The Anticancer Effects of Atractylenolide III Associate With the Downregulation of Jak3/Stat3-Dependent IDO Expression.

Objective: Indoleamin-2,3-dioxygenase-1 (IDO) has been identified as a checkpoint protein involved in generating the immunosuppressive tumor microenvironment that supports tumor growth. It has been reported that atractylenolide III (ATLIII) has anticancer and immune modulatory effects. This study is to determine the anticancer effects of ATLIII with the Jak3/Stat3-dependent IDO inactivation. Methods: We assessed the cytotoxicity of ATLIII and IFN-γ on lung cancer cells by MTT. We determined the efficacy of ATLIII on IFN-γ-induced IDO expression by RT-PCR and Western blot. We also determined the efficacy of ATLIII on Jak3/Stat3 pathway expression induced by IFN-γ and Jak3/Stat3-dependent IDO activation. Further molecular docking assay predicted the binding activity and site of ATLIII to Jak3 protein. Additional immunofluorescence staining was used to measure the Stat3 intracellular localization. Finally, we performed mouse animal experiments to observe changes in the expression of IDO, p-Jak3, p-Stat3, and tryptophan/kynurenine after ATLIII administration. Results: ATLIII showed no cytotoxicity at a wide of dosage range. ATLIII reduced the phosphorylation level of Jak3 and Stat3 in response to IFN-γ stimulation, then remarkably reduced the nuclear translocation of p-Stat3 by IFN-γ. Lastly, ATLIII significantly downregulated the expression level of IDO at a wide dosage range. Molecular docking assay showed that the oxygen atom on the five-membered ring of ATLIII was capable of forming a hydrogen bond with Leu905-NH2 site of Jak3 protein. Further evidence showed that though IFN-γ had normal capacity to trigger Stat3 phosphorylation, nuclear translocation, and promoter luciferase activity, ATLIII failed to trigger efficacy on reducing these changes under forced Jak3-Leu905 mutant expression condition. Finally, we confirmed this view in in vivo experiments. Conclusion: ATLIII has shown significant efficacy to inhibit IFN-γ-triggered Jak3/Stat3 pathway-dependent IDO activation, and do so through a direct binding to Jak3 protein. This study elucidated a new mechanism for the anticancer effect of ATLIII, which may provide a feasible target for the clinical immunotherapy of malignant tumors.

Pleiotropic effects of herbs characterized with blood-activating and stasis-resolving functions on angiogenesis.

Accumulative evidences have underpinned the nature candidates from Chinese medicine (CM), particularly CM served as blood activating and stasis resolving (BASR, Huoxue Huayu in Chinese) by targeting tumor-associated angiogenesis. However, recent experiment research on the therapeutic angiogenesis by BASR-CM attracts wide attention and discussion. This opinion review focused on the underlying link between two indications and anticipated that (1) BASR-CM might emphasize on a balanced multi-cytokines network interaction; (2) BASR-CM might address on the nature of diseases prior to differently affecting physiological and pathological angiogenesis; (3) BASR-CM might mainly act on perivascular cells, either promotes arteriogenesis by increasing arteriogenic factors in ischemic diseases, or simultaneously keep a quiescent vasculature to impede angiogenesis in tumor context.

Effect of aqueous extracts of several kinds of herbs on human platelet aggregation and expression of P-selectin in vitro.

OBJECTIVE: To study the effect of aqueous extract of several kinds of herbs on human platelet aggregation and expression of P-selectin in vitro. METHODS: Blood was collected from volunteers. Effects of the prepared water extracts of herbs on platelet aggregation were monitored on a Packs-4 aggregometer. The fluorescence intensity of water extracts of Caulis Spatholobi, Flos Carthami and Rhizoma Curcumae on the expression of P-selectin in human platelets of healthy persons was measured with flow cytometry. RESULTS: Out of several herbs investigated, Flos Carthami and Rhizoma Curcumae potently inhibited platelet aggregation after incubation with platelet-rich plasma (PRP) for 15 min. Caulis Spatholobi Flos Carthami and Rhizoma Curcumae inhibited adenosine-5'-diphosphate (ADP) or platelet activating factor (PAF)-induced platelet aggregation in PRP in a dose-dependent manner. In contrast to Flos Carthami and Rhizoma Curcumae, Caulis Spatholobi could not inhibit thrombin-induced platelet aggregation. Despite its inability to inhibit thrombin-induced platelet aggregation in PRP, Caulis Spatholobi had a greater anti-aggregating activity in PRP induced by ADP or PAF. Caulis Spatholobi and Flos Carthami showed significant inhibitory effects on the expression of P-selectin. CONCLUSIONS: Caulis Spatholobi, Flos Carthami and Rhizoma Curcumae have potent anti-platelet properties, and their inhibitory actions are mediated via different mechanisms. Caulis Spatholobi inhibited ADP-induced platelet aggregation but not by thrombin, indicating that its mechanism of action might be independent of the thromboxane pathway. The effect of Caulis Spatholobi and Flos Carthami were associated with suppressing the expression of P-selectin.

PKM2 regulates hepatocellular carcinoma cell epithelial-mesenchymal transition and migration upon EGFR activation.

Pyruvate kinase isozyme type M2(PKM2) was first found in hepatocellular carcinoma(HCC), and its expression has been thought to correlate with prognosis. A large number of studies have demonstrated that epithelial-mesenchymal transition (EMT) is a crucial event in hepatocellular carcinoma (HCC) and associated metastasis, resulting in enhanced malignancy of HCC. However, the roles of PKM2 in HCC EMT and metastasis remain largely unknown. The present study aimed to determine the effects of PKM2 in EGF-induced HCC EMT and elucidate the molecular mechanisms in vitro. Our results showed that EGF promoted EMT in HCC cell lines as evidenced by altered morphology, expression of EMT-associated markers, and enhanced invasion capacity. Furthermore, the present study also revealed that nuclear translocation of PKM2, which is regulated by ERK pathway, regulated ß-catenin-TCF/LEF-1 transcriptional activity and associated EMT in HCC cell lines. These discoveries provide evidence of novel roles of PKM2 in the progression of HCC and potential therapeutic target for advanced cases.

[Tumor microenvironment elicits primary resistance to afatinib through HGF secretion].

OBJECTIVE: To observe the effects of hepatocyte growth factor (HGF) derived from tumor microenvironment and/or afatinib on the growth of human lung adenocarcinoma H1975 cells and explore the potential mechanisms by which HGF induces primary resistance to afatinib. METHODS: The effects of HGF, TGF-α and afatinib on the growth of H1975 cells were evaluated by MTT assay. The HGF concentrations of normal human fetal lung fibroblasts MRC-5 cells and human lung adenocarcinoma H1975 cells co-cultured or separately cultured were determined by ELISA assay. Western blot was used to detect the expressions of EGFR and Met signal pathway-related proteins and epithelial-mesenchymal transition (EMT) markers in H1975 cells treated with HGF and/or afatinib. RESULTS: The MTT assay showed that H1975 cells were hyposensitive to afatinib in the presence of HGF. The ELISA assay showed that HGF production by H1975 cells was less than 0.1 ng/2.0×10(6) cells, but HGF production by MRC-5 cells was (151.37 ± 2.07)ng/2.0×10(6) cells incubated for 48 h. When H1975 cells and MRC-5 cells were co-cultured for 72 h, the concentration of HGF in the culture supernatant was (61.13 ± 16.21)ng/ml. In the presence of HGF, the expression of p-Met, p-Akt and p-ERK proteins in the H1975 cells was markedly up-regulated. afatinib inhibited p-EGFR, but did not affect the expression of p-Met, p-Akt and p-ERK proteins. In the presence of afatinib, HGF up-regulated the expression of vimentin and down-regulated the expression of E-cadherin. CONCLUSIONS: HGF secreted by stromal cells in the tumor micro-environment may confer resistance to afatinib in H1975 cells by activation of the Met/PI3K/Akt and Met/MAPK/ERK signaling pathways, and is involved in the epithelial-mesenchymal transition process.

Luteolin reduces the invasive potential of malignant melanoma cells by targeting ß3 integrin and the epithelial-mesenchymal transition.

AIM: To investigate whether luteolin, a highly prevalent flavonoid, reverses the effects of epithelial-mesenchymal transition (EMT) in vitro and in vivo and to determine the mechanisms underlying this reversal. METHODS: Murine malignant melanoma B16F10 cells were exposed to 1% O(2) for 24 h. Cellular mobility and adhesion were assessed using Boyden chamber transwell assay and cell adhesion assay, respectively. EMT-related proteins, such as E-cadherin and N-cadherin, were examined using Western blotting. Female C57BL/6 mice (6 to 8 weeks old) were injected with B16F10 cells (1×10(6) cells in 0.2 mL per mouse) via the lateral tail vein. The mice were treated with luteolin (10 or 20 mg/kg, ip) daily for 23 d. On the 23rd day after tumor injection, the mice were sacrificed, and the lungs were collected, and metastatic foci in the lung surfaces were photographed. Tissue sections were analyzed with immunohistochemistry and HE staining. RESULTS: Hypoxia changed the morphology of B16F10 cells in vitro from the cobblestone-like to mesenchymal-like strips, which was accompanied by increased cellular adhesion and invasion. Luteolin (5-50 µmol/L) suppressed the hypoxia-induced changes in the cells in a dose-dependent manner. Hypoxia significantly decreased the expression of E-cadherin while increased the expression of N-cadherin in the cells (indicating the occurrence of EMT-like transformation), which was reversed by luteolin (5 µmol/L). In B16F10 cells, luteolin up-regulated E-cadherin at least partly via inhibiting the ß3 integrin/FAK signal pathway. In experimental metastasis model mice, treatment with luteolin (10 or 20 mg/kg) reduced metastatic colonization in the lungs by 50%. Furthermore, the treatment increased the expression of E-cadherin while reduced the expression of vimentin and ß3 integrin in the tumor tissues. CONCLUSION: Luteolin inhibits the hypoxia-induced EMT in malignant melanoma cells both in vitro and in vivo via the regulation of ß3 integrin, suggesting that luteolin may be applied as a potential anticancer chemopreventative and chemotherapeutic agent.