Isoalantolactone exerts anti-melanoma effects via inhibiting PI3K/AKT/mTOR and STAT3 signaling in cell and mouse models.

BACKGROUND AND AIM: Although the anti-cancer activity of isoalantolactone (IATL) has been extensively studied, the anti-melanoma effects of IATL are still unknown. Here, we have investigated the anti-melanoma effects and mechanism of action of IATL. MTT and crystal violet staining assays were performed to detect the inhibitory effect of IATL on melanoma cell viability. Apoptosis and cell cycle arrest induced by IATL were examined using flow cytometry. The molecular mechanism of IATL was explored by Western blotting, confocal microscope analysis, molecular docking, and cellular thermal shift assay (CETSA). A B16F10 allograft mouse model was constructed to determine the anti-melanoma effects of IATL in vivo. The results showed that IATL exerted anti-melanoma effects in vitro and in vivo. IATL induced cytoprotective autophagy in melanoma cells by inhibiting the PI3K/AKT/mTOR signaling. Moreover, IATL inhibited STAT3 activation both in melanoma cells and allograft tumors not only by binding to the SH2 domain of STAT3 but also by suppressing the activity of its upstream kinase Src. These findings demonstrate that IATL exerts anti-melanoma effects via inhibiting the STAT3 and PI3K/AKT/mTOR signaling pathways, and provides a pharmacological basis for developing IATL as a novel phytotherapeutic agent for treating melanoma clinically.

A traditional prescription comprising Astragali Radix and Schisandra chinensis Fructus induces apoptosis and protective autophagy in hepatocellular carcinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatocellular carcinoma (HCC) poses a growing challenge to global health efforts. The 5-year survival rate of HCC patients is still dismal. A traditional prescription Qi-Wei-Wan (QWW) comprising Astragali Radix and Schisandra chinensis Fructus has traditionally been used for HCC treatment according to traditional Chinese medicine theory, but the pharmacological basis is not clear. AIM OF THE STUDY: This study aims to investigate the anti-HCC effects of an ethanolic extract of QWW (hereafter, QWWE) and the mechanism of action. MATERIALS AND METHODS: An UPLC-Q-TOF-MS/MS method was developed to control the quality of QWWE. Two human HCC cell lines (HCCLM3 and HepG2) and a HCCLM3 xenograft mouse model were employed to investigate the anti-HCC effects of QWWE. The anti-proliferative effect of QWWE in vitro was determined by MTT, colony formation and EdU staining assays. Apoptosis and protein levels were examined by flow cytometry and Western blotting, respectively. Nuclear presence of signal transducer and activator of transcription 3 (STAT3) was examined by immunostaining. Transient transfection of pEGFP-LC3 and STAT3C plasmids was performed to assess autophagy and determine the involvement of STAT3 signaling in QWWE's anti-HCC effects, respectively. RESULTS: We found that QWWE inhibited the proliferation of and triggered apoptosis in HCC cells. Mechanistically, QWWE inhibited the activation of SRC and STAT3 at Tyr416 and Tyr705, respectively; inhibited the nuclear translocation of STAT3; lowered Bcl-2 protein levels, while increased Bax protein levels in HCC cells. Over-activating STAT3 attenuated the cytotoxic and apoptotic effects of QWWE in HCC cells. Moreover, QWWE induced autophagy in HCC cells by inhibiting mTOR signaling. Blocking autophagy with autophagy inhibitors (3-methyladenine and chloroquine) enhanced the cytotoxicity, apoptotic effect and the inhibitory effect on STAT3 activation of QWWE. Intragastric administration of QWWE at 10 mg/kg and 20 mg/kg potently repressed tumor growth and inhibited STAT3 and mTOR signaling in tumor tissues, but did not significantly affect mouse body weight. CONCLUSION: QWWE exhibited potent anti-HCC effects. Inhibiting the STAT3 signaling pathway is involved in QWWE-mediated apoptosis, while blocking mTOR signaling contributes to QWWE-mediated autophagy induction. Blockade of autophagy enhanced the anti-HCC effects of QWWE, indicating that the combination of an autophagy inhibitor and QWWE might be a promising therapeutic strategy for HCC management. Our findings provide pharmacological justifications for the traditional use of QWW in treating HCC.

Gracillin exerts anti-melanoma effects in vitro and in vivo: role of DNA damage, apoptosis and autophagy.

BACKGROUND: Melanoma is an aggressive cancer. Gracillin has been reported to treat various types of cancer, such as colorectal and lung cancer. However, there is a paucity of research on the anti-melanoma effects of gracillin. PURPOSE: The aim of this study was to assess the anti-melanoma effects and mechanisms of action of gracillin in vitro and in vivo. METHODS: Cell viability was detected using MTT and crystal violet staining assays. Cell proliferation was examined by EdU staining assays. Cell cycle arrest and apoptosis were analyzed by flow cytometry. Autophagic flux was monitored under a confocal microscope. Protein levels were determined by immunoblotting. LY294002 and rapamycin (Rapa) were used to determine the involvement of PI3K/AKT/mTOR signaling in gracillin-mediated autophagy. Signal transducer and activator of transcription 3 (STAT3) was overactivated to explore the contribution of the STAT3 signaling pathway in the anti-melanoma effects of gracillin. A B16F10 allograft mouse model was developed to evaluate the anti-melanoma effects of gracillin in vivo. RESULTS: We demonstrated that in melanoma cells, gracillin inhibited proliferation, induced G0/G1 phase cell cycle arrest, evoked apoptosis, and triggered autophagic cell death. Gracillin induced DNA damage in melanoma cells. Moreover, it suppressed the phosphorylation/activation of PI3K, AKT, mTOR, and 4E-BP1 in melanoma cells. Inhibiting PI3K/AKT and mTOR activity using LY294002 and Rapa, respectively, increased the protein level of LC3B-II in gracillin-treated melanoma cells. Furthermore, gracillin downregulated the protein levels of p-JAK2 (Tyr1007/1008), p-Src (Tyr416), and p-STAT3 (Tyr705) in melanoma cells. Over-expression of STAT3 in A375 cells significantly mitigated the cytotoxic and apoptotic effects of gracillin. In vivo studies showed that gracillin (1 mg/kg or 8 mg/kg, administered intraperitoneally for 16 consecutive days) suppressed B16F10 tumor growth and Src/STAT3 and AKT/mTOR signaling in tumors. No overt toxicity was observed in mice. CONCLUSION: Induction of DNA damage, inhibition of PI3K/AKT/mTOR signaling and suppression of STAT3 signaling are involved in gracillin-mediated cell cycle arrest, autophagic cell death and apoptosis, respectively, in melanoma cells. These findings provide novel insights into the anti-melanoma molecular mechanisms of gracillin, and suggest a potential role of gracillin in melanoma management.

Dihydrotanshinone I inhibits the growth of hepatoma cells by direct inhibition of Src.

BACKGROUND: Liver cancer is one of the leading causes of cancer-related death worldwide. Dihydrotanshinone I (DHI) was shown to inhibit the growth of several types of cancer. However, research related to hepatoma treatment using DHI is limited. PURPOSE: Here, we explored the inhibitory effect of DHI on the growth of hepatoma cells, and investigated the underlying molecular mechanisms. METHODS: The proliferation of Hep3B, SMCC-7721 and SK-Hep1 hepatoma cells was evaluated using the MTS and Edu staining assay. Hepatoma cell death was analyzed with a LIVE/DEAD Cell Imaging Kit. The relative expression and phosphorylation of proto-oncogene tyrosine-protein kinase Src (Src) and signal transducer and activator of transcription-3 (STAT3) proteins in hepatoma cells, as well as the expression of other protein components, were measured by western blotting. The structural interaction of DHI with Src proteins was evaluated by molecular docking, molecular dynamics simulation, surface plasmon resonance imaging and Src kinase inhibition assay. Src overexpression was achieved by infection with an adenovirus vector encoding human Src. Subsequently, the effects of DHI on tumor growth inhibition were further validated using mouse xenograft models of hepatoma. RESULTS: In vitro studies showed that treatment with DHI inhibited the proliferation and promoted cell death of Hep3B, SMCC-7721 and SK-Hep1 hepatoma cells. We further identified and verified Src as a direct target of DHI by using molecular stimulation, surface plasmon resonance image and Src kinase inhibition assay. Treatment with DHI reduced the in vitro phosphorylation levels of Src and STAT3, a transcription factor regulated by Src. In the xenograft mouse models, DHI dose-dependently suppressed tumor growth and Src and STAT3 phosphorylation. Moreover, Src overexpression partly abrogated the inhibitory effects of DHI on the proliferation and cell death in hepatoma cells. CONCLUSION: Our results suggest that DHI inhibits the growth of hepatoma cells by direct inhibition of Src.

Dauricine inhibits proliferation and promotes death of melanoma cells via inhibition of Src/STAT3 signaling.

Melanoma is the most common type of skin cancer. Signal transducer and activator of transcription 3 (STAT3) signaling has been demonstrated to be a therapeutic target for melanoma. Dauricine (Dau), an alkaloid compound isolated from the root of Menispermum dauricum DC., has shown tumor-suppressing effects in multiple human cancers, but its potential in melanoma remains unexplored. In this study, we demonstrated that Dau significantly inhibited the viability and proliferation of A375 and A2058 melanoma cells. Death of melanoma cells was also markedly promoted by Dau. Moreover, Dau inhibited phosphorylation-mediated activation of STAT3 and Src in a dose-dependent manner. Notably, constitutive activation of Src partially abolished the antiproliferative and cytotoxic activities of Dau on melanoma cells. Molecular docking showed that Dau could dock on the kinase domain of Src with a binding energy of -10.42 kcal/mol. Molecular dynamics simulations showed that Src-Dau binding was stable. Surface plasmon resonance imaging analysis also showed that Dau has a strong binding affinity to Src. In addition, Dau suppressed the growth of melanoma cells and downregulated the activation of Src/STAT3 in a xenograft model in vivo. These data demonstrated that Dau inhibits proliferation and promotes cell death in melanoma cells by inhibiting the Src/STAT3 pathways.

[Effects of lead stress on net photosynthetic rate, SPAD value and ginsenoside production in Ginseng (Panax ginseng)].

The paper aimed to evaluate the effects of lead stress on photosynthetic performance and ginsenoside content in ginseng (Panax ginseng). To accomplish this, three years old ginseng were cultivated in pot and in phytotron with different concentrations of lead, ranging from 0 to 1000 mg x kg(-1) soil for a whole growth period (about 150 days). The photosynthetic parameters in leaves and ginsenoside content in roots of ginseng were determined in green fruit stage and before withering stage, respectively. In comparison with the control, net photosynthetic rate and SPAD value in ginseng leaves cultivated with 100 and 250 mg x kg(-1) of lead changed insignificantly, however, ginseng supplied with 500 and 1 000 mg x kg(-1) of lead showed a noticeably decline in the net rate of photosynthesis and SPAD value (P < 0.05), the lowest net photosynthetic rate and SPAD value showed in the treatment supplied with 1 000 mg x kg(-1) of lead, with decline of 57.8%,11.0%, respectively. Total content of ginsenoside in ginseng roots cultivated with 100 mg x kg(-1) of lead showed insignificantly change compared to the control, but the content increased remarkably in treatments supplied with 250, 500, 1 000 mg x kg(-1) of lead (P < 0.05), and highest content appeared in these ginsengs exposed to 1000 mg x kg(-1) of lead. The net photosynthetic rate and SPAD value in leaves of ginseng both showed significantly negative linear correlations with lead stress level (P < 0.01), and significant positive linear correlations between total content of ginsenoside and lead concentration was also observed (P < 0.05). These results strongly indicate that exposing to high level of lead negatively affects photosynthetic performance in ginseng leaves, but benefits for accumulation of secondary metabolism (total content of ginsenoside) in ginseng root.

[The content dynamics of glycyrrhizic acid and soluble sugar in glycyrrhizae radix et rhizoma].

OBJECTIVE: To study the content dynamics of glycyrrhizic acid and soluble sugar in Glycyrrhizae Radix et Rhizoma. METHODS: Adopted the PVP root canal and the field experiment, the active ingredient content of glycyrrhizic acid and soluble sugar 2 years old cultivated Glycyrrhiza uralensis in the samples was studied. RESULTS: The content of glycyrrhizic acid was high in the root morphology of the bottom of the vertical distribution; The content of glycyrrhizic acid was decreased in the rhizome from the root to the direction away from the main root; in one year, the content of glycyrrhizic acid was varied with the Glycyrrhiza uralensis developmental stages. The accumulation peak was in early July, the peak of soluble sugar content was the period in mid-July to early August. CONCLUSION: The optimum harvest period is the late September to early October.