[Effects of electroacupuncture on NLRP3/Caspase-1/GSDMD axis and neurological function in rats with cerebral ischemic reperfusion].

OBJECTIVE: To investigate the neuroprotective effect of electroacupuncture (EA) at "Quchi" (LI 11) and "Zusanli" (ST 36) in the rats with cerebral ischemic reperfusion and the potential mechanism of microglia pyroptosis. METHODS: Sixty SD rats were randomly divided into a sham-operation group, a model group and an EA group, with 20 rats in each group. The Zea Longa method was employed to establish the rat model of the middle cerebral artery occlusion and reperfusion (MACO/R) in the left brain. In the EA group, since the 2nd day of modeling, EA was given at "Quchi" (LI 11) and "Zusanli" (ST 36) of right side with disperse-dense wave, 4 Hz/20 Hz in frequency and 0.2 mA in current intensity, 30 min each time, once a day for lasting 7 consecutive days. The reduction rate of cerebral blood flow was measured with laser Doppler flowmetry during operation. The neurological function of rats was observed using Zea Longa neurobehavioral score. The cerebral infarction volume was detected by TTC staining method. The microglia positive expression in the ischemic side of the cortex was detected with the immunofluorescence method. Under transmission electron microscope, the ultrastructure of cell in the ischemic cortex was observed. The mRNA expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cysteinyl aspartate specific proteinase-1 (Caspase-1) and gasdermin D (GSDMD) in the ischemic cortex were detected using real-time PCR. RESULTS: Compared with the sham-operation group, in the model group, the reduction rate of cerebral blood flow was increased during operation (P<0.001); Zea Longa neurobehavional score and the percentage of cerebral infarction volume were increased (P<0.001), the numbers of M1-type microglia marked by CD68+ and M2-type microglia marked by TMEM119+ were elevated in the ischemic cortex (P<0.001), the mRNA expression of NLRP3, ASC, Caspase-1 and GSDMD was increased (P<0.001, P<0.01); the cytomembrane structure was destroyed, with more cell membrane pores formed in the ischemic cortex. Compared with the model group, after intervention, Zea Longa neurobehavioral score and the percentage of cerebral infarction volume were reduced (P<0.05), the number of M1-type microglia marked by CD68+ was reduced (P<0.05) and the number of M2-type microglia marked by TMEM119+ was increased (P<0.05); and the mRNA expression of NLRP3, ASC, Caspase-1 and GSDMD was decreased (P<0.01, P<0.05) in the EA group. Even though the cytomembrane structure was incomplete, there were less membrane pores presented in the ischemic cortex in the EA group after intervention. CONCLUSION: The intervention with EA attenuates the neurological dysfunction and reduces the volume of cerebral infarction in the rats with cerebral ischemic reperfusion. The underlying mechanism is related to the inhibition of microglia pyroptosis through modulating NLRP3/Caspase-1/GSDMD axis.

Pan-Cancer Study of the Prognosistic Value of Selenium Phosphate Synthase 1.

Objective: This study sought to determine the mean prognostic usefulness of seleniumphosphate synthase (SEPHS1) by investigating its expression in 33 human malignancies and its relationship to tumor immunity.Methods: The expression of selenophosphate synthase 1 (SEPHS1) in 33 human malignant tumors was examined using the Genotype-Tissue Expression (GTEx), Cancer Genome Atlas (TCGA), and TIMER databases. Furthermore, the TCGA cohort was used to investigate relationships between SEPHS1 and immunological checkpoint genes (ICGs), tumor mutation burden (TMB), microsatellite instability (MSI), and DNA mismatch repair genes (MMRs). To establish independent risk factors and calculate survival probabilities for liver hepatocellular carcinoma (LIHC) and brain lower-grade glioma (LGG), Cox regression models and Kaplan-Meier curves were utilized. Eventually, the Genomics of Cancer Drug Sensitivity (GDSC) database was used to evaluate the drug sensitivity in LGG and LIHC patients with high SEPHS1 expression.Results: Overall, in numerous tumor tissues, SEPHS1 was highly expressed, and it significantly linked with the prognosis of LGG, ACC, and LIHC (P < .05). Furthermore, in numerous cancers, SEPHS1 expression was linked to tumor-infiltrating immune cells (TIICs), TMB, MSI, and MMRs. According to univariate and multivariate Cox analyses, SEPHS1 expression was significant for patients with LGG and LIHC.Conclusion: High SEPHS1 expression has a better prognosis for LGG, while low SEPHS1 expression has a better prognosis for LIHC. Chemotherapy was advised for LGG patients, particularly for those with high SEPHS1 expression because it can predict how responsive patients will be to 5-Fluorouracil and Temozolomide. This interaction between SEPHS1 and chemoradiotherapy has a positive clinical impact and may be used as evidence for chemotherapy for LGG and LIHC patients.

Dandelion extract inhibits triple-negative breast cancer cell proliferation by interfering with glycerophospholipids and unsaturated fatty acids metabolism.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with limited treatment options and a poor prognosis. TNBC exists widely reprogrammed lipid metabolism, and its metabolic-associated proteins and oncometabolites are promising as potential therapeutic targets. Dandelion (Taraxacum mongolicum) is a classical herbal medicine used to treat breast diseases based on traditional Chinese medicine theory and was reported to have antitumor effects and lipid regulatory capacities. Our previous study showed that dandelion extract was effective against TNBC. However, whether dandelion extract could regulate the lipid metabolisms of TNBC and exert its antitumor effects via interfering with lipids metabolism remained unclear. In this study, an integrated approach combined with network pharmacology and multi-omics techniques (including proteomics, metabolomics, and lipidomics) was performed to investigate the potential regulatory mechanisms of dandelion extract against TNBC. We first determined the antitumor effects of dandelion extract in vitro and in vivo. Then, network pharmacology analysis speculated the antitumor effects involving various metabolic processes, and the multi-omics results of the cells, tumor tissues, and plasma revealed the changes in the metabolites and metabolic-associated proteins after dandelion extract treatment. The alteration of glycerophospholipids and unsaturated fatty acids were the most remarkable types of metabolites. Therefore, the metabolism of glycerophospholipids and unsaturated fatty acids, and their corresponding proteins CHKA and FADS2, were considered the primary regulatory pathways and biomarkers of dandelion extract against TNBC. Subsequently, experimental validation showed that dandelion extract decreased CHKA expression, leading to the inhibition of the PI3K/AKT pathway and its downstream targets, SREBP and FADS2. Finally, the molecular docking simulation suggested that picrasinoside F and luteolin in dandelion extract had the most highly binding scores with CHKA, indicating they may be the potential CHKA inhibitors to regulate glycerophospholipids metabolisms of TNBC. In conclusion, we confirmed the antitumor effects of dandelion extract against TNBC cells in vitro and demonstrated that dandelion extract could interfere with glycerophospholipids and unsaturated fatty acids metabolism via downregulating the CHKA expression and inhibiting PI3K/AKT/SREBP/FADS2 axis.

Marsdenia tenacissima extract disturbs the interaction between tumor-associated macrophages and non-small cell lung cancer cells by targeting HDGF.

ETHNOPHARMACOLOGICAL RELEVANCE: Marsdenia tenacissima (Roxb.) Wight et Arn. is a traditional Chinese herbal medicine, and its water-soluble ingredient Marsdenia tenacissima extract (MTE), was widely used for cancer treatment. The multi-pharmacological efficacies and mechanisms of MTE in directly inhibiting tumor cells have been extensively studied. However, the anti-tumor effects of MTE in the tumor-associated macrophages (TAMs) microenvironment remain unclear. AIM OF THE STUDY: To uncover the role of hepatoma-derived growth factor (HDGF) in the interaction between TAMs and non-small cell lung cancer (NSCLC) cells. To evaluate the anti-tumor effects of MTE on the vicious crosstalk between TAMs and NSCLC by targeting HDGF. MATERIALS AND METHODS: HDGF-overexpression PC-9 and H292 NSCLC cell lines were constructed and verified. RNA-sequencing (RNA-seq) was performed in HDGF-overexpression PC-9 cells to probe the differential expression of genes. THP-1-derived macrophages were characterized using specific markers after stimulation with phorbol-12-myristate 13-acetate (PMA) and rhIL-4 or rhHDGF. The role of HDGF both in NSCLC cells and TAMs was determined using approaches like Western blot, qRT-PCR, ELISA, and flow cytometry. The interaction between tumor cells and TAMs were assessed by indirect co-culture H1975, PC-9 cells with M2 type macrophages. The effects of MTE on anti-tumor and macrophage polarization were evaluated in vitro and in vivo. RESULTS: RNA-seq results identified IL-4 as a critical response to HDGF in NSCLC. HDGF induced macrophages polarizing toward M2 type, and promoted NSCLC cells proliferation, migration and invasion in vitro. On the one hand, HDGF dose-dependently promoted IL-4 expression in NSCLC cells. On the other hand, HDGF induced M2 macrophage polarization through the IL-4/JAK1/STAT3 signaling pathway. MTE treatment significantly decreased the expression and secretion of HDGF in NSCLC cells. Meanwhile, MTE treatment led to M2 macrophage repolarization, as evidenced by decreased expression of M2 markers and increased levels of M1 markers. Importantly, MTE treatment significantly suppressed tumor development in C57BL/6 mice bearing Lewis lung cancer (LLC) cells in vivo, accompanied by decreased plasma HDGF levels, reduced M2 macrophages infiltration and increased M1 macrophages proportion in mice tumor tissues. CONCLUSIONS: HDGF upregulated IL-4 expression in NSCLC cells, and promoted M2 polarization by the IL-4/JAK1/STAT3 signaling pathway in macrophages. MTE disturbed the interaction between NSCLC and TAMs in vitro, and inhibited tumor growth in vivo, at least in part, by suppressing HDGF. Therefore, our present study revealed a novel anti-tumor mechanism of MTE through inhibiting HDGF expression and enhancing macrophage polarization from M2 to M1 phenotype.

Metabolic reprogramming by traditional Chinese medicine and its role in effective cancer therapy.

Metabolic reprogramming, characterized by alterations of cellular metabolic patterns, is fundamentally important in supporting the malignant behaviors of cancer cells. It is considered as a promising therapeutic target against cancer. Traditional Chinese medicine (TCM) and its bioactive components have been used in cancer therapy for an extended period, and they are well-known for their multi-target pharmacological functions and fewer side effects. However, the detailed and advanced mechanisms underlying the anticancer activities of TCM remain obscure. In this review, we summarized the critical processes of cancer cell metabolic reprogramming, including glycolysis, mitochondrial oxidative phosphorylation, glutaminolysis, and fatty acid biosynthesis. Moreover, we systemically reviewed the regulatory effects of TCM and its bioactive ingredients on metabolic enzymes and/or signal pathways that may impede cancer progress. A total of 46 kinds of TCMs was reported to exert antitumor effects and/or act as chemosensitizers via regulating metabolic processes of cancer cells, and multiple targets and signaling pathways were revealed to contribute to the metabolic-modulating functions of TCM. In conclusion, TCM has its advantages in ameliorating cancer cell metabolic reprogramming by its poly-pharmacological actions. This review may shed some new light on the explicit recognition of the mechanisms of anticancer actions of TCM, leading to the development of natural antitumor drugs based on reshaping cancer cell metabolism.

Bu-Fei decoction and modified Bu-Fei decoction inhibit the growth of non-small cell lung cancer, possibly via inhibition of apurinic/apyrimidinic endonuclease 1.

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a ubiquitous multifunctional protein, which possesses DNA repair and redox activities. High levels of APE1 are associated with chemo­ and radioresistance, and poor prognosis in various types of cancer, including non­small cell lung cancer (NSCLC). Bu­Fei decoction (BFD) is a traditional Chinese herbal formula, which is believed to supplement Qi, clear away heat and nourish the lungs. BFD and modified Bu­Fei decoction (MBFD) have been used in China to treat patients with lung cancer. The present study aimed to evaluate the potential antitumor effects of BFD and MBFD on NSCLC in vitro and in vivo. In addition, the possible contribution of APE1 was examined. MTT assay was used to investigated the anti-tumor activity of BFD and MBFD on H1975 and H292 NSCLC cell lines. The DNA damage of cells in the control and the experimental groups was detected using comet assay. The in vivo anti-tumor effects of BFD and MBFD were evaluated in a NSCLC tumor nude mouse xenograft model. Polymerase chain reaction (PCR), reverse transcription­quantitative PCR (RT­qPCR) analysis and western blot analysis were applied to analyze the mRNA and protein expression levels of APE1 in H1975 and H292 cells, so as to the xenograft tumor tissues. The concentration of APE1 in mice plasma was determined using enzyme linked immunosorbent assay (ELISA). In vitro, BFD and MBFD inhibited the growth of cultured H1975 and H292 NSCLC cells. The results of a comet assay revealed that BFD and MBFD increased DNA damage. Furthermore, the expression levels of APE1 were decreased in response to BFD and MBFD at the mRNA and protein levels. In mice carrying NSCLC xenografts, BFD and MBFD inhibited tumor growth and decreased APE1 expression. In addition, in normal human lung bronchial epithelial BEAS­2B cells, the half maximal inhibitory concentrations of BFD and MBFD were much higher compared with in NSCLC cells, and they had no effect on DNA damage. These results suggested that BFD and MBFD may inhibit the growth of NSCLC, possibly by inhibiting APE1 expression.

Shugan Liangxue Decoction () Down-Regulates Estrogen Receptor α Expression in Breast Cancer Cells.

OBJECTIVE: To observe the effect of Shugan Liangxue Decoction (, SGLXD) on estrogen receptor α (ERα) in human breast cancer cells. METHODS: The effect of SGLXD (0.85-5.10 mg/mL) on the proliferation of breast cancer cells were evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The nuclear ERα protein levels in MCF-7, T47D and ZR-75-1 cells which treated by SGLXD for 24 h were examined by western blot and immunofluorescence assay. MCF-7 and MDA-MB-231 cells were treated by 17ß-estradiol (E2) with or without SGLXD, for 24 h, and the E2 targeted genes c-myc and bcl-2 protein product was evaluated by western blot. RESULTS: SGLXD showed dose-dependent inhibition on the proliferation of MCF-7, T47D and ZR-75-1 cells, but did not inhibit the proliferation of MDA-MB-231 cells. Furthermore, the promotive effect on cell growth induced by E2 was also significantly inhibited by SGLXD treatment. With the treatment of 1.70, 3.40, 5.10 mg/mL SGLXD, the nuclear ERα protein level was reduced to 88.1%, 70.4% and 60.9% in MCF-7 cells, and was decreased to 43.0%, 38.4% and 5.9% in ZR-75-1 cells as compared with the control group. In T47D cells, the nuclear ERα protein was down-regulated to 51.3% and 4.3% by 3.40 and 5.10 mg/mL SGLXD treatment. The down-regulative effect of SGLXD on nuclear ERα was confirmed by immunofluorescence assay. SGLXD decreased the protein product of c-myc and bcl-2. CONCLUSIONS: SGLXD may exhibit selective inhibition effect on the proliferation of ER positive breast cancer cells. SGLXD reduced the nuclear ERα expression and the protein product of E2 target gene c-myc and bcl-2.

Taraxacum mongolicum extract induced endoplasmic reticulum stress associated-apoptosis in triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive and deadly breast cancer subtype with limited treatment options. It is necessary to seek complementary strategies for TNBC management. Taraxacum mongolicum, commonly named as dandelion, is a herb medicine with anti-cancer activity and has been utilized to treat mammary abscess, hyperplasia of mammary glands from ancient time in China, but the scientific evidence and action mechanisms still need to be studied. AIM OF THE STUDY: This study was intended to investigate the therapeutic effect and molecular mechanisms of dandelion extract in TNBC cell line. METHODOLOGY: Dandelion extract was prepared and purified, and then its chemical composition was determined. Cell viability was evaluated by MTT assay. Analysis of cell apoptosis and cell cycle was assessed by flow cytometry. The expression levels of mRNA and proteins were determined by real-time PCR and Western blotting, respectively. Caspase inhibitor Z-VAD-FMK and CHOP siRNA were used to confirm the cell apoptosis induced by dandelion extract. RESULTS: Dandelion extract significantly decreased MDA-MB-231cell viability, triggered G2/M phase arrest and cell apoptosis. Concurrently, it caused a markedly increase of cleaved caspase-3 and PARP proteins. Caspase inhibitor Z-VAD-FMK abolished the apoptosis triggered by dandelion extract. The three ER stress-related signals were strongly induced after dandelion treatment, including increased mRNA expressions of ATF4, ATF6, XBP1s, GRP78 and CHOP genes, elevated protein levels of phosphorylated PERK, eIF-2α, IRE1, as well as the downstream molecules of CHOP and GRP78. MDA-MB-231 cells transfected with CHOP siRNA significantly reduced apoptosis induced by dandelion extract. The underlying mechanisms at least partially ascribe to the strong activation of PERK/p-eIF2α/ATF4/CHOP axis. CONCLUSION: ER stress related cell apoptosis accounted for the anti-cancer effect of dandelion extract, and these findings support dandelion extract might be a potential therapeutic approach to treat TNBC.

Chinese medicine Bu-Fei decoction attenuates epithelial-mesenchymal transition of non-small cell lung cancer via inhibition of transforming growth factor ß1 signaling pathway in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine Bu-Fei decoction (BFD) has been utilized to treat patients with Qi deficiency for decades, with the advantages of invigorating vital energy, clearing heat-toxin and moistening lung, etc. According to previous clinical experience and trials, BFD has been found to indeed improve life quality of lung cancer patients and prolong survival time. Nevertheless, little is known on its potential mechanisms so far. Being regarded as a pivotal cytokine in the tumor microenvironment, transforming growth factor ß (TGF-ß) stands out as a robust regulator of epithelial-mesenchymal transition (EMT), which is closely linked to tumor progression. AIM OF THE STUDY: The present study was designed to explore whether BFD antagonized EMT via blocking TGF-ß1-induced signaling pathway, and then help contribute to create a relatively steady microenvironment for confining lung cancer. MATERIALS AND METHODS: This experiment was performed in lung adenocarcinoma A549 cells both in vitro and in vivo. In detail, the influences mediated by TGF-ß1 alone or in combination with different concentrations of BFD on migration were detected by wound healing and transwell assays, and the effects of BFD on cell viability were determined by cell counting kit-8 (CCK-8) assay. TGF-ß1, EMT relevant proteins and genes were evaluated by western blotting, confocal microscopy, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC) and enzyme-linked immuno sorbent assay (ELISA). Female BALB/C nude mice were subcutaneously implanted A549 cells and given BFD by gavage twice daily for 28 days. The tumor volume was monitored every 4 days to draw growth curve. The tumor weight, expression levels of EMT-related protein in tumor tissues and TGF-ß1 serum level were evaluated, respectively. RESULTS: BFD only exerted minor effects on A549 cell proliferation and this was in accordance with the in vivo result, which showed that the tumor growth and weight were not be restrained by BFD administration. However, the data elucidated that BFD could dose-dependently suppress EMT induced by TGF-ß1 in vitro via attenuating canonical Smad signaling pathway. In the A549 xenograft mouse model, BFD also inhibited protein markers that are associated with EMT and TGF-ß1 secretion into serum. CONCLUSIONS: Based on these above data, the conclusion could be put forward that BFD probably attenuated TGF-ß1 mediated EMT in A549 cells via decreasing canonical Smad signaling pathway both in vitro and in vivo, which may help restrain the malignant phenotype induced by TGF-ß1 in A549 cells to some extent.

High-performance liquid chromatography coupled with tandem mass spectrometry technology in the analysis of Chinese Medicine Formulas: A bibliometric analysis (1997-2015).

There is a recognized challenge in analyzing traditional Chinese medicine formulas because of their complex chemical compositions. The application of modern analytical techniques such as high-performance liquid chromatography coupled with a tandem mass spectrometry has improved the characterization of various compounds from traditional Chinese medicine formulas significantly. This study aims to conduct a bibliometric analysis to recognize the overall trend of high-performance liquid chromatography coupled with tandem mass spectrometry approaches in the analysis of traditional Chinese medicine formulas, its significance and possible underlying interactions between individual herbs in these formulas. Electronic databases were searched systematically, and the identified studies were collected and analyzed using Microsoft Access 2010, Graph Pad 5.0 software and Ucinet software package. 338 publications between 1997 and 2015 were identified, and analyzed in terms of annual growth and accumulated publications, top journals, forms of traditional Chinese medicine preparations and highly studied formulas and single herbs, as well as social network analysis of single herbs. There is a significant increase trend in using high-performance liquid chromatography coupled with tandem mass spectrometry related techniques in analysis of commonly used forms of traditional Chinese medicine formulas in the last 3 years. Stringent quality control is of great significance for the modernization and globalization of traditional Chinese medicine, and this bibliometric analysis provided the first and comprehensive summary within this field.

Recent highlights of Chinese medicine for advanced lung cancer.

Owing to its unique superiority in improving quality of life and prolonging survival time among advanced lung cancer patients, Chinese medicine (CM) has, in recent years, received increased attentions worldwide. We utilized a bibliometric statistical method based on MEDLINE/GoPubMed to conduct a comprehensive analysis of the current application status of CM in lung cancer, by including annual and accumulated publications, origin distribution of countries and journals, and keywords with a higher frequency score. Then the relevant clinical trials and mechanistic studies were systematically summarized within the field according to research types. We have raised potential problems and provided potentially useful reference information that could guide similar studies in the future. The basic experimental results are highly consistent with clinical trials, leading us to conclude that CM can offer better overall therapeutic benefits when used in combination with routine Western medicine for patients with advanced lung cancer.

Injectable Chinese herbal formula Kang'ai for nonsmall cell lung cancer: Trial sequential analysis of 2,259 participants from 31 randomized controlled trials.

OBJECTIVE: The aim was to evaluate the efficacy and safety of Kang' ai (KA) injection for patients with nonsmall cell lung cancer (NSCLC). Furthermore, to identify if more trials are needed before reliable conclusions could be drawn with regard to these outcomes. MATERIALS AND METHODS: We searched the Cochrane library, PubMed, EMBASE, VIP, CBMdisc, and CNKI in September 2012, and then an additional updated search was conducted in January 2013. Only relevant randomized controlled trials (RCTs) on KA injection plus first-line cisplatin-based chemotherapy in the treatment of NSCLC were identified. Trials' data was reviewed and extracted by two reviewers independently. The quality of included studies was assessed according to a statement from Cochrane Handbook. RevMan 5 Software and Trial sequential analysis (TSA) software were applied for data analyses. RESULTS: A total of 31 RCTs involving 2259 patients were included. The results of meta-analysis showed that compared with chemotherapy alone, the combination of KA injection plus chemotherapy had a statistically significant benefit in improving clinical response rate (relative risk [RR] =1.29, 95% confidence interval [CI]: 1.17-1.41, P < 0.00001), clinical benefit rate (RR = 1.19, 95% CI: 1.14-1.25, P < 0.00001) and quality of life (RR = 1.79, 95% CI: 1.63-1.98, P < 0.00001); hematological toxicity (white blood cell) (RR = 0.71, 95% CI: 0.66-0.76, P < 0.00001) and nonhematological toxicity (nausea and vomiting) (RR = 0.73, 95% CI: 0.65-0.83, P < 0.00001) were improved as well. TSA showed that all cumulative Z-score crossed their monitoring boundaries, demonstrating that no more trials are needed before reliable conclusions could be drawn. CONCLUSION: Current evidence presented that KA injection might improve the therapeutic effect when combined with chemotherapy. Moreover, no more trials are needed in future according to TSA. Nevertheless, additional randomized studies investigating KA injection are needed to be further evaluated.

Metabolic profiling of tenacigenin B, tenacissoside H and tenacissoside I using UHPLC-ESI-Orbitrap MS/MS.

Marsdenia tenacissima, which is widely used as an anticancer herb in traditional Chinese medicine, has been shown to possess anticancer activity. However, its metabolic profile is poorly investigated. Tenacigenin B is the major steroidal skeleton of C-21 steroids in M. tenacissima. Tenacissoside H and Tenacissoside I are detected at relatively high levels in M. tenacissima. Therefore, we studied their metabolic characteristics in human liver microsomes by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry. Fourteen metabolites were tentatively identified by accurate mass measurement and MS/MS fragmentation behavior. It was found that hydroxylation reactions were the major metabolic pathway of Tenacissoside H and Tenacissoside I in human liver microsomes, whereas the metabolic pathway of Tenacigenin B involved dehydrogenation reactions. This is the first time that the metabolic profile of C-21 steroids from M. tenacissima has been explored in human liver microsomes, which is of great significance for subsequent pharmacokinetic and interaction research. Biotransformation in vivo or in vitro may influence the structure of a compound and change its activity. Identification of their fragmentation behaviors and metabolites provides valuable and new information for further understanding the anti-tumor activity of M. tenacissima. Copyright © 2016 John Wiley & Sons, Ltd.

Recent highlights of experimental research for inhibiting tumor growth by using Chinese medicine.

To give an overview of contemporary experimental research using Chinese medicine (CM) for the treatment of cancer. As an integral part of mainstream medicine in the People's Republic of China, CM emphasizes improvements in holistic physical condition instead of merely killing tumor cells, which is consistent with the current medical model that advocates patient-oriented treatment. Great progress has been made in experimental research, and the principle aspects include anti-tumor angiogenesis, inducing apoptosis and differentiation, reversing multidrug resistance, and improving immune function. As a current hot topic in cancer research, tumor microenvironment (TME) highlights the mutual and interdependent interaction between tumor cells and their surrounding tissues, and the CM treatment concept bears a striking resemblance to it. To date, primary points of TME include extracellular matrix remodeling, inflammation, hypoxia, and angiogenesis, but trials using CM with a focus on TME are rare. Despite considerable recent development, experimental research on CM for solving cancer issues appears insufficient. Greater efforts in this field are urgently needed.

Marsdenia tenacissima extract enhances gefitinib efficacy in non-small cell lung cancer xenografts.

PURPOSE: The stem of Marsdenia tenacissima (Roxb.) Wight et Arn. has long been used as a medicine to treat cancer in China. Our previous in vitro results showed that Marsdenia tenacissima extract (MTE) overcomes gefitinib resistance in non-small cell lung cancer (NSCLC) cells. However, it is unknown whether MTE could enhance gefitinib efficacy in vivo. The present study was intended to investigate the in vivo anti-tumour activity of MTE combined with gefitinib. METHODS: Human NSCLC H460 (K-ras mutation) or H1975 cells (EGFR T790M mutation) were subcutaneously inoculated into nude mice. Tumour volume and body weight were measured regularly. Resected tumours were weighed after the animals were sacrificed. Immunoblotting or immunohistochemistry was used to assess the cellular proliferation and apoptosis in xenograft tumour tissue. Expression of the EGFR downstream pathways and c-Met were measured with western blot analysis to explore possible mechanisms. RESULTS: MTE (5, 10, 20 g/kg) dose-dependently reduced tumour growth and induced cell apoptosis. MTE suppressed EGFR related signals, and 20 g/kg was the most effective dose. Low-dose MTE (5 g/kg) significantly enhanced gefitinib efficacy in resistant H460 and H1975 xenografts. The combination inhibited tumour proliferation and induced cell apoptosis in both resistant NSCLC xenografts. Constitutive activation of the PI3K/Akt and MEK/ERK pathways is related to EGFR-TKI resistance. Accordingly, phosphorylation of PI3K/Akt/mTOR and ERK1/2 was suppressed after combination treatment. Simultaneously, cross-talked c-Met and EGFR were also prominently lowered in the presence of MTE combined with gefitinib. CONCLUSION: The present results suggest that the combination of MTE and gefitinib may be a promising therapeutic approach to enhance gefitinib efficacy in resistant NSCLC.

[Prevention and treatment of aromatase inhibitor-associated bone loss by shugan jiangu recipe in postmenopausal women with breast cancer: a clinical study].

OBJECTIVE: To study the effect of Shugan Jiangu Recipe (SJR) on bone mineral density (BMD) and serum bone metabolic biochemical markers in postmenopausal breast cancer patients with osteopenia. METHODS: Totally 38 patients of postmenopausal women with breast cancer, who received aromatase inhibitors (AIs), were assigned to the treatment group (21 cases) and the control group (17 cases) by using random digit table. All patients took Caltrate D Tablet (containing Ca 600 mg and Vit D3 125 IU), one tablet daily. Patients in the treatment group took SJR, 6 g each time, twice daily for 6 successive months. The bone mineral density (BMD) level was detected before treatment and at months 6 after treatment. Levels of bone alkaline phosphatase (BALP), bone gla protein (BGP), tartrate-resistant acid phosphatase (TRAP), and C-terminal telopeptide of type II collagen (CTX-II) were detected by enzyme linked immunosorbent assay (ELISA). The drug safety was also assessed. RESULTS: Compared with before treatment, BMD of L2-4 and femur neck obviously increased in the treatment group at month 6 after treatment (P < 0.01), serum BALP and TRAP decreased (P < 0.05). Compared with before treatment, BMD of L2-4 and femur neck obviously decreased in the control group at month 6 after treatment (P < 0.05), serum BALP and TRAP increased (P < 0.01). Compared with the control group, lumbar and femur neck BMD obviously increased, serum levels of BGP and BALP obviously decreased, and serum levels of CTX-II and TRAP obviously increased in the treatment group at month 6 after treatment (P < 0.01). No serious adverse event occurred during the treatment period. Bone fracture occurred in one case of the control group (5.8%). CONCLUSION: SJR could attenuate bone loss of postmenopausal women with breast cancer who received AIs, increase BMD and improve abnormal bone metabolism.

Enhancement of gefitinib-induced growth inhibition by Marsdenia tenacissima extract in non-small cell lung cancer cells expressing wild or mutant EGFR.

BACKGROUND: Non-small cell lung cancer (NSCLC) expressed high levels of epidermal growth factor receptor (EGFR). Gefitinib (Iressa) has demonstrated clinical efficacy in NSCLC patients harboring EGFR mutations or refractory to chemotherapy. However, most of NSCLC patients are with wild type EGFR, and showed limited response to gefitinib. Therefore, to develop new effective therapeutic interventions for NSCLC is still required. Our previous study showed Marsdenia tenacissima extract (MTE) restored gefitinib efficacy in the resistant NSCLC cells, but whether MTE acts in the gefitinib-sensitive NSCLC cells is the same as it in the resistant one is unknown. METHODS: Dose response curves for gefitinib and MTE were generated for two sensitive NSCLC cell lines with mutant or wild type EGFR status. Three different sequential combinations of MTE and gefitinib on cell growth were evaluated using IC50 and Combination Index approaches. The flow cytometric method was used to detect cell apoptosis and cell cycle profile. The impact of MTE combined with gefitinib on cell molecular network response was studied by Western blotting. RESULTS: Unlike in the resistant NSCLC cells, our results revealed that low cytotoxic dose of MTE (8 mg/ml) combined gefitinib with three different schedules synergistically or additively enhanced the growth inhibition of gefitinib. Among which, MTE→MTE+gefitinib treatment was the most effective one. MTE markedly prompted cell cycle arrest and apoptosis caused by gefitinib both in EGFR mutant (HCC827) and wild type of NSCLC cells (H292). The Western blotting results showed that MTE→MTE+gefitinib treatment further enhanced the suppression of gefitinib on cell growth and apoptosis pathway such as ERK1/2 and PI3K/Akt/mTOR. This combination also blocked the activation of EGFR and c-Met which have cross-talk with each other. Unlike in gefitinib-resistant NSCLC cells, MTE alone also demonstrated certain unexpected modulation on EGFR related cell signal pathways in the sensitive cells. CONCLUSION: Our results suggest that MTE is a promising herbal medicine to improve gefitinib efficacy in NSCLC regardless of EGFR status. However, why MTE acted differently between gefitinib-sensitive and -resistant NSCLC cells needs a further research.

Anti-tumor effect of Shu-Gan-Liang-Xue decoction in breast cancer is related to the inhibition of aromatase and steroid sulfatase expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Shu-Gan-Liang-Xue Decoction (SGLXD), a traditional Chinese herbal formula used to ameliorate the hot flushes in breast cancer patients, was reported to have anti-tumor effect on breast cancer. Estrogen plays a critical role in the genesis and evolution of breast cancer. Aromatase and steroid sulfatase (STS) are key estrogen synthesis enzymes that predominantly contribute to the high local hormone concentrations. The present study was to evaluate the anti-tumor effect of SGLXD on estrogen receptor (ER) positive breast cancer cell line ZR-75-1, and to investigate its underlying mechanisms both in vitro and in vivo. MATERIALS AND METHODS: The anti-tumor activity of SGLXD in vitro was investigated using the MTT assay. The in vivo anti-tumor effect of SGLXD was evaluated in non-ovariectomized and ovariectomized athymic nude mice. The effect of SGLXD on enzymatic activity of aromatase and STS was examined using the dual-luciferase reporter (DLR) based on bioluminescent measurements. Aromatase and STS protein level were assessed using Western blot assay. RESULTS: SGLXD showed dose-dependent inhibitory effect on the proliferation of ZR-75-1 cells with IC50 value of 3.40 mg/mL. It also suppressed the stimulating effect on cell proliferation of testosterone and estrogen sulfates (E1S). Oral administration of 6 g/kg of SGLXD for 25 days resulted in a reduction in tumor volume in non-ovariectomized and ovariectomized nude mice. The bioluminescent measurements confirmed that SGLXD has a dual-inhibitory effect on the activity of aromatase and STS. Western blot assay demonstrated that the treatment of SGLXD resulted in a decrease in aromatase and STS protein levels both in vitro and in vivo. CONCLUSION: Our results suggested that SGLXD showed anti-tumor effect on breast cancer cells both in vitro and in vivo. The anti-tumor activity of SGLXD is related to inhibition of aromatase and STS via decreasing their expression. SGLXD may be considered as a novel treatment for ER positive breast cancer.

Clinical study on long-term overall survival of advanced non-small-cell lung cancer patients treated with Chinese medicine and Western medicine.

OBJECTIVE: To investigate the prognostic influence on long-term overall survival (OS) from treatment with Chinese medicine (CM) and chemotherapy or targeted therapy in advanced non-small-cell lung cancer (NSCLC) patients. METHODS: The clinical data of 206 advanced NSCLC patients who were treated with CM and Western medicine in Beijing Cancer Hospital from April 1999 to July 2013 were retrospectively analyzed. Long-term survivors were defined as OS ≥ 3 years after treatment with CM and chemotherapy. Twenty-eight patients had OS ≥ 3 years, 178 had OS < 3 years, and all clinical data were statistically analyzed with the Cox model. Variables were gender, age, smoking status, performance status (PS) score, pathological type, clinical stage, first-line chemotherapy, targeted therapy, and use of CM. Univariate survival analysis was performed using the Kaplan-Meier method and log-rank sequential inspection. Multivariate survival analysis was used to analyze the meaningful factors of univariate survival analysis with the Cox model. RESULTS: The survival rate of patients with OS ≥ 3 years was 13.6% (28/206). Cox multivariate regression analysis showed that PS score, clinical stage, disease control rate to first-line chemotherapy, and use of CM were independent factors of longterm OS (all <0.05). However, gender, age, smoking, and use of epidermal growth factor receptor tyrosine-kinase inhibitor were not significant (P>0.05). CONCLUSION: PS score, clinical stage, disease control rate to first-line chemotherapy, and use of CM are probably independent prognostic factors for long-term OS in patients with advanced NSCLC.

Marsdenia tenacissima extract inhibits gefitinib metabolism in vitro by interfering with human hepatic CYP3A4 and CYP2D6 enzymes.

ETHNOPHARMACOLOGICAL RELEVANCE: The stem of Marsdenia tenacissima (Roxb.) Wight et Arn. is mainly produced in Yunnan China and has long been used as a medicine to treat cancer in China. Xiao-Ai-Ping injection, the water-soluble part of the stem of Marsdenia tenacissima, is administrated as an anti-cancer agent in clinics for decades. Our previous study showed that Marsdenia tenacissima extract (MTE) restored gefitinib sensitivity in gefitinib-resistant non-small cell lung cancer (NSCLC) cells, but the mechanism involved is unknown. Gefitinib undergoes hepatic metabolism predominantly through human cytochrome P450 (CYP) 3A4 and CYP2D6 enzymes. This study aims to evaluate whether MTE interferes with gefitinib metabolism via human hepatic P450 enzymes. MATERIAL AND METHODS: A cocktail-substrate assay was used to test the effect of MTE on major CYP enzyme activities by incubation of pooled human liver microsomes with specific substrate probes of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in the absence and presence of MTE. Recombinant human CYP450 enzymes were used to predict in vitro gefitinib metabolic clearance in the absence and presence of MTE. The metabolites of the substrate probes and gefitinib were detected by high-performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS). Human hepatoma HepG2 cells were used to investigate the effect of gefitinib alone or in combination with MTE on CYP3A4 and CYP2D6 mRNA and protein expression. RESULTS: The cocktail-substrate assay showed that MTE inhibited CYP450 activities in human liver microsomes with the inhibition rate of 3A4>2C9>2C19>1A2>2D6. The co-administration of MTE with gefitinib significantly decreased the in vitro intrinsic clearance (Clint) of gefitinib by 2.6 and 4.0-fold for CYP2D6 and CYP3A4, respectively, but did not affect other CYP450s. CYP2D6 and CYP3A4 mRNA and protein expression in human hepatoma HepG2 cells were greatly reduced in the combined gefitinib and MTE treatment. CONCLUSION: We demonstrate that MTE inhibits gefitinib metabolism by interfering with CYP3A4 and CYP2D6. Meanwhile, MTE combined with gefitinib down-regulates the mRNA and protein expression of CYP3A4 and CYP2D6 in the HepG2 cells. Thus, these data suggest that MTE is a promising herbal medicine to enhance gefitinib efficacy through improving its metabolic stability.