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Ethyl Acetate Fraction from Hedyotis diffusa plus Scutellaria barbata Exerts Anti-Breast Cancer Effect via miR-200c-PDE7B/PD-L1-AKT/MAPK Axis.

ABSTRACT: Background:Hedyotis diffusa (HD) Willd. and Scutellaria barbata (SB) D. Don in different ratios have been frequently used to treat various cancers in clinical Traditional Chinese Medicine prescriptions. However, the optimal ratio, active fraction, and molecular mechanisms associated with the anti-breast cancer role of this herbal couplet have not been elaborated. Methods:To screen out the optimal ratio of this herbal couplet, we compare aqueous extracts of HD, SB, or HD plus SB in different weight ratios (HS11, HS12, HS21) for their anticancer effects on murine breast cancer 4T1 cells in vitro and in vivo. EA11, the ethyl acetate fraction from HS11 (the aqueous extract of the couplet at an equal weight ratio), is further assessed for its antiproliferative effect as well as the antitumorigenic impact with the aid of immunocompetent mice. Colony formation, flow cytometry, western blot, ELISA, and qRT-PCR are used to elucidate mechanisms underlying EA11-led effects. Results:HS11 presents the most potential suppression of 4T1 cell proliferation and tumor growth among these aqueous extracts. The comparison results show that EA11 is more effective than HS11 in vitro and in vivo. EA11 inhibits colony formation and induces apoptosis in a concentration-dependent manner. EA11 reduces the protein expressions of PDE7B, PD-L1, ?-catenin, and cyclin D1 while elevating the concentration of cellular cAMP and miR-200c expression in 4T1 cells. Additionally, EA11 exerts its anticancer effect partially via the inactivation of MAPK and AKT signaling pathways. Conclusions:This study implicates that EA11 prevents breast tumor development by interfering with the miR-200c-PDE7B/PD-L1-AKT/MAPK axis. EA11 may represent a potential therapeutic candidate for breast cancer.

SUBMITTER: Yang Y

PROVIDER: S-EPMC7453271 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Ethyl Acetate Fraction from <i>Hedyotis diffusa</i> plus <i>Scutellaria barbata</i> Exerts Anti-Breast Cancer Effect via miR-200c-PDE7B/PD-L1-AKT/MAPK Axis.

Yang Yue Y Fang Ting T Cao Yi-Lan YL Lv Ya-Xin YX Chang Qing-Qi QQ Zhang Dan-Dan DD

Evidence-based complementary and alternative medicine : eCAM 20200819

<h4>Background</h4><i>Hedyotis diffusa</i> (HD) Willd. and <i>Scutellaria barbata</i> (SB) D. Don in different ratios have been frequently used to treat various cancers in clinical Traditional Chinese Medicine prescriptions. However, the optimal ratio, active fraction, and molecular mechanisms associated with the anti-breast cancer role of this herbal couplet have not been elaborated.<h4>Methods</h4>To screen out the optimal ratio of this herbal couplet, we compare aqueous extracts of HD, SB, or ...[more]

PMID: 32922506

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Project description:Background Hepatocellular carcinoma (HCC) is characterized by poor diagnosis and high mortality. Novel and efficient therapeutic agents are urgently needed for the treatment. Hedyotis diffusa Willd (HDW) is used to treat cancers, especially HCC in China. Purpose The study aimed to identify the main anti-HCC extract in HDW and to explore the mechanism of the active extract. Materials and Methods The high-performance liquid chromatography-quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS) method was used for the simultaneous determination of main compounds in the ethyl acetate fraction of HDW (EHDW). The toxicity test of different HDW fractions was carried out on larvae at 2 day-post-fertilization (dpf) for 72 h. The in vivo anti-HCC effect of different HDW fractions was evaluated on a zebrafish tumor model by immersion administration. The antiproliferative effect of HDW fractions was determined with MTT assay, as well as hematoxylin and eosin (HE) staining assay. Hoechst 33258 staining was used to observe changes in nucleus morphology. Flow cytometry analysis was used to investigate apoptosis induction. Western blot analysis was used to examine apoptosis-related proteins, and key proteins in JNK/Nur77 signaling pathway. SP600125 was served to validate the apoptotic mechanism. Results EHDW showed the strongest tumor cell growth inhibitory effect on zebrafish tumor model. Further study revealed that EHDW induced apoptosis in zebrafish tumor model and in cultured Hep3B cells. Meanwhile, it has been shown that the levels of BCL2-associated X (Bax), cytochrome c (cyto c), cleaved-caspase 3, and poly-ADP-ribose polymerase (PARP) cells were upregulated. In contrast, the level of antiapoptotic B cell lymphoma-2 (Bcl-2) was downregulated in Hep3B cells. Additionally, EHDW activated JNK/Nur77 pathway by increasing the levels of p-JNK(Thr183/Tyr185) and p-Nur77(Ser351). Further study showed that blockage of JNK by SP600125 reversed EHDW-induced JNK/Nur77 pathway and the downstream apoptotic proteins. Conclusion In conclusion, EHDW exerted the anti-HCC effect, which may be attributed to the activation of JNK/Nur77 pathway. This study supported the rationale of HDW as an HCC therapeutic agent.

Project description:Hedyotis Diffusa Willd, used in Traditional Chinese Medicine, is a treatment for various diseases including cancer, owing to its mild effectiveness and low toxicity. The aim of this study was to identify the main anticancer components in Hedyotis Diffusa Willd, and explore mechanisms underlying their activity. Hedyotis Diffusa Willd was extracted and fractionated using ethyl acetate to obtain the H-Ethyl acetate fraction, which showed higher anticancer activity than the other fractions obtained against HepG2 cells with sulforhodamine B assays. The active component of the H-Ethyl acetate fraction was identified to be 1,3-dihydroxy-2-methylanthraquinone (DMQ) with much high inhibitory rate up to 48.9 ± 3.3% and selectivity rate up to 9.4 ± 4.5 folds (p<0.01) at 125 μmol/L. HepG2 cells treated with the fraction and DMQ visualized morphologically using light and fluorescence microscopy. Annexin V--fluorescein isothiocyanate / propidium iodide staining flow cytometry, DNA ladder and cell cycle distribution assays. Mechanistic studies showed up-regulation of caspase-3, -8, and -9 proteases activities (p<0.001), indicating involvement of mitochondrial apoptotic and death receptor pathways. Further studies revealed that reactive oxygen species in DMQ and the fraction treated HepG2 cells increased (p<0.01) while mitochondrial membrane potential reduced significantly (p<0.001) compared to the control by flow cytometry assays. Western blot analysis showed that Bax, p53, Fas, FasL, p21 and cytoplasmic cytochrome C were up-regulated (p<0.01), while Bcl-2, mitochondrial cytochrome C, cyclin E and CDK 2 were down-regulated dose-dependently (p<0.01). The reverse transcriptase-polymerase chain reaction showed that mRNA expressions of p53 and Bax increased (p<0.001) while that of Bcl-2 decreased (p<0.001). Pre-treatment with caspase-8 inhibitor Z-IETD-FMK, or caspase-9 inhibitor Z-LEHD-FMK, attenuated the growth-inhibitory and apoptosis-inducing effects of DMQ and the fraction on HepG2 cells. These results suggested that DMQ and the H-Ethyl acetate fraction of Hedyotis Diffusa Willd showed potential anticancer effects. Furthermore, the mechanisms of action may involve mitochondrial apoptotic and death receptor pathways.

Dataset Information

Ethyl Acetate Fraction from Hedyotis diffusa plus Scutellaria barbata Exerts Anti-Breast Cancer Effect via miR-200c-PDE7B/PD-L1-AKT/MAPK Axis.

Publications

Ethyl Acetate Fraction from <i>Hedyotis diffusa</i> plus <i>Scutellaria barbata</i> Exerts Anti-Breast Cancer Effect via miR-200c-PDE7B/PD-L1-AKT/MAPK Axis.

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