen-RGD alone diminished K562 and SKOV3 cell viability through the cAMP/PKA pathway by inhibiting PDE1 and CaM. These two compounds showed anti-inflammatory effects on both cell lines. Conclusions: This study indicated for the first time that combining ketoprofen-RGD and Nar can be a promising anti-inflammatory therapeutic regimen for treating leukemia and ovarian cancer.

Curcumin potentiates the anti-inflammatory effects of Tehranolide by modulating the STAT3/NF-κB signaling pathway in breast and ovarian cancer cell lines.

BACKGROUND: Studies have demonstrated that natural products, such as curcumin and artemisinin, possess anti-inflammatory effects, which can be beneficial for cancer treatment. Tehranolide, as a novel natural product, has a wide range of biological activities, including anti-cancer effects. However, many properties of Tehranolide, like its anti-inflammatory activity and its combination with curcumin, have not been investigated yet. This investigation examined the anti-inflammatory activity of Tehranolide, either alone or in combination with curcumin, via modulating the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and STAT3 (signal transducer and activator of transcription 3) signaling pathways in MDA-MB-231 and SKOV3, breast and ovarian cancer cell lines. METHODS: ELISA-based methods were employed to measure the pro-inflammatory cytokine levels and the NF-κB activity in lipopolysaccharide (LPS)-induced cells. The real-time PCR experiment and Griess test were performed to evaluate inducible nitric oxide synthase (iNOS) gene expression and nitrite levels, respectively. The STAT3 and NF-κB signaling pathways were investigated by Western blotting analysis. Tehranolide's anti-cancer activity was also assessed in a mouse model of breast cancer using the TUNEL (terminal deoxynucleotidyl transferase nick-end labeling) assay. RESULTS: Tehranolide diminished levels of pro-inflammatory cytokines in cancer cells. Additionally, it suppressed NF-κB DNA binding and STAT3 phosphorylation, reducing iNOS gene expression and nitrite production. Moreover, Western blotting showed that Tehranolide enhanced the inhibitory κB (IκBα) and Bcl-2 (B-cell lymphoma 2)-associated X (BAX) expression, and downregulated the expression of Bcl-2 proteins. Furthermore, the TUNEL assay demonstrated that Tehranolide induced apoptosis in a breast cancer mouse model. Curcumin potentiated all the anti-inflammatory effects of Tehranolide. CONCLUSION: This investigation indicated for the first time that Tehranolide, either alone or in combination with curcumin, exerted its anti-inflammatory effects by suppressing NF-κB and STAT3 signaling pathways in SKOV3 and MDA-MB-231 cells.

A novel combination of metformin and resveratrol alleviates hepatic steatosis by activating autophagy through the cAMP/AMPK/SIRT1 signaling pathway.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent liver disorder that is associated with the accumulation of triglycerides (TG) in hepatocytes. Resveratrol (RSV), as a natural product, and metformin have been reported to have potential lipid-lowering effects for the treatment of NAFLD via autophagy, but the combined effects of both have not yet been studied. The current study aimed to investigate the role of autophagy in the lipid-lowering effects of RSV, alone and in combination with metformin, on the hepatic steatosis model of HepG2 cells and elucidate the mechanism of action. Triglyceride measurement and real-time PCR showed that RSV-metformin reduced lipid accumulation and the expression of lipogenic genes in palmitic acid (PA)-induced HepG2 cells. Additionally, the LDH release assay indicated that this combination protected HepG2 cells against PA-induced cell death through autophagy. The western blotting analysis revealed that RSV-metformin induced autophagy by reducing the expression of p62 and increasing LC3-I and LC3-II proteins. This combination also enhanced cAMP, phosphorylated AMP-activated protein kinase (p-AMPK), and Beclin-1 levels in HepG2 cells. Furthermore, SIRT1 inhibitor treatment inhibited autophagy induced by RSV-metformin, which indicated the autophagy induction is SIRT1-dependent. This study demonstrated for the first time that RSV-metformin reduced hepatic steatosis by triggering autophagy via the cAMP/AMPK/SIRT1 signaling pathway.

STAT3-mediated Apoptotic-enhancing Function of Sclareol Against Breast Cancer Cells and Cell Sensitization to Cyclophosphamide.

Sclareol is an organic compound with potential anti-tumor effects against various cancer types. However, its precise molecular mechanism in the suppression of tumor growth has not been fully elucidated. In the present study, the anti-proliferative and apoptosis-inducing effects of sclareol with cyclophosphamide were investigated in breast cancer cells and the involvement of the JAK/STAT pathway was evaluated. For this purpose, MCF-7 breast cancer cells were cultured and treated with various concentrations of sclareol to determine its IC50. Cell viability was measured by MTT assay and apoptosis was assessed by flow cytometric analysis of annexin V binding. Gene and protein expression were examined by real-time PCR and Western blotting, respectively. The activity of caspase enzymes was also measured. The results showed that sclareol significantly reduced cell viability and triggered cell death and its co-administration with cyclophosphamide enhanced its anti-cancer properties. Additionally, sclareol up-regulated the expression of p53 and BAX and reduced the expression of Bcl-2. Docking studies indicated an interaction between sclareol and STAT3 which was proved by attenuation of STAT3 phosphorylation after treatment of the cells with sclareol. Sclareol was also capable of suppressing the function of IL-6 in modulating the expression of apoptosis-associated genes. Altogether these data suggest the potential of sclareol as an anti-cancer agent and demonstrate that a combination of sclareol with cyclophosphamide might serve as an effective chemotherapeutic approach resulting in improvements in the treatment of breast cancer.