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.

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.

Tehranolid and Artemisinin Effects on Ameliorating Experimental Autoimmune Encephalomyelitis by Modulating Inflammation and Remyelination.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system. Artemisinin (ART) is a natural sesquiterpene lactone with an endoperoxide bond that is well-known for its anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS. Tehranolide (TEH) is a novel compound with structural similarity to ART. In this study, we aimed to investigate the ameliorating effect of TEH on EAE development by targeting proteins and genes involved in this process and compare its effects with ART. Female C57BL/6 mice were immunized with MOG35-55. Twelve days post-immunization, mice were treated with 0.28 mg/kg/day TEH and 2.8 mg/kg/day ART for 18 consecutive days, and the clinical score was measured daily. The levels of pro-inflammatory and anti-inflammatory cytokines were assessed in mice serum and splenocytes by ELISA. We also evaluated the mRNA expression level of cytokines, as well as genes involved in T cell differentiation and myelination in the spinal cord tissue by qRT-PCR. Administration of TEH and ART significantly alleviated EAE signs. A significant reduction in IL-6 and IL-17 secretion and IL-17 and IL-1 gene expression in spinal cord were observed in the TEH-treated group. ART had similar or less significant effects. Moreover, TGF-ß, IL-4, and IL-10 genes were stimulated by ART and TEH in the spinal cord, while the treatments did not affect IFN-γ expression. Both treatments dramatically increased the expression of FOXP3, GATA3, MBP, and AXL. Additionally, the T-bet gene was reduced after TEH administration. The compounds made no changes in RORγt, nestin, Gas6, Tyro3, and Mertk mRNA expression levels in the spinal cord. The study revealed that both TEH and ART can effectively modulate the genes responsible for inflammation and myelination that play a crucial role in EAE. Interestingly, TEH demonstrated a greater potency compared to ART and hence may have the potential to be evaluated in interventions for the management of MS.

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.

Attenuation of Inflammatory Responses in Breast and Ovarian Cancer Cells by a Novel Chalcone Derivative and Its Increased Potency by Curcumin.

Background: Breast and ovarian cancers are two common malignancies in women and a leading cause of death globally. The aim of the present study was to explore the effects of a novel chalcone derivative 1-(4-(methylsulfonyl)phenyl)-3-(phenylthio)-3-(p-tolyl)propane-1-one (MPP) individually or combined with curcumin, a well-known herbal medicine with anticancer properties, as a new combination therapy on inflammatory pathways in breast and ovarian cancer cell lines. Methods: LPS-induced NF-κB DNA-binding activity and the levels of proinflammatory cytokines were measured in the MPP- and MPP-curcumin combination-treated MDA-MB-231 and SKOV3 cells by ELISA-based methods. The expression of COX2, INOS, and MMP9 genes and nitrite levels was also evaluated by real-time qRT-PCR and Griess method, respectively. IκB levels were evaluated by Western blotting. Results: MPP significantly inhibited the DNA-binding activity of NF-κB in each cell line and subsequently suppressed the expression of downstream genes including COX2, MMP9, and INOS. The levels of proinflammatory cytokines, as well as NO, were also decreased in response to MPP. All the effects of MPP were enhanced by the addition of curcumin. MPP, especially when combined with curcumin, caused a remarkable increase in the concentration of IκB. Conclusion: MPP and its coadministration with curcumin effectively reduced the activity of the NF-κB signaling pathway, leading to a reduced inflammatory response in the environment of cancer cells. Thus, MPP, either alone or combined with curcumin, might be considered an effective remedy for the suppression of inflammatory processes in breast and ovarian cancer cells.

Resveratrol promotes liver cell survival in mice liver-induced ischemia-reperfusion through unfolded protein response: a possible approach in liver transplantation.

BACKGROUND: Ischemia-reperfusion (I/R) of the liver is a multifactorial condition that happens during transplantation and surgery. The deleterious effects of I/R result from the acute production of reactive oxygen species (ROS), which can trigger immediate tissue damage and induce a series of destructive cellular responses, including apoptosis organ failure and inflammation. The production of ROS in the I/R process can damage the antioxidant system and cause liver damage. Resveratrol has been shown to have antioxidant properties in several investigations. Here, we address the therapeutic effect of resveratrol on I/R-induced liver injury by focusing on unfolded protein response (UPR) signaling pathway. METHODS: Five minutes before reperfusion, resveratrol was injected into the tail vein of mice. They were ischemic for 1 h and then re-perfused for 3 h before being slaughtered (I/R). The activity of liver enzymes and the expression levels of genes involved in the unfolded protein response pathway were used to measure the hepatic damage. RESULTS: Our results revealed that the low dose of resveratrol (0.02 and 0.2 mg/kg) post-ischemic treatment significantly reduced the ALT and AST levels. In addition, compared with the control group, the expression of UPR pathway genes GRP78, PERK, IRE1α, CHOP, and XBP1 was significantly reduced in the resveratrol group. In the mice that received lower doses of resveratrol (0.02 and 0.2 mg/kg), the histopathological changes induced by I/R were significantly improved; however, the highest dose (2 mg/kg) of resveratrol could not significantly protect and solve the I/R damage. CONCLUSION: The findings of this study suggest that hepatic ischemia occurs after liver transplantation and that receiving low-dose resveratrol treatment before reperfusion may promote graft survival through inhibition of UPR arms, especially PERK and IRE1α.

Novel and emerging concepts in the role of steroids in thyroid cancer promotion and progression.

Thyroid cancer (TC) is becoming more common all over the world. It is, however, frequently neglected from a scientific standpoint, as they rarely result in a fatal conclusion. The female gender is disproportionately affected, with a frequency of 3 to 5 times higher depending on the severity of the condition, which may or may not cause severe physical discomfort but has a significant influence on life quality. The breakdown of equilibrium between the multiple components that maintain cellular homeostasis may be the cause of the illnesses. On the contrary, excessive or uncontrolled exposure to different hormones, particularly steroids, has been shown to impact the development of thyroid illnesses. The goal of this review is to look at the function of steroids in the expansion and progression of thyroid malignancy (Ref. 54). Keywords: thyroid cancer, signaling, steroid hormone.

Naringenin and cryptotanshinone shift the immune response towards Th1 and modulate T regulatory cells via JAK2/STAT3 pathway in breast cancer.

BACKGROUND: Use of natural products has been proposed as an efficient method in modulation of immune system and treatment of cancers. The aim of this study was to investigate the potential of cryptotanshinone (CPT), naringenin, and their combination in modulating the immune response towards Th1 cells and the involvement of JAK2/STAT3 signaling pathway in these effects. METHODS: Mouse models of delayed type hypersensitivity (DTH) were produced and treated with naringenin and CPT. The proliferation of spleen cells were assessed by Bromodeoxyuridine (BrdU) assay. Flowcytometry and enzyme-linked immunosorbent assay (ELISA) tests were employed to evaluate subpopulation of T-lymphocytes and the levels of cytokines, respectively. The JAK/STAT signaling pathway was analyzed by Western blotting. RESULTS: We showed higher DTH, increased lymphocyte proliferation, decreased tumor growth and reduced JAK2/STAT3 phosphorylation in mice treated with naringenin and CPT. Moreover, a significant decline in the production of IL-4 and an upsurge in the production of IFN-γ by splenocytes were observed. Additionally, the population of intra-tumor CD4+CD25+Foxp3+ T cells was significantly lower in naringenin + CPT treated animals than that in controls. CONCLUSION: Naringenin-CPT combination could exert immunomodulatory effects, suggesting this combination as a novel complementary therapeutic regimen for breast cancer.

Naringenin enhances anti-proliferation effect of 1-ferrocenyl-3-(4-methylsulfonylphenyl) propen-1-one on two different cells via targeting calmodulin signaling pathway.

BACKGROUND: FMSP is a synthesized ferrocene derivative with anti-cancer characteristics on tumor cells. Naringenin is a polyphenolic flavonoid with anti-tumor ability. METHODS: Cell viability and proliferation of two cancer cells and a normal cell line after treatment with these agents were determined with MTT assay. To predict the possible interaction between calmodulin (CaM) and FMSP and naringenin, docking studies were performed. By using fluorescence emission spectra, the effects of FMSP and naringenin on CaM structure and activity were studied. CaM-dependent activation of phosphodiesterase 1 (PDE1) by FMSP in relation to naringenin and their combination were compared. Effects of these compounds on PDE1 inhibition, cAMP accumulation, and cAMP-dependent protein kinase A (PKA) activation were assayed. RESULTS: The combination of FMSP and naringenin had more inhibitory effects on CaM structure than FMSP and naringenin alone. Results of docking analyses also confirmed efficient interaction of the two compounds with a hydrophobic pocket of calmodulin active site. Kinetic analyses of these agents' interaction with CaM showed FMSP and naringenin both competitively inhibited PDE1 activation without changing the Vmax parameter. FMSP and naringenin synergistically increased Km values at a higher level compared to FMSP or naringenin alone. The combination of these two agents also had more cytotoxic effects on cancer cells than FMSP alone. CONCLUSIONS: It was shown that mechanism of proliferation inhibition in both cancer cells by these compounds is based on CaM and consequent PDE inhibition followed by intracellular cAMP level elevation and increased PKA activity in a dose-dependent manner.

Anticancer properties of novel zinc oxide quantum dot nanoparticles against breast cancer stem-like cells.

Cancer stem cells (CSCs) play an essential role in cancer development, metastasis, relapse, and resistance to treatment. In this article, the effects of three synthesized ZnO nanofluids on proliferation, apoptosis, and stemness markers of breast cancer stem-like cells are reported. The antiproliferative and apoptotic properties of ZnO nanoparticles were evaluated on breast cancer stem-like cell-enriched mammospheres by MTS assay and flowcytometry, respectively. The expression of stemness markers, including WNT1, NOTCH1, ß-catenin, CXCR4, SOX2, and ALDH3A1 was assessed by real-time PCR. Western blotting was used to analyze the phosphorylation of Janus kinase 2 (JAK2) and Signal Transducer and Activator of Transcription 3 (STAT3). Markers of stemness were significantly decreased by ZnO nanofluids, especially sample (c) with code ZnO-148 with a different order of addition of polyethylene glycol solution at the end of formulation, which considerably decreased all the markers compared to the controls. All the studied ZnO nanofluids considerably reduced viability and induced apoptosis of spheroidal and parental cells, with ZnO-148 presenting the most effective activity. Using CD95L as a death ligand and ZB4 as an extrinsic apoptotic pathway blocker, it was revealed that none of the nanoparticles induced apoptosis through the extrinsic pathway. Results also showed a marked inhibition of the JAK/STAT pathway by ZnO nanoparticles; confirmed by downregulation of Mcl-1 and Bcl-XL expression. The present data demonstrated that ZnO nanofluids could combat breast CSCs via decreasing stemness markers, stimulating apoptosis, and suppressing JAK/STAT activity.

Effects of Ziziphus Jujuba Extract Alone and Combined with Boswellia Serrata Extract on Monosodium Iodoacetate Model of Osteoarthritis in Mice.

Background: As a chronic joint condition, osteoarthritis (OA) is a common problem among older people. Pain, aching, stiffness, swelling, decreased flexibility, reduced function, and disability are the symptoms of arthritis. Objectives: In this study, we tested the extracts of Ziziphus jujuba (ZJE) and Boswellia serrata (BSE) to reduce OA symptoms as an alternative treatment. Methods: NMRI mice were administered an intra-articular injection of monosodium iodoacetate (MIA; 1 mg/10 mL) in the left knee joint cavity for the induction of OA. Hydroalcoholic extracts of ZJE (250 and 500 mg/kg), BSE (100 and 200 mg/kg), and combined ZJE and BSE were orally administered daily for 21 days. Following behavioral tests, plasma samples were collected to detect inflammatory factors. To screen for general toxicity, acute oral toxicity was evaluated. Results: Oral administration of all the hydroalcoholic extracts significantly increased the locomotor activity, pixel values of the foot-print area, paw withdrawal threshold, the latency of the withdrawal response to heat stimulation, and decreased the difference between pixel values of hind limbs compared to the vehicle group. Also, the elevated levels of IL-1ß, IL-6, and TNF-α were reduced. As tested in this study, ZJE and BSE were practically nontoxic and had a high degree of safety. Conclusions: This study demonstrated that the oral administration of ZJE and BSE slows the progression of OA through anti-nociceptive and anti-inflammatory properties. Oral co-administration of ZJE and BSE extracts can be used as herbal medicine to inhibit OA progression.

Vitamin D Receptor Gene Polymorphisms and the Risk of Metabolic Syndrome (MetS): A Meta-Analysis.

BACKGROUND: Several studies have assessed the association between the vitamin D receptor (VDR) polymorphism and the risk of metabolic syndrome (MetS). However, the results were inconsistent and inconclusive. Therefore, we conducted a meta-analysis to clarify the exact association between the vitamin D receptor (VDR) polymorphisms and the risk of MetS. METHODS: All accessible studies reporting the association between the FokI (rs2228570) or/and TaqI (rs731236) or/and BsmI (rs1544410) or/and ApaI (rs7975232 polymorphisms of the Vitamin D Receptor and susceptibility to MetS published prior to February 2019 were systematically searched in Web of Science, Scopus, and PubMed. After that, Odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were estimated to evaluate the strength of the association in five genetic models. RESULTS: A total of 9 articles based on four gene variations, and comprising 3348 participants with 1779 metabolic syndrome patients were included. The overall results suggested a significant association between BsmI (rs1544410) polymorphism and MetS susceptibility in recessive model (OR, 0.72, 95% CI, 0.55-0.95, fixed effect model), allelic model (OR, 0.83, 95% CI, 0.72-0.95, fixed effect model), and bb vs BB (OR, 0.65, 95% CI, 0.46-0.93, fixed effect). However, no significant association was identified between TaqI (rs731236) polymorphism, ApaI (rs7975232) polymorphism, and FokI (rs2228570) polymorphism and MetS. CONCLUSION: This meta-analysis suggested an association between the BsmI (rs1544410) polymorphism and MetS. Indeed, BsmI (rs1544410) acts as a protective factor in the MetS. As a result, the VDR gene could be regarded as a promising pharmacological and physiological target in the prevention or treatment of the MetS.

Anti-Breast Cancer Activities of Ketoprofen-RGD Conjugate by Targeting Breast Cancer Stem-Like Cells and Parental Cells.

BACKGROUND: Cancer Stem Cells (CSCs) play an important role in various stages of cancer development, advancement, and therapy resistance. Ketoprofen-RGD has been revealed to act as an anti-cancer agent against some tumors. OBJECTIVE: We aimed to explore the effects of a novel Ketoprofen-RGD compound on the suppression of Breast Cancer Stem-like Cells (BCSCs) and their parental cells. METHODS: Mammospheres were developed from MCF-7 cells and assessed by CSC surface markers through flowcytometry. The anti-proliferative and pro-apoptotic activities of Ketoprofen-RGD were measured by MTS assay and flowcytometry. The expression levels of stemness markers and JAK2/STAT proteins were measured by quantitative Real Time-PCR (qRT-PCR) and western blotting, respectively. Intracellular Reactive Oxygen Species (ROS) was measured using a cell permeable, oxidant-sensitive fluorescence probe (carboxy-H2DCFDA). RESULTS: Ketoprofen-RGD significantly reduced the mammosphere formation rate and the expression of three out of six stemness markers and remarkably decreased viability and induced apoptosis of spheroidal and parental cells compared to controls. Further experiments using CD95L, as a death ligand, and ZB4 antibody, as an extrinsic apoptotic pathway blocker, showed that Ketoprofen-RGD induced intrinsic pathway, suggesting a mechanism by which Ketoprofen-RGD triggers apoptosis. ROS production was also another way to induce apoptosis. Results of western blot analysis also revealed a marked diminish in the phosphorylation of JAK2 and STAT proteins. CONCLUSION: Our study, for the first time, elucidated an anti-BCSC activity for Ketoprofen-RGD via declining stemness markers, inducing toxicity, and apoptosis in these cells and parental cells. These findings may suggest this compound as a promising anti-breast cancer.

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.

A Ferrocene Derivative Reduces Cisplatin Resistance in Breast Cancer Cells through Suppression of MDR-1 Expression and Modulation of JAK2/STAT3 Signaling Pathway.

BACKGROUND: Breast cancer is the most common kind of cancer among women in the world. Despite major cancer therapy successes in recent years, cancer cells usually develop mechanisms to survive chemotherapy- induced cell death. Therefore, new strategies are needed to reverse cancer chemoresistance. OBJECTIVE: The aim of this study was to investigate the effect of a recently-synthesized ferrocene derivative named 1-ferrocenyl-3-(4-methylsulfonylphenyl)propen-1-one (FMSP) on cisplatin resistance in MCF-7 cells, focusing on its inhibitory effects on Multi-Drug Resistance-1 (MDR-1) and inflammatory-related STAT3 pathway. METHODS: Cisplatin-resistant MCF-7 cells were developed and the effect of cisplatin and FMSP on cell viability was examined by MTT assay. RT-PCR and Western blotting analyses were performed to assess the gene and protein expression of MDR-1 as well as phosphorylation of JAK2 and STAT3. RESULTS: Overexpression of MDR1 as well as a marked increase in the level of phosphorylated STAT3 was observed in cisplatin-resistant MCF-7 (MCF-7R) cells. FMSP successfully reduced the MCF-7R cell viability and reversed both MDR1 expression and STAT3 phosphorylation status through which sensitivity of MCF-7R cells to cisplatin treatment was regained. CONCLUSION: Our results indicated that FMSP may be considered as a promising therapeutic agent for the prevention and management of chemoresistance in breast cancer cells.

Anti-Breast Cancer Activities of 8-Hydroxydaidzein by Targeting Breast Cancer Stem-Like Cells.

PURPOSE: Cancer stem cells (CSCs) play an important role in various stages of cancer development and therapy refractoriness. 8-Hydroxydaidzein (8-OHD) has revealed anti-cancer activity in different tumors. Accordingly, we aimed to assess the effects of 8-OHD on the suppression of breast cancer stem-like cells (BCSCs). METHODS: The anti-proliferative and pro-apoptotic properties of 8-OHD were examined by MTS assay and flowcytometry. The expression levels of stemness markers and JAK2/STAT proteins were measured by quantitative real time-PCR (qRT-PCR) and western blotting, respectively. RESULTS: 8-OHD significantly decreased three out of six stemness markers and remarkably reduced viability and induced apoptosis of spheroidal and parental cells compared to controls. Further experiments using CD95L, as a death ligand, and ZB4 antibody, as an extrinsic apoptotic pathway blocker, showed that 8-OHD induced apoptosis through the intrinsic pathway, proposing a mechanism by which 8-OHD triggers apoptosis. Results of western blot analysis also revealed a marked decline in the phosphorylation of JAK2 and STAT proteins. CONCLUSION: Our study, for the first time, elucidated an anti-BCSC activity for 8-OHD via decreasing stemness markers, inducing toxicity and stimulating apoptosis in these cells and parental cells. Our results also suggested a novel mechanism by which 8-OHD induces apoptosis in BCSCs.

The Effect of a Newly Synthesized Ferrocene Derivative against MCF-7 Breast Cancer Cells and Spheroid Stem Cells through ROS Production and Inhibition of JAK2/STAT3 Signaling Pathway.

BACKGROUND: Breast Cancer Stem Cells (BCSCs) possess the ability of self-renewal and cellular heterogeneity, and therefore, play a key role in the initiation, propagation and clinical outcome of breast cancer. It has been shown that ferrocene complexes have remarkable potential as anticancer drugs. OBJECTIVE: The present study was conducted to investigate the effects of a novel ferrocene complex, 1- ferrocenyl-3-(4-methylsulfonylphenyl)propen-1-one (FMSP) on MCF-7 breast cancer cell line and its derived mammospheres with cancer stem cell properties. METHODS: Mammospheres were developed from MCF-7 cells and validated by the evaluation of CD44 and CD24 cell surface markers by flow cytometry as well as of the expression of genes that are associated with stem cell properties by real-time PCR. Cells viability was assessed by a soluble tetrazolium salt (MTS) after the treatment of cells with various concentrations of FMSP. Apoptosis was evaluated by flow cytometry analysis of annexin V and PI labeling of cells. Reactive Oxygen Species (ROS) production was measured using a cellpermeable, oxidant-sensitive fluorescence probe (carboxy-H2DCFDA). The involvement of the JAK2/STAT3 pathway was also investigated by western blotting. RESULTS: FMSP could successfully prevent mammosphere formation from differentiated MCF-7 cells and significantly down-regulated the expression of genes involved in the production of the stem cell properties including Wnt1, Notch1, ß -catenin, SOX2, CXCR4 and ALDH1A1. FMSP decreased cell viability in both MCF-7 cells and spheroid cells, although MCF-10A cells were unaffected by this compound. Apoptosis was also dramatically induced by FMSP, via ROS production but independent of CD95 activation. Phosphorylation levels of JAK2 and STAT3 were also found to be significantly attenuated even in the presence of IL-6, the putative activator of the JAK/STAT pathway. CONCLUSION: FMSP can effectively target BCSCs via ROS production and modulation of major signaling pathways that contribute to the stemness of breast cancer cells, and therefore, might be considered a promising anticancer agent after in vivo studies.