Senescence Inducer Shikonin ROS-Dependently Suppressed Lung Cancer Progression.

Lung adenocarcinoma (LAC), predominant subclassfication of lung cancer, leads high incidence and mortality annually worldwide. During the premalignant transition from lung adenomas to LAC, cellular senescence is regard as a critical physiological barrier against tumor progression. Nevertheless, the role of senescence in tumorigenesis is controversial and few senescence inducers are extensively determined. In this study, we used two classical cell lines A549 and H1299 and two NSCLC xenograft models on Balb/c-nude mice to reveal the pro-senescence effects of shikonin and the corresponding underlying mechanism in LAC. Shikonin, a pure compound isolated from the herbal medicine Lithospermum erythrorhizon, remarkably stimulated cellular senescence including increased SAHF formation, enlarged cellular morphology, and induced SA-ß-Gal positive staining. Further mechanism study revealed that the pro-senescence effect of shikonin was dependent on the increased intercellular ROS generation, which subsequently triggered DNA damage-p53/p21waf axis without activating oncogenes such as Ras and MEK-1. Meanwhile, Kdm2b, an H3K36me2-specific demethylase effectively suppressed ROS generation, was also notably suppressed by shikonin treatment. Moreover, shikonin at 10 mg/kg significantly inhibited tumor weights by 55.84% and 50.98% in A549 and H1299 xenograft model, respectively (P < 0.05) through activating cellular senescence. Our study suggested that shikonin, a ROS-dependent senescence inducer, could serve as a promising agent for further lung cancer treatment.

Camptosorus sibiricus rupr aqueous extract prevents lung tumorigenesis via dual effects against ROS and DNA damage.

ETHNOPHARMACOLOGICAL RELEVANCE: Camptosorus sibiricus Rupr (CSR) is a widely used herbal medicine with antivasculitis, antitrauma, and antitumor effects. However, the effect of CSR aqueous extract on B[a]P-initiated tumorigenesis and the underlying mechanism remain unclear. Moreover, the compounds in CSR aqueous extract need to be identified and structurally characterized. AIM OF THE STUDY: We aim to investigate the chemopreventive effect of CSR and the underlying molecular mechanism. MATERIALS AND METHODS: A B[a]P-stimulated normal cell model (BEAS.2B) and lung adenocarcinoma animal model were established on A/J mice. In B[a]P-treated BEAS.2B cells, the protective effects of CSR aqueous extract on B[a]P-induced DNA damage and ROS production were evaluated through flow cytometry, Western blot, real-time quantitative PCR, single-cell gel electrophoresis, and immunofluorescence. Moreover, a model of B[a]P-initiated lung adenocarcinoma was established on A/J mice to determine the chemopreventive effect of CSR in vivo. The underlying mechanism was analyzed via immunohistochemistry and microscopy. Furthermore, the new compounds in CSR aqueous extract were isolated and structurally characterized using IR, HR-ESI-MS, and 1D and 2D NMR spectroscopy. RESULTS: CSR effectively suppressed ROS production by re-activating Nrf2-mediated reductases HO-1 and NQO-1. Simultaneously, CSR attenuated the DNA damage of BEAS.2B cells in the presence of B[a]P. Moreover, CSR at 1.5 and 3 g/kg significantly suppressed tumorigenesis with tumor inhibition ratios of 36.65% and 65.80%, respectively. The tumor volume, tumor size, and multiplicity of B[a]P-induced lung adenocarcinoma were effectively decreased by CSR in vivo. After extracting and identifying the compounds in CSR aqueous extract, three new triterpene saponins were isolated and characterized structurally. CONCLUSIONS: CSR aqueous extract prevents lung tumorigenesis by exerting dual effects against ROS and DNA damage, suggesting that CSR is a novel and effective agent for B[a]P-induced carcinogenesis. Moreover, by isolating and structurally characterizing three new triterpene saponins, our study further standardized the quality of CSR aqueous extract, which could widen CSR clinical applications.

Aconitum alkaloids, the major components of Aconitum species, affect expression of multidrug resistance-associated protein 2 and breast cancer resistance protein by activating the Nrf2-mediated signalling pathway.

BACKGROUND: Aconitum alkaloids from Aconitum species are often used to treat arthritis and rheumatic diseases but have the drawback of high toxicity. Identifying their pharmacokinetic behaviour is important for the safe clinical application of Aconitum species. Efflux transporters (ETs), including P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP), have important functions in regulating the pharmacokinetic behaviours of drugs and in herb-herb or herb-drug interactions (HDIs). The Aconitum alkaloids regulate P-gp expression and function, but their effects on MRP2 and BCRP expression remain unknown. PURPOSE: To determine the effects of three Aconitum alkaloids, aconitine (AC), benzoylaconine (BAC), and aconine, on MRP2 and BCRP. METHODS: The levels of the protein and mRNA expression of MRP2 and BCRP in vivo and in vitro were measured via Western blotting and real-time PCR, respectively. Fluorescence signals of MRP2 and BCRP were detected via confocal fluorescence microscopy. A reporter assay using HepG2-C8 cells, which were generated by transfecting plasmids containing the antioxidant response element (ARE)-luciferin gene into HepG2 cells, was used to examine the ARE-luciferin activity. The transport activities of MRP2 and BCRP were tested via flow cytometry using substrate probes. RESULTS: The Aconitum alkaloids significantly up-regulated MRP2 and BCRP expression, accompanied by a marked increase in nuclear factor E2-related factor-2 (Nrf2) expression in the jejunum, ileum, and colon of FVB mice, in the order AC < BAC < aconine. In the in vitro model, the Aconitum alkaloids increased MRP2 and BCRP expression in Caco-2 and LS174T cells, in the order AC < BAC < aconine. Additionally, these alkaloids promoted the translocation of Nrf2 from the cytoplasm to the nucleus and significantly increased ARE-luciferin activity in HepG2-C8 cells. Luteolin, a potent inhibitor of Nrf2, markedly prevented MRP2 and BCRP expression from being induced by the three Aconitum alkaloids. The efflux activity of MRP2 was also significantly increased in cells receiving the same treatment. CONCLUSIONS: The tested Aconitum alkaloids significantly increased the expression of MRP2 and BCRP by activating the Nrf2-mediated signalling pathway and enhanced the efflux activity of MRP2. The potential for herb-herb interactions or HDIs exists when Aconitum species are co-administered with substrate drugs that are transported via MRP2 and BCRP. Therefore, the Aconitum alkaloids may be used as quality indicators for the herbs of Aconitum species.

Regulation of the kynurenine metabolism pathway by Xiaoyao San and the underlying effect in the hippocampus of the depressed rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoyao San (XYS) is a classic Chinese herbal formula for treatment of depression. The present study aimed to investigate the antidepressant effects of XYS in a rat model of chronic unpredictable mild stress (CUMS) and the underlying mechanisms. MATERIALS AND METHODS: A CUMS rat model of depression was established via 4 weeks of unpredictable stimulation. Then the rats were orally administered paroxetine and XYS for 2 weeks with continued stress. Behavioral assessments, including an open field test (OFT), sucrose preference test (SPT) and forced swim test (FST), were conducted to evaluate the antidepressant effects of XYS. The concentrations in rat plasma of tryptophan (Trp) and its metabolic products, including kynurenine (Kyn) and quinolinic acid (QUIN), were determined using high performance liquid chromatography tandem mass spectrometry with electrochemical detection (HPLC-MS/MS). The mRNA and protein levels in rat hippocampus of depression-related brain derived neurotrophic factor (BDNF), cyclic AMP response element binding protein (CREB) and nerve cell adhesion molecule (NCAM) were determined by real-time qPCR and Western blot, respectively. Enzyme Linked Immunosorbent Assay (ELISA) was used to detect the activities of indoleamine 2,3-dioxygenase (IDO) and kynurenine-3-monooxygenase (KMO) in rat plasma. RESULTS: The results showed that a successful CUMS rat model was established through 4 weeks of continuous unpredictable stimulation, as indicated by the significant decrease in locomotor activity and increase in immobility time in the OFT, reduction in body weight and food intake etc. Compared with the normal group, the concentrations of Kyn and QUIN had significantly (p < 0.05) decreased at day 28 in the control group, but then improved after drug treatment with paroxetine and XYS. There were no obvious changes in the activities of IDO and KMO. Compared with the normal group, the mRNA of NCAM, CREB and BDNF were significantly down-regulated (p < 0.001) in the control group, BDNF gene was up-regulated by paroxetine or XYS treatment, NCAM and CREB gene did not change in XYS group, protein expressions of BDNF and CREB were significantly increased, and NCAM was significantly reduced (p < 0.05). CONCLUSIONS: XYS reversed the abnormalities of the tryptophan-kynurenine metabolic pathways in depressed rats and achieved an excellent antidepressant effect. Its direct impact may be observed as changes in biological indicators in rat hippocampus tissue.

Induction of P-glycoprotein expression and activity by Aconitum alkaloids: Implication for clinical drug-drug interactions.

The Aconitum species, which mainly contain bioactive Aconitum alkaloids, are frequently administered concomitantly with other herbal medicines or chemical drugs in clinics. The potential risk of drug-drug interactions (DDIs) arising from co-administration of Aconitum alkaloids and other drugs against specific targets such as P-glycoprotein (P-gp) must be evaluated. This study focused on the effects of three representative Aconitum alkaloids: aconitine (AC), benzoylaconine (BAC), and aconine, on the expression and activity of P-gp. We observed that Aconitum alkaloids increased P-gp expression in LS174T and Caco-2 cells in the order AC > BAC > aconine. Nuclear receptors were involved in the induction of P-gp. AC and BAC increased the P-gp transport activity. Strikingly, intracellular ATP levels and mitochondrial mass also increased. Furthermore, exposure to AC decreased the toxicity of vincristine and doxorubicin towards the cells. In vivo, AC significantly up-regulated the P-gp protein levels in the jejunum, ileum, and colon of FVB mice, and protected them against acute AC toxicity. Taken together, the findings of our in vitro and in vivo experiments indicate that AC can induce P-gp expression, and that co-administration of AC with P-gp substrate drugs may cause DDIs. Our findings have important implications for Aconitum therapy in clinics.

The significant inhibition on CYP3A caused by radix Aconiti single herb is not observed in the Wutou decoction: The necessity of combination therapy of radix Aconiti.

ETHNOPHARMACOLOGICAL RELEVANCE: Wutou (WT, Radix Aconiti), the mother root of Aconitum carmichaelii Debx., is a famous Chinese herb against rheumatoid arthritis. In Chinese clinics, PWT is often prepared as a decoction in combination with other herbs, such as Wutou decoction (WTD). The present study aimed to compare the effects of PWT single herb and WTD on CYP3A activity ex vivo and in vivo. MATERIALS AND METHODS: In the ex vivo study, CYP3A activity was determined by using testosterone (Tes) as a specific probe. Levels of Tes and its metabolite 6ß-hydroxytestosterone (6ß-OH-Tes) were measured using a validated ultra-performance liquid chromatography (UPLC) method. CYP3A protein and mRNA levels were measured by using Western blot and real-time PCR, respectively. In the in vivo study, CYP3A activity was determined by using buspirone (BP) as a specific probe. The plasma concentrations of BP and its primary metabolites, namely, 1-(2-pyrimidinyl) piperazine (1-PP) and 6'-hydroxybuspirone (6'-OH-BP), were determined using a validated UPLC-tandem mass spectrometry (UPLC/MS/MS) method. RESULTS: Compared with the control group, the formation rates of 6ß-OH-Tes from Tes ex vivo significantly decreased in groups treated with PWT at the tested doses, and this decrease was accompanied by a striking decrease in CYP3A protein and mRNA levels. However, a significant increase was observed in the ratios in the WTD groups compared with PWT single herb groups. In vivo, both formation ratios of 6'-OH-BP and 1-PP from BP showed no significant change in the WTD group. CONCLUSIONS: PWT can significantly inhibit CYP3A activity ex vivo at the tested doses because of the down-regulation of CYP3A protein and mRNA expression levels. WTD can significantly reverse the inhibition caused by PWT. WTD also had no significant effect on CYP3A activity in vivo. Results implied that the use of PWT as a part of the WTD prescription rather than PWT single herb is more appropriate in clinics.

Pinelliae rhizoma, a toxic chinese herb, can significantly inhibit CYP3A activity in rats.

Raw Pinelliae Rhizoma (RPR) is a representative toxic herb that is widely used for eliminating phlegm or treating cough and vomiting. Given its irritant toxicity, its processed products, including Pinelliae Rhizoma Praeparatum (PRP) and Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine (PRPZA), are more commonly applied and administered concomitantly with other chemical drugs, such as cough medications. This study aimed to investigate the effects of RPR, PRP, and PRPZA on CYP3A activity. Testosterone (Tes) and buspirone (BP) were used as specific probe substrates ex vivo and in vivo, respectively. CYP3A activity was determined by the metabolite formation ratios from the substrates. Ex vivo results show that the metabolite formation ratios from Tes significantly decreased, indicating that RPR, PRP, and PRPZA could inhibit CYP3A activity in rats. CYP3A protein and mRNA levels were determined to explore the underlying mechanism. These levels showed marked and consistent down-regulation with CYP3A activity. A significant decrease in metabolite formation ratios from BP was also found in PRPZA group in vivo, implying that PRPZA could inhibit CYP3A activity. Conclusively, co-administration of PR with other CYP3A-metabolizing drugs may cause drug-drug interactions. Clinical use of PR-related formulae should be monitored carefully to avoid adverse interactions.