"Golgi-customized Trojan horse" nanodiamonds impair GLUT1 plasma membrane localization and inhibit tumor glycolysis.

Nutrient or energy deprivation, especially glucose restriction, is a promising anticancer therapeutic approach. However, establishing a precise and potent deprivation strategy remains a formidable task. The Golgi morphology is crucial in maintaining the function of transport proteins (such as GLUT1) driving glycolysis. Thus, in this study, we present a "Golgi-customized Trojan horse" based on tellurium loaded with apigenin (4',5,7-trihydroxyflavone) and human serum albumin, which was able to induce GLUT1 plasma membrane localization disturbance via Golgi dispersal leading to the inhibition of tumor glycolysis. Diamond-shaped delivery system can efficiently penetrate into cells as a gift like Trojan horse, which decomposes into tellurite induced by intrinsically high H2O2 and GSH levels. Consequently, tellurite acts as released warriors causing up to 3.8-fold increase in Golgi apparatus area due to the down-regulation of GOLPH3. Further, this affects GLUT1 membrane localization and glucose transport disturbance. Simultaneously, apigenin hinders ongoing glycolysis and causes significant decrease in ATP level. Collectively, our "Golgi-customized Trojan horse" demonstrates a potent antitumor activity because of its capability to deprive energy resources of cancer cells. This study not only expands the applications of tellurium-based nanomaterials in the biomedicine but also provides insights into glycolysis restriction for anticancer therapy.

The CDK1/TFCP2L1/ID2 cascade offers a novel combination therapy strategy in a preclinical model of bladder cancer.

Aberrant activation of embryogenesis-related molecular programs in urothelial bladder cancer (BC) is associated with stemness features related to oncogenic dedifferentiation and tumor metastasis. Recently, we reported that overexpression of transcription factor CP2-like protein-1 (TFCP2L1) and its phosphorylation at Thr177 by cyclin-dependent kinase-1 (CDK1) play key roles in regulating bladder carcinogenesis. However, the clinical relevance and therapeutic potential of this novel CDK1-TFCP2L1 molecular network remain elusive. Here, we demonstrated that inhibitor of DNA binding-2 (ID2) functions as a crucial mediator by acting as a direct repressive target of TFCP2L1 to modulate the stemness features and survival of BC cells. Low ID2 and high CDK1 expression were significantly associated with unfavorable clinical characteristics. TFCP2L1 downregulated ID2 by directly binding to its promoter region. Consistent with these findings, ectopic expression of ID2 or treatment with apigenin, a chemical activator of ID2, triggered apoptosis and impaired the proliferation, suppressed the stemness features, and reduced the invasive capacity of BC cells. Combination treatment with the specific CDK1 inhibitor RO-3306 and apigenin significantly suppressed tumor growth in an orthotopic BC xenograft animal model. This study demonstrates the biological role and clinical utility of ID2 as a direct target of the CDK1-TFCP2L1 pathway for modulating the stemness features of BC cells.

Screening of bioactive ingredients of Tsantan Sumtang in ameliorating H9c2 cells injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Tsantan Sumtang (TS), a traditional Tibetan medicine, has been used in the clinic for the treatment of myocardial ischemia (MI) for ages, however, the bioactive ingredients that are responsible for improving MI remain unknown. AIM OF THE STUDY: This study investigated the chemical components of TS and their medicinal efficacies at cell levels, in order to expound the bioactive ingredients in TS. MATERIALS AND METHODS: First, a response-surface methodology was employed to determine the optimum ethanol reflux extraction process of polyphenols in TS (PTS) due to their close correlation with MI improvement. Second, a serum pharmacochemistry technique was used to analyze the compounds of PTS absorbed into the blood of rats. Third, hypoxia-, H2O2-, and adriamycin (ADM)-induced H9c2 cell injury models were used to investigate the cardioprotective effects of these compounds in vitro. Fourth, protective effects of isovitexin, quercitrin, and isoeugenol on mitochondrial function were further tested. RESULTS: The optimum extraction conditions for obtaining PTS were an ethanol concentration of 78.22%, an extraction time of 67.4 min, and a material-liquid ratio of 1:72.60 mL/g. Serum pharmacochemistry analysis detected 21 compounds, of which 11 compounds were always present in the blood within 5 h. Cytotoxicity and the protective effect of 11 compounds in hypoxia-, H2O2-, and ADM-induced H9c2 cell injury models shown that isovitexin, quercitrin, and isoeugenol had almost no cytotoxicity, and they could elevate the survival rate in injured H9c2 cells. Furthermore, isovitexin, quercitrin, and isoeugenol could decrease mitochondrial reactive oxygen species (ROS) releasion, inhibite mitochondrial permeability transition pore (mPTP) opening, ameliorate the change of mitochondrial membrane potential (MMP) to exert mitochondrial protection effect. CONCLUSION: Isovitexin, quercitrin, and isoeugenol exhibited cardioprotective effect at cell levles, these three compounds might be the bioactive ingredients in TS. These findings elucidate the pharmacodynamic substances and mechanisms of TS, guiding its clinical use.

The Zebrafish Embryo as a Model to Test Protective Effects of Food Antioxidant Compounds.

The antioxidant activity of food compounds is one of the properties generating the most interest, due to its health benefits and correlation with the prevention of chronic disease. This activity is usually measured using in vitro assays, which cannot predict in vivo effects or mechanisms of action. The objective of this study was to evaluate the in vivo protective effects of six phenolic compounds (naringenin, apigenin, rutin, oleuropein, chlorogenic acid, and curcumin) and three carotenoids (lycopene B, ß-carotene, and astaxanthin) naturally present in foods using a zebrafish embryo model. The zebrafish embryo was pretreated with each of the nine antioxidant compounds and then exposed to tert-butyl hydroperoxide (tBOOH), a known inducer of oxidative stress in zebrafish. Significant differences were determined by comparing the concentration-response of the tBOOH induced lethality and dysmorphogenesis against the pretreated embryos with the antioxidant compounds. A protective effect of each compound, except ß-carotene, against oxidative-stress-induced lethality was found. Furthermore, apigenin, rutin, and curcumin also showed protective effects against dysmorphogenesis. On the other hand, ß-carotene exhibited increased lethality and dysmorphogenesis compared to the tBOOH treatment alone.

Protective mechanism of Erigeron breviscapus injection on blood-brain barrier injury induced by cerebral ischemia in rats.

This study investigates the protective effect of Erigeron breviscapus injection, a classic traditional Chinese medicine most typically used by Chinese minority to treat stroke, on cerebral ischemia-reperfusion injury and the related signaling pathways. Use network pharmacology methods to study the relationship between E. breviscapus (Vant.) Hand-Mazz. and ischemic stroke, predict the mechanism and active ingredients of E. breviscapus (Vant.) Hand-Mazz. in improving ischemic stroke disease. We study the protective effect of E. breviscapus injection on blood-brain barrier (BBB) injuries induced by cerebral ischemia in rats by regulating the ROS/RNS-MMPs-TJs signaling pathway. The rat model of focal cerebral ischemia-reperfusion injury has been prepared using the wire-suppository method. Firstly, the efficacy of E. breviscapus injection, Scutellarin and 3,5-dicaffeoylquinic acid in protecting BBB injury caused by cerebral ischemia has been evaluated. Secondly, the following two methods have been used to study the mechanism of E. breviscapus injection in regulating the ROS/RNS-MMPS-TJS signaling pathway: real-time PCR and western blot for the determination of iNOS, MMP-9, claudin-5, occludin, ZO-1 mRNA and protein expression in brain tissue. We find that PI3K-Akt signaling pathway predicted by network pharmaology affects the blood-brain barrier function, so we chose the blood-brain barrier-related MMP-9, claudin-5, iNOS, occludin and ZO-1 proteins are used for research. The results of our research show that 3 drugs can reduce the rate of cerebral infarction in rats, relieve the abnormal neuroethology of rats, reduce the degree of brain tissue lesion, increase the number of the Nissl corpuscle cells and repair the neuron ultrastructure in injured rats. At the same time, it can obviously reduce the ultrastructure damage of the BBB in rats. All three drugs significantly reduced the content of Evans blue in the ischemic brain tissue caused by cerebral ischemia in rats with BBB injury. In addition, E. breviscapus injection, Scutellarin and 3,5-dicaffeoylquinic acid can decrease the protein expression of iNOS and MMP-9 in rat ischemic brain tissue. In addition, 3,5-dicaffeoylquinic acid can increase the protein expression of claudin-5. We conclude that E. breviscapus injection, Scutellarin and 3,5-dicaffeoylquinic acid have obvious therapeutic effects on BBB and neuron injury induced by cerebral ischemia in rats. Our results from studying the mechanism of action show that E. breviscapus injection and Scutellarin inhibited the activation of MMP-9 by inhibiting the synthesis of iNOS, 3,5-dicaffeoylquinic acid inhibits the expression and activation of MMP-9 by inhibiting the activation of iNOS and reducing the generation of free radicals, thus reducing the degradation of important cytoskeleton connexin claudin-5 in the tight junction (TJ) structure by inhibiting the expression and activation of MMP-9. Finally BBB structure integrity was protected.

Prophylactic effect of myricetin and apigenin against lipopolysaccharide-induced acute liver injury.

BACKGROUND: Liver has an important role in the initiation and progression of multiple organ failure that occurs in sepsis. Many natural active substances can be used to reduce the liver injury caused by sepsis. For this aim, the effects of myricetin and apigenin on mice model of acute liver injury was evaluated in this study. METHODS AND RESULTS: Thirty-six mice were randomly divided into six groups as; control, lipopolysaccharide (LPS) (5 mg/kg), LPS + myricetin (100 mg/kg), LPS + myricetin (200 mg/kg), LPS + apigenin (100 mg/kg), and LPS + apigenin (200 mg/kg) groups. Myricetin and apigenin were administered orally for 7 days, and LPS was administered intraperitoneally only on the 7th day of the study. 24 h after LPS application, all animals were sacrificed and serum biochemical parameters, histopathology and oxidative stress and inflammation markers of liver tissue were examined. Myricetin and apigenin pre-treatments increased serum albumin and total protein levels, liver GSH level and catalase and SOD activities and decreased serum ALT, AST, ALP, γ-GT, CRP, total and direct bilirubin levels, liver MPO activity, MDA, NOx, PGE2, TNF-α, IL-1ß, and IL-6 levels, iNOS and COX-2 mRNA levels, phosphorylation of NF-κB p65, IκB, and IKK proteins but not p38, ERK, and JNK proteins in LPS-treated mice. Myricetin and apigenin administration also regained the hepatic architecture disrupted during LPS application. CONCLUSION: Myricetin and apigenin pre-treatments led to reduction of liver injury indices and oxidative stress and inflammatory events and these flavonoids has probably hepatoprotective effects in acute liver injury.

Apigenin ameliorates hyperuricemic nephropathy by inhibiting URAT1 and GLUT9 and relieving renal fibrosis via the Wnt/ß-catenin pathway.

BACKGROUND: Hyperuricemia (HUA) is characterized by abnormal serum uric acid (UA) levels and demonstrated to be involved in renal injury leading to hyperuricemic nephropathy (HN). Apigenin (API), a flavonoid naturally present in tea, berries, fruits, and vegetables, exhibits various biological functions, such as antioxidant and anti-inflammatory activity. PURPOSE: To investigate the effect of API treatment in HN and to reveal its underlying mechanisms. METHODS: The mice with HN were induced by potassium oxonate intraperitoneally and orally administered for two weeks. The effects of API on renal function, inflammation, fibrosis, and uric acid (UA) metabolism in mice with HN were evaluated. The effects of API on urate transporters were further examined in vitro. RESULTS: The mice with HN exhibited abnormal renal urate excretion and renal dysfunction accompanied by increased renal inflammation and fibrosis. In contrast, API reduced the levels of serum UA, serum creatinine (CRE), blood urea nitrogen (BUN) and renal inflammatory factors in mice with HN. Besides, API ameliorated the renal fibrosis via Wnt/ß-catenin pathway suppression. Furthermore, API potently promoted urinary UA excretion and inhibited renal urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) in mice with HN. In vitro, API competitively inhibited URAT1 and GLUT9 in a dose-dependent manner, with IC50 values of 0.64 ± 0.14 µM and 2.63 ± 0.69 µM, respectively. CONCLUSIONS: API could effectively attenuate HN through co-inhibiting UA reabsorption and Wnt/ß-catenin pathway, and thus it might be a potential therapy to HN.

Apigenin-Loaded PLGA-DMSA Nanoparticles: A Novel Strategy to Treat Melanoma Lung Metastasis.

The flavone apigenin (APG), alone as well as in combination with other chemotherapeutic agents, is known to exhibit potential anticancer effects in various tumors and inhibit growth and metastasis of melanoma. However, the potential of apigenin nanoparticles (APG-NPs) to prevent lung colonization of malignant melanoma has not been well investigated. APG-loaded PLGA-NPs were surface-functionalized with meso-2,3-dimercaptosuccinic acid (DMSA) for the treatment of melanoma lung metastasis. DMSA-conjugated APG-loaded NPs (DMSA-APG-NPs) administered by an oral route exhibited sustained APG release and showed considerable enhancement of plasma half-life, Cmax value, and bioavailability compared to APG-NPs both in plasma and the lungs. DMSA-conjugated APG-NPs showed comparably higher cellular internalization in B16F10 and A549 cell lines compared to that of plain NPs. Increased cytotoxicity was observed for DMSA-APG-NPs compared to APG-NPs in A549 cells. This difference between the two formulations was lower in B16F10 cells. Significant depolarization of mitochondrial transmembrane potential and an enhanced level of caspase activity were observed in B16F10 cells treated with DMSA-APG-NPs compared to APG-NPs as well. Western blot analysis of various proteins was performed to understand the mechanism of apoptosis as well as prevention of melanoma cell migration and invasion. DMSA conjugation substantially increased accumulation of DMSA-APG-NPs given by an intravenous route in the lungs compared to APG-NPs at 6 and 8 h. This was also corroborated by scintigraphic imaging studies with radiolabeled formulations administered by an intravenous route. Conjugation also allowed comparatively higher penetration as evident from an in vitro three-dimensional tumor spheroid model study. Finally, the potential therapeutic efficacy of the formulation was established in experimental B16F10 lung metastases, which suggested an improved bioavailability with enhanced antitumor and antimetastasis efficacy of DMSA-conjugated APG-NPs following oral administration.

Cytotoxicity of apigenin toward multiple myeloma cell lines and suppression of iNOS and COX-2 expression in STAT1-transfected HEK293 cells.

BACKGROUND: Apigenin is one of the most abundant dietary flavonoids that possesses multiple bio-functions. PURPOSE: This study was designed to determine the influence of apigenin on gene expressions, cancer cells, as well as STAT1/COX-2/iNOS pathway mediated inflammation and tumorigenesis in HEK293-STAT1 cells. Furthermore, the cytotoxic activity toward multiple myeloma (MM) cell lines was investigated. METHODS: Bioinformatic analyses were used to predict the sensitivity and resistance of tumor cells toward apigenin and to determine cellular pathways influenced by this compound. The cytotoxic and ferroptotic activity of apigenin was examined by the resazurin reduction assay. Additionally, we evaluated apoptosis, and cell cycle distribution, induction of reactive oxygen species (ROS) and loss of integrity of mitochondrial membrane (MMP) by using the flow cytometry analysis. DAPI staining was used to detect characteristic apoptotic features. Furthermore, we verified its anti-inflammatory and additional mechanism of cell death by western blotting. RESULTS: COMPARE and hierarchical cluster analyses exhibited that 29 of 55 tumor cell lines were sensitive against apigenin (p < 0.001). The Ingenuity Pathway Analysis data showed that important bio-functions affected by apigenin were: gene expression, cancer, hematological system development and function, inflammatory response, and cell cycle. The STAT1 transcription factor was chosen as target protein on the basis of gene promoter binding motif analyses. Apigenin blocked cell proliferation of wild-type HEK293 and STAT1 reporter cells (HEK293-STAT1), promoted STAT1 suppression and subsequent COX-2 and iNOS inhibition. Apigenin also exhibited synergistic activity in combination with doxorubicin toward HEK293-STAT1 cells. Apigenin exerted excellent growth-inhibitory activity against MM cells in a concentration-dependent manner with the greatest activity toward NCI-H929 (IC50 value: 10.73 ± 3.21 µM). Apigenin induced apoptosis, cell cycle arrest, ferroptosis and autophagy in NCI-H929 cells. CONCLUSION: Apigenin may be a suitable candidate for MM treatment. The inhibition of the STAT1/COX-2/iNOS signaling pathway by apigenin is an important mechanism not only in the suppression of inflammation but also in induction of apoptosis.

Cyclodextrin pendant polymer as an efficient drug carrier for scutellarin.

A novel ß-cyclodextrin pendant polymer (ε-PL-CD), composed of poly(ε-lysine) (ε-PL) main chain and glycine-ß-cyclodextrin (Gly-CD) side chains, was prepared by a simple two-step procedure. The ε-PL-CD was investigated as a drug carrier of hydrophobic drug scutellarin (SCU). The characterization and complexation mode of the SCU:ε-PL-CD were researched in both solution and solid state by means of photoluminescence spectroscopy, 1H and 2D NMR, X-Ray powder diffraction (XRPD), thermal gravimetric analysis, Particle size and Zeta potential. The solubility test indicated that the solubilizing ability of SCU:ε-PL-CD was significantly improved compared with SCU:ß-CD and free SCU. Besides, in vitro cell experiment, it was found that SCU:ε-PL-CD has a strong inhibitory effect on the growth and invasion of tumor cells. The present study provides useful information for ε-PL-CD as a drug carrier material.

Apigenin-Loaded Solid Lipid Nanoparticle Attenuates Diabetic Nephropathy Induced by Streptozotocin Nicotinamide Through Nrf2/HO-1/NF-kB Signalling Pathway.

BACKGROUND: Apigenin is known to have a broad-spectrum efficacy in oxidative stress and conditions due to inflammation, although weak absorption, fast metabolic rate and a fast elimination (systemic) limit the pharmacological efficacy of this drug. Hence, we propose the usage of highly bioavailable Apigenin-solid lipid nanoparticles (SLNPs) to recognize such limitations. The defensive function of Apigenin-SLNPs on renal damage induced by streptozotocin (STZ) in animals was studied. MATERIALS AND METHODS: We initially injected the rats with 35 mg kg-1 streptozocin intraperitoneally, and after 7 days, the rats were then injected 150 mg kg-1 of metformin intragastrically followed by a once-daily intragastric dose of Apigenin-SLNP (25 or 50 mg kg-1) for a continuous period of 30 days. We then measured the level of insulin and blood glucose, superoxide dismutase, catalase and malondialdehyde in the tissues of the kidney. We also observed messenger-RNA expression of Interleukin-1ß, Interleukin-6 and Tumor Necrosis Factor-alpha in renal tissue through RT-PCR technique. Moreover, H&E staining and Western blotting observed the histopathological variations and protein expression of nuclear factor erythroid 2-related factor 2/heme oxygenase/Nuclear Factor-κB signaling pathway, respectively. RESULTS: An enhancement in the expressing of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 and a suppression in the expression of Nuclear Factor-κB occurred due to Apigenin-SLNPs treatment, which was a result of the protective mechanism of Apigenin-SLNPs which is because of not only its anti-inflammatory function (by inhibition of release of inflammatory factors) but also their anti-oxidant activity (through reduction of lipid peroxidation production). CONCLUSION: We found that a protective effect on diabetic nephropathy was shown due to Apigenin-SLNPs, in rats induced with streptozocin maybe through the pathway of nuclear factor erythroid 2-related factor 2/heme oxygenase-1/Nuclear Factor-κB.

Apigenin by targeting hnRNPA2 sensitizes triple-negative breast cancer spheroids to doxorubicin-induced apoptosis and regulates expression of ABCC4 and ABCG2 drug efflux transporters.

Acquired resistance to doxorubicin is a major hurdle in triple-negative breast cancer (TNBC) therapy, emphasizing the need to identify improved strategies. Apigenin and other structurally related dietary flavones are emerging as potential chemo-sensitizers, but their effect on three-dimensional TNBC spheroid models has not been investigated. We previously showed that apigenin associates with heterogeneous ribonuclear protein A2/B1 (hnRNPA2), an RNA-binding protein involved in mRNA and co-transcriptional regulation. However, the role of hnRNPA2 in apigenin chemo-sensitizing activity has not been investigated. Here, we show that apigenin induced apoptosis in TNBC spheroids more effectively than apigenin-glycoside, owing to higher cellular uptake. Moreover, apigenin inhibited the growth of TNBC patient-derived organoids at an in vivo achievable concentration. Apigenin sensitized spheroids to doxorubicin-induced DNA damage, triggering caspase-9-mediated intrinsic apoptotic pathway and caspase-3 activity. Silencing of hnRNPA2 decreased apigenin-induced sensitization to doxorubicin in spheroids by diminishing apoptosis and partly abrogated apigenin-mediated reduction of ABCC4 and ABCG2 efflux transporters. Together these findings provide novel insights into the critical role of hnRNPA2 in mediating apigenin-induced sensitization of TNBC spheroids to doxorubicin by increasing the expression of efflux transporters and apoptosis, underscoring the relevance of using dietary compounds as a chemotherapeutic adjuvant.

Flavonoids potentiated anticancer activity of cisplatin in non-small cell lung cancer cells in vitro by inhibiting histone deacetylases.

AIMS: Cisplatin is the mainstay of first-line treatment for advanced non-small cell lung cancer (NSCLC). Accumulating evidence suggests that flavonoids inhibit histone deacetylase (HDAC) to mediate their anticancer effect in various cancer types. The study was conducted to investigate the inhibition of HDAC and the modulation of apoptotic and cell cycle regulatory genes by selected flavonoids to potentiate the anticancer effect of cisplatin. MAIN METHODS: Combinations of cisplatin and selected flavonoids were investigated in three NSCLC cell lines (A549, H460, and H1299). Sulforhodamine B assay was used to evaluate cytotoxicity of drug combinations. Western blot analysis was conducted to evaluate histone acetylation. Flow cytometric assays were used to investigate the apoptotic and cell cycle effect. Chromatin immunoprecipitation assay was performed to elucidate the binding of transcription factors to promoters of selected apoptotic and cell cycle regulatory genes. KEY FINDINGS: Apigenin was found to exhibit the strongest HDAC inhibitory effect among all flavonoids tested. Cisplatin-apigenin combination was shown to produce significantly more S phase prolongation and G2/M cell cycle arrest, and apoptosis compared with cisplatin or apigenin alone, by inducing p21 and PUMA, respectively. More pronounced effect was observed in p53-proficient than p53-null NSCLC cells. Mechanistically, apigenin was found to reduce the binding of HDAC1 but increase the association of RNA polymerase II and Sp1 to p21 and PUMA promoters. SIGNIFICANCE: Our findings provide a better insight about the mechanism contributing to the HDAC inhibitory effect of apigenin to potentiate anticancer effect of cisplatin by inducing apoptosis and cell cycle arrest.

Overcoming the hypoxia-induced drug resistance in liver tumor by the concurrent use of apigenin and paclitaxel.

The chemotherapeutic efficacy of paclitaxel against hypoxic tumors is usually unsatisfactory, which is partially due to the so-called hypoxia-induced drug resistance. The mechanism of hypoxia-induced resistance is primarily associated with hypoxia-inducible factor 1α (HIF-1α), which is an oxygen-sensitive transcriptional activator coordinating the cellular response to hypoxia. Apigenin is a natural occurring HIF-1α inhibitor that can suppress the expression of HIF-1α through multiple pathways and reverse the hypoxia-induced resistance found in cancer cells. Here we report that the use of apigenin can suppress the HIF-1α expression in hypoxic tumors through the simultaneous inhibition of the AKT/p-AKT pathway and HSP90, which is beneficial for enhancing the anticancer activity of the co-administered paclitaxel. The potential synergistic effect of apigenin and paclitaxel was further validated on HepG2 cell line and tumor-bearing mouse models.

Investigation of possible effects of apigenin, sorafenib and combined applications on apoptosis and cell cycle in hepatocellular cancer cells.

Hepatocellular carcinoma (HCC) is the most common type of liver tumors. There is only one chemodrug for treatment called sorafenib that is an effective multikinase inhibitor. However, most of the patients gain resistance to sorafenib treatment in six months. Thus, there is a limitation for treatment of HCC. Apigenin is a natural flavonoid that has been used for many years as an antioxidant and anti-inflammatory agent. The aim of this study is to investigate the combined therapeutic effects of sorafenib and apigenin upon apoptosis and cell cycle on HepG2 cell line. Cytotoxic effects of sorafenib and apigenin on HepG2 cells were determined by XTT assay. Effects of single and combined treatment on cell migration, invasion and colony formation were analysed by wound healing, transwell matrigel invasion assay and colony formation assay, respectively. TUNEL assay was performed for analyse apoptosis rates. Expression changes of genes related with apoptosis and cell cycle were analysed by quantitative real-time PCR. Combined treatment of sorafenib and apigenin has more decreasing effects on cell viability than single treatment groups. Also, combination group caused significant increase of apoptotic cells. Migration and invasion capability of cells in combined treatment group are decreased. Lastly, quantitative real-time PCR results showed that combination of both drugs arrested cell cycle and increased apoptotic gene expressions more than single treatment groups. This is the first study that investigating the combined treatment of sorafenib and apigenin on HCC in vitro. By combined treatment, apigenin potentiates sorafenib cytotoxicity on HepG2 cells. Effects of combined treatment on migration, invasion, apoptosis and gene expressions showed that may sorafenib and apigenin have synergistic effect.

Vitexin ameliorates preeclampsia phenotypes by inhibiting TFPI-2 and HIF-1α/VEGF in a l-NAME induced rat model.

Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality with few safe, effective, and minimally invasive therapeutics. Inflammation, oxidative stress, and angiogenic imbalance have been reported to contribute to PE pathogenesis. Vitexin (VI) possesses various pharmacological activities including the potent regulation of the above biological processes in different conditions. This study aims to investigate whether VI has therapeutic potential to PE and the underlying mechanisms. Sprague-Dawley pregnant rats pretreated with or without VI were fed with l-NAME-containing water to induce experimental PE. Results showed that VI decreased high systolic blood pressure and urinary protein in PE rats time- and dose-dependently. Meanwhile, VI of higher dosage (45, 60 mg/kg) corrected abnormal pregnancy outcomes, including low pup weight and low pups/placenta ratio. In addition, VI of high dosage (60 mg/kg) decreased sFlt-1, increased PlGF and alleviated oxidative stress both in blood and placental samples compared with nontreated PE group. Furthermore, VI alleviated placental TFPI-2, HIF 1α, and VEGF in PE rats. In short, the present study suggests that the inhibition of placental TFPI-2 and HIF-1α/VEGF might be one of the potential mechanisms underlying the protective effects of VI to experimental PE induced by l-NAME.

Isovitexin reduces carcinogenicity and stemness in hepatic carcinoma stem-like cells by modulating MnSOD and FoxM1.

BACKGROUND: Manganese superoxide dismutase (MnSOD) upregulating FoxM1 have previously been demonstrated promoting lung cancer stemness. Isovitexin exhibits antitumor activities in various cancers. This study aimed to assess whether isovitexin inhibits hepatic carcinoma stem-like cells (HCSLCs) features via regulating MnSOD and FoxM1 expression. METHODS: Second-generation spheres from the hepatic carcinoma cell lines, respectively, were used as HCSLCs. Protein amounts of MnSOD, FoxM1 and stemness-associated markers (CD133, CD44, ALDH1, Bmi1, Nanog and Oct4) were determined by immunoblotting. In vitro carcinogenicity was evaluated by sphere- and colony-formation assays. The effects of isovitexin on HCSLC carcinogenicity and stemness were examined in vitro and in xenograft models. An adenoviral delivery system was employed to manipulate MnSOD and/or FoxM1. Luciferase reporter assay was performed to verify isovitexin downregulated FoxM1 by inhibiting MnSOD-mediated effects of E2F1 and/or Sp1 on activation of FoxM1 promoter. RESULTS: FoxM1 upregulation by MnSOD contributed to carcinogenicity and stemness, with increased sphere- and colony-formation capabilities, upregulated stemness-associated markers and CD133+ subpopulation as well as elevated oncogenicity in vivo in HCSLCs compared with hepatic carcinoma cells. Isovitexin substantially decreased sphere and colony formation rates, and stemness-associated markers in cultured HCSLCs by suppressing MnSOD and FoxM1 expression. Importantly, isovitexin significantly inhibited tumor growth of in nude mice bearing HCSLCs and reduced CD133 protein expression of xenograft in nude mice. MnSOD or FoxM1 knockdown enhanced the effects of isovitexin suppression on carcinogenicity and stemness in HCSLC. MnSOD or FoxM1 overexpression attenuated the effects of isovitexin. Additionally, isovitexin and MnSOD knockdown could inhibit FoxM1 reporter activity via a decreased binding of E2F1 and/or Sp1 onto FoxM1 promoter. FoxM1 overexpression reversed the effects of isovitexin combined with MnSOD knockdown, without affecting MnSOD expression. Moreover, MnSOD knockdown plus thiostrepton, a FoxM1 specific inhibitor, cooperated with isovitexin to repress xenograft tumor growth and downregulate MnSOD and FoxM1 in nude mice bearing HCSLCs from MHCC97H cells. CONCLUSIONS: Isovitexin inhibits carcinogenicity and stemness in HCSLCs by downregulating FoxM1via inhibition of MnSOD.

Vitexin ameliorates high fat diet-induced obesity in male C57BL/6J mice via the AMPKα-mediated pathway.

Vitexin, a bioactive compound isolated from hawthorn leaf extracts, has been reported to exhibit many biological activities, such as anticancer, antioxidation, and adipogenesis inhibition activities. The current study explored the effects of vitexin on high fat diet (HFD)-induced obesity/adipogenesis in male C57BL/6J mice and 3T3-L1 adipocytes, as well as the underlying mechanisms thereof. Vitexin significantly mitigated HFD-induced body weight gain and adiposity. Vitexin also partially normalized serum, hepatic lipid contents, and decreased adipocyte size induced by the HFD. Consistently, there were significant effects of vitexin on important regulators of lipid metabolism, including AMP-activated protein kinase-α (AMPKα), CAATT element binding protein-α (C/EBPα), and fatty acid synthase (FAS) in white adipose tissue. Moreover, vitexin significantly inhibited fat accumulation in 3T3-L1 adipocytes, and this was totally abolished by compound C (an AMPKα inhibitor). These results suggest that vitexin may prevent HFD-induced obesity/adipogenesis via the AMPKα mediated pathway.

Scutellarein selectively targets multiple myeloma cells by increasing mitochondrial superoxide production and activating intrinsic apoptosis pathway.

OBJECTIVES: Scutellarein is a flavonoid monomer found in traditional Chinese medicine such as Scutellaria barbata. This study aimed to investigate the cytotoxic effect of scutellarein treatment on multiple myeloma (MM) cells. METHODS: circulating B lymphocytes (CBL) isolated from healthy donors' peripheral blood served as control for MM.1R and IM-9 MM cells. CLB and MM cells were treated with various concentrations of scutellarein before their cell viability and apoptosis being evaluated. Nude mice burdened with MM xenograft tumor were intravenously injected with different concentrations of scutellarein, and their tumor burden change were monitored. Apoptosis of MM cells or CBL after scutellarein treatment was assayed by measuring caspase-3, -8 and -9 activities. FADD or APAF1 gene knockdown in MM cells was achieved by lentiviral transfection. Amount of Cytochrome C in cytosol or mitochondria as well as that of Bax and Bcl-2 protein were evaluated by Western blot. Mitochondria-induced apoptosis was assayed by measuring mitochondrial membrane potential change. Production of general reactive oxygen species and mitochondrial superoxide in MM or CBL was detected after scutellarein treatment, which was reduced by MitoTEMPO or apocynin treatment, respectively. RESULTS: Scutellarein treatment showed potent cytotoxicity on MM cells but not on viable CBL, and intravenous injection of scutellarein significantly reduced MM xenograft tumor burden in nude mice. Scutellarein treatment in MM cells activated the mitochondrial-mediated intrinsic apoptosis pathway by increasing the production of mitochondrial superoxide, which was reduced to ROS by NADPH, but this effect was weakened in healthy CBL. Co-treatment with scutellarein synergized with bortezomib in inducing apoptosis in MM cells in vitro and in reducing tumor volume in MM xenografted nude mice. CONCLUSIONS: Scutellarein induced mitochondrial-mediated intrinsic apoptosis selectively on malignant cells comparing to healthy cells.

Apigenin suppresses PD-L1 expression in melanoma and host dendritic cells to elicit synergistic therapeutic effects.

BACKGROUND: The PD-L1/PD-1 pathway blockade-mediated immune therapy has shown promising efficacy in the treatment of multiple cancers including melanoma. The present study investigated the effects of the flavonoid apigenin on the PD-L1 expression and the tumorigenesis of melanoma. METHODS: The influence of flavonoids on melanoma cell growth and apoptosis was investigated using cell proliferation and flow cytometric analyses. The differential IFN-γ-induced PD-L1 expression and STAT1 activation were examined in curcumin and apigenin-treated melanoma cells using immunoblotting or immunofluorescence assays. The effects of flavonoid treatment on melanoma sensitivity towards T cells were investigated using Jurkat cell killing, cytotoxicity, cell viability, and IL-2 secretion assays. Melanoma xenograft mouse model was used to assess the impact of flavonoids on tumorigenesis in vivo. Human peripheral blood mononuclear cells were used to examine the influence of flavonoids on PD-L1 expression in dendritic cells and cytotoxicity of cocultured cytokine-induced killer cells by cell killing assays. RESULTS: Curcumin and apigenin showed growth-suppressive and pro-apoptotic effects on melanoma cells. The IFN-γ-induced PD-L1 upregulation was significantly inhibited by flavonoids, especially apigenin, with correlated reductions in STAT1 phosphorylation. Apigenin-treated A375 cells exhibited increased sensitivity towards T cell-mediated killing. Apigenin also strongly inhibited A375 melanoma xenograft growth in vivo, with enhanced T cell infiltration into tumor tissues. PD-L1 expression in dendritic cells was reduced by apigenin, which potentiated the cytotoxicity of cocultured cytokine-induced killer cells against melanoma cells. CONCLUSIONS: Apigenin restricted melanoma growth through multiple mechanisms, among which its suppression of PD-L1 expression exerted a dual effect via regulating both tumor and antigen presenting cells. Our findings provide novel insights into the anticancer effects of apigenin and might have potential clinical implications.