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Introduction: Selaginella doederleinii Hieron is a traditional Chinese herbal medicine, the ethyl acetate extract from Selaginella doederleinii (SDEA) showed favorable anticancer potentials. However, the effect of SDEA on human cytochrome P450 enzymes (CYP450) remains unclear. To predict the herb-drug interaction (HDI) and lay the groundwork for further clinical trials, the inhibitory effect of SDEA and its four constituents (Amentoflavone, Palmatine, Apigenin, Delicaflavone) on seven CYP450 isoforms were investigated by using the established CYP450 cocktail assay based on LC-MS/MS. Methods: Appropriate substrates for seven tested CYP450 isoforms were selected to establish a reliable cocktail CYP450 assay based on LC-MS/MS. The contents of four constituents (Amentoflavone, Palmatine, Apigenin, Delicaflavone) in SDEA were determined as well. Then, the validated CYP450 cocktail assay was applied to test the inhibitory potential of SDEA and four constituents on CYP450 isoforms. Results: SDEA showed strong inhibitory effect on CYP2C9 and CYP2C8 (IC50 ≈ 1 µg/ml), moderate inhibitory effect against CYP2C19, CYP2E1 and CYP3A (IC50 < 10 µg/ml). Among the four constituents, Amentoflavone had the highest content in the extract (13.65%) and strongest inhibitory effect (IC50 < 5 µM), especially for CYP2C9, CYP2C8 and CYP3A. Amentoflavone also showed time-dependent inhibition on CYP2C19 and CYP2D6. Apigenin and Palmatine both showed concentration-dependent inhibition. Apigenin inhibited CYP1A2, CYP2C8, CYP2C9, CYP2E1 and CYP3A. Palmatine inhibited CYP3A and had a weak inhibitory effect on CYP2E1. As for Delicaflavone, which has the potential to develop as an anti-cancer agent, showed no obvious inhibitory effect on CYP450 enzymes. Conclusion: Amentoflavone may be one of the main reasons for the inhibition of SDEA on CYP450 enzymes, the potential HDI should be considered when SDEA or Amentoflavone were used with other clinical drugs. On the contrast, Delicaflavone is more suitable to develop as a drug for clinical use, considering the low level of CYP450 metabolic inhibition.

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Structural modification of natural products is the effective option to improve their pharmacological effects and drug properties. DLF is a lead compound of antitumor drug, which is a broad-spectrum, low toxic and high-efficient component isolated from Selaginella doederleinii Hieron by our research group. Here, we report the structural modification method of this component, and find that the acetylated product of C4'''- OH (C4'''-acetyl-delicaflavone, 4'''ADLF) has better inhibitory effect on the selected cancer cell lines, including, lung, liver, colon and cervical cancer cell lines. Since the increased water solubility of 4'''ADLF may lead to higher absorption rate and activity, we evaluate the pharmacodynamics in vitro and in vivo, and the pharmacokinetic of 4'''ADLF. It shows that 4'''ADLF inhibit the proliferation and induce cycle arrest in tumor cells, and had better anticancer activity and bioavailability than DLF.

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AIM: To study the effect and mechanism of Delicaflavone on migration and invasion of gefitinib-resistant lung cancer cell line PC-9/GR. METHODS: MTT assay was used to detect cell viability. Transwell and scratch assays were used to detect cell invasion and migration abilities. Western blotting was used to detect the expressions of MMP-9, MMP-2, E-cadherin, N-cadherin, Vimentin and PI3K/Akt/mTOR pathway-related proteins in PC-9/GR cells. RESULTS: Compared with control group, 20 mg/L Delicaflavone could significantly inhibit the viability of PC-9/GR cells for 24 h (P<0.05), while Delicaflavone below 10 mg/L had no significant effect on cell proliferation. The number of invasive cells and migrated cells were decreased significantly by Delicaflavone in a concentration-dependent way (P<0.05 and P<0.01). Delicaflavone could concentration-dependently reduce the expression of MMP-9, MMP-2, N-cadherin, vimentin (P<0.01), meanwhile up-regulate the expression of E-cadherin (P<0.01). In addition, Delicaflavone also decreased the expression of p-PI3K, p-Akt and p-mTOR in a concentration-dependent manner (P<0.01). CONCLUSION: Delicaflavone can inhibit the migration and invasion of PC-9/GR cells by regulating epithelial-mesenchymal transition via PI3K/Akt/mTOR pathway.

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Delicaflavone (DF), a natural active ingredient from Selaginella doederleinii Hieron, has been reported to have favorable anticancer effects and is thus considered a potential anticancer agent. However, its pharmacokinetics and plasma protein binding properties remain unknown. Here, we investigated the pharmacokinetic profile of DF in rats using a validated HPLC-MS/MS methods, as well as its human serum albumin (HSA) binding properties through multi-spectroscopic and in silico methods. The results showed that DF was rapidly eliminated and had a widespread tissue distribution after intravenous administration. DF showed linear dynamics in the dose range of 30-60 mg/kg and poor oral bioavailability. The major distribution tissues of DF were the liver, lungs, and kidneys. Ultraviolet and fluorescence spectroscopy and molecular docking demonstrated that DF had a static quenching effect on HSA, with one binding site, and relatively strong binding constants. Thermodynamic analysis of the binding data revealed that hydrogen bonding and van der Waals interactions played major roles in binding. The results of this study further our understanding of the pharmacokinetic and plasma protein binding properties of the potential anticancer agent DF and shed light on pharmacological strategies that may be useful for the development of novel cancer therapeutics.

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Here, we review the molecular mechanism and pharmacokinetics of flavonoids in the treatment of resistant EGF receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC) and particularly the possible mechanism(s) of delicaflavone, a biflavonoid extracted from Selaginella doederleinii Hieron. EGFR TK inhibitors (EGFR-TKI) are ubiquitously used in the treatment of NSCLC bearing EGFR mutations. However, patients treated with EGFR-TKI inevitably and continuously develop resistance. In laboratory studies, flavonoids, as potential adjuvants for cancer chemotherapy, exhibited anti-cancer properties such as inhibition of chemoresistance by interference with ABC transporters-induced drug efflux, curbing of c-MET amplification, or reversal of T790M mutation-mediated resistance. The current review aims at summarizing the association between the anti-cancer potentials of flavonoids and their possible regulatory roles in certain types of mutation that could trigger EGFR-TKI resistance in NSCLC. Potential practical applications of these phytochemicals, as well as the relevant pharmacokinetics, are also discussed.

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BACKGROUND: The incidence and mortality of lung cancer continue to increase around the world; in 2018, new lung cancer cases accounted for 11.6% of all cancer cases, and lung cancer deaths accounted for 18.4% of cancer deaths. Cisplatin (DDP) is a first-line chemotherapy drug for lung cancer; however, DDP resistance can lead to a poor prognosis in patients with lung cancer. Therefore, reversing DDP resistance is a treatment goal. MATERIALS AND METHODS: Cell counting kit-8 (CCK8) assays, wound healing analyses, Transwell assays, in vitro tumor xenografts, and flow cytometry were used to detect the proliferation, migration, invasion, and apoptosis of multidrug resistant A549/DDP and PC9/DDP cells, respectively. Western blot was performed to detect protein levels of cleaved caspase-3, CHOP, and GRP78. RESULTS: Delicaflavone inhibited DDP resistance of lung cancer cells and decreased proliferation in a dose- and time-dependent manner. It also decreased migration and invasion and enhanced apoptosis. Western blots showed that delicaflavone overcame DDP resistance by increasing the expression of GRP78 and CHOP and the apoptosis-related protein cleaved caspase-3. CONCLUSION: Delicaflavone can reverse DDP resistance in A549/DDP and PC9/DDP cells by inhibiting cell proliferation and migration and enhancing apoptosis and cleaved caspase-3 levels by increasing the expression of CHOP and GRP78 protein via the endoplasmic reticular stress pathway. It could be a useful therapeutic adjunct to treat DDP-resistant lung cancer.

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Searching for potential anticancer agents from natural sources is an effective strategy for developing novel chemotherapeutic agents. In this study, data supporting the in vitro and in vivo anticancer effects of delicaflavone, a rarely occurring biflavonoid from Selaginella doederleinii, were reported. Delicaflavone exhibited favorable anticancer properties, as shown by the MTT assay and xenograft model of human non-small cell lung cancer in male BALB/c nude mice without observable adverse effect. By transmission electron microscopy with acridine orange and Cyto-ID®Autophagy detection dyes, Western blot analysis, and RT-PCR assay, we confirmed that delicaflavone induces autophagic cell death by increasing the ratio of LC3-II to LC3-I, which are autophagy-related proteins, and promoting the generation of acidic vesicular organelles and autolysosomes in the cytoplasm of human lung cancer A549 and PC-9 cells in a time- and dose-dependent manner. Delicaflavone downregulated the expression of phospho-Akt, phospho-mTOR, and phospho-p70S6K in a time- and dose-dependent manner, suggesting that it induced autophagy by inhibiting the Akt/mTOR/p70S6K pathway in A549 and PC-9 cells. Delicaflavone is a potential anticancer agent that can induce autophagic cell death in human non-small cell lung cancer via the Akt/mTOR/p70S6K signaling pathway. Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer. KEY MESSAGES: Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer.

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