Ping-Ping Li

Effects of combined Chinese drugs and chemotherapy in treating advanced non-small cell lung cancer

ObjectiveTo evaluate the efficacy and side effects of combined Chinese drugs and chemotherapy in treating advanced non-small cell lung cancer (NSCLC).MethodsSixty-three patients with stage III B and IV NSCLC hospitalized from October 2001 to October 2008 were enrolled and assigned to two groups using a randomizing digital table, with 33 patients in the treatment group and 30 in the control group. They were all treated with the Navelbine and Cisplatin (NP) chemotherapy, but to the treatment group the Chinese drugs Shengmai Injection (生脉注射液) by intravenous dripping and Gujin Granule (固金颗粒冲剂) by oral intake were given additionally. The main observation indexes were response rate (RR), median survival time, 1-year survival rate and median time to progression (TTP); secondary observation indexes were side effects and cycles of chemotherapy.ResultsAltogether, 61 patients (33 from the treatment group and 28 from the control group) completed the observation and were assessable. RR was 48.5% (16/33) in the treatment group and 32.2% (9/28) in the control group, and the median survival time were 13 months and 9 months, respectively; the difference between the two groups was significant (P=0.0373 and P=0.014 respectively). However, the differences between groups were insignificant in terms of 1-year survival rate [51.5% (17/33) vs 46.4% (13/28), P=0.4042], median TTP (5.95 months vs 4.64 months, P=0.3242), grade III or IV bone marrow inhibition occurrence rate [33.3% (11/33) vs 39.3% (11/28), P=0.3500], and mean cycles of chemotherapy applied (2.94±0.94 cycles vs 2.75±0.75 cycles, P=0.4100).ConclusionCombined Chinese drugs and chemotherapy can enhance the short-term therapeutic efficacy in the treatment of NSCLC and prolong patients’ median survival time, but show no evident impact on TTP.

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.

MicroRNA-33a-3p suppresses cell migration and invasion by directly targeting PBX3 in human hepatocellular carcinoma

MicroRNAs (miRNAs) have been shown to function as either oncogenes or tumor suppressors by negatively regulating target genes involved in tumor initiation and progression. In this study, we demonstrated that down-regulation of miR-33a-3p in human primary hepatocellular cancer (HCC) specimens was significantly associated with metastases and poor survival. Over-expression of miR-33a-3p in HepG2 cells remarkably suppressed not only cell growth, migration and invasion, but also tumor growth and metastases in the chick embryo chorioallantoic membrane (CAM) assay, and down-regulated Pre-B-Cell Leukemia Homeobox 3 (PBX3) expression. Conversely, inhibition of miR-33a-3p in Bel-7402 cells resulted in increased of cell growth, spreading and invasion. Furthermore, rescue experiments by over-expression PBX3 completely eliminated the inhibitory effects of miR-33a-3p on tumor growth and metastasis, both in vitro and in vivo. The luciferase assay showed that 3′-untranslated regions (3′-UTRs) of PBX3 were inhibited significantly by miR-33a-3p, while mutations in the miR-33a-3p pairing residues rescued the luciferase expression. Taken together, our findings suggest that miR-33a-3p suppressed the malignant phenotype while also inhibiting PBX3 expression in hepatocellular cancer, implying that miR-33a-3p may be a promising biomarkers and therapy target for HCC intervention.

Shu-Gan-Liang-Xue Decoction, a Chinese herbal formula, down-regulates the expression of steroid sulfatase genes in human breast carcinoma MCF-7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE Shu-Gan-Liang-Xue Decoction (SGLXD), a traditional Chinese herbal formula, has been used to ameliorate hot flushes symptom in breast cancer patients for decades. AIM OF THE STUDY Steroid sulfatase (STS) has a crucial role in regulating the estrogen biosynthesis within breast tumors. We aimed to investigate whether SGLXD could modulate STS expression in human breast carcinoma MCF-7 cells. MATERIALS AND METHODS By semi-quantitative/quantitative reverse transcription-PCR, we investigated the transcript level of STS in MCF-7 cells treated with various concentrations of SGLXD. By using transient cotransfection with estrogen dependent plasmid pERE-TK-Luc and internal control plasmid pRL-TK in MCF-7 cells and dual luciferase reporter (DLR) based bioluminescent measurements, we evaluated the enzymatic activity of STS after SGLXD treatment. RESULTS By RT-PCR and real time PCR, the mRNA level of STS was decreased by SGLXD treatment, in the dose-dependent manner, compared to negative control (p<0.01). By DLR assay, different concentrations of SGLXD significantly inhibited the enzymatic activity of STS in MCF-7 cells dose-dependently (p<0.05). CONCLUSIONS SGLXD could act as a selective estrogen enzyme modulator by down-regulating the STS expression in MCF-7 cells.

Evaluation of estrogenic potential of Shu-Gan-Liang-Xue Decoction by dual-luciferase reporter based bioluminescent measurements in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE Shu-Gan-Liang-Xue Decoction (SGLXD), a cipher prescription of traditional Chinese medicine, has been used to ameliorate hot flushes symptom in breast cancer patients for several decades. However, estrogenic activity of SGLXD is remaining unclear. AIM OF THE STUDY To evaluate the estrogenic potential of SGLXD and each of the component herbs; to investigate the effect of SGLXD on cell viability of MCF-7 cells. MATERIALS AND METHODS We evaluated the estrogenic potential of SGLXD and each of the component herbs with dual-luciferase reporter assay and bioluminescent measurements, by using transient cotransfection with estrogen dependent plasmid pERE-TK-Luc and internal control plasmid pRL-TK in MCF-7 cells. We investigated the effect of SGLXD on cell viability of MCF-7 by MTT assay. RESULTS SGLXD and each of the component herbs did not demonstrate estrogenic activity compared to negative control, even at high concentration (p > 0.05). By MTT assay, different concentrations of SGLXD significantly inhibited the growth of MCF-7 cells dose-dependently (p < 0.05). CONCLUSIONS SGLXD and single herbs do not manifest estrogenic activity, while SGLXD has weak inhibitory effects on MCF-7 cell viability.

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.

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.

Simultaneous determination of gefitinib and its major metabolites in mouse plasma by HPLC–MS/MS and its application to a pharmacokinetics study

Gefitinib (Iressa) is the first oral EGFR tyrosine kinase inhibitor and it brings benefits to non-small cell lung cancer patients with EGFR mutation. In this study, a simple, rapid and credible high performance liquid chromatography-tandem mass spectrometry method was established and validated for the simultaneous quantification of gefitinib and its main metabolites M523595, M537194, M387783 and M608236 in NSCLC tumor-bearing mouse plasma. Sample extraction was done by protein precipitation using acetonitrile containing dasatinib as the internal standard. The chromatography run time was 6min using an Agilent RRHD SB-C18 column with a gradient of acetonitrile and water (0.1% formic acid, v/v). The mass analysis was performed by a triple quadrupole mass spectrometry in positive multiple reaction monitoring mode. The calibration range was 0.5-100ng/mL for M608236 and 1-200ng/mL for other analytes with the correlation coefficients (r(2))≥0.99. For quality control samples, inter- and intra-assay precision was less than 15% and accuracies ranged from 92.6% to 107.58% for all analytes. The extraction recoveries were in the range of 86-105% and no significant matrix effect was observed. This simple and reproducible high-throughput method was successfully applied to the pharmacokinetic study of gefitinib and its major metabolites in mouse.

Pharmacokinetics of Gefitinib: Roles of Drug Metabolizing Enzymes and Transporters

Gefitinib (Iressa, AstraZeneca) has been widely used for the treatment of locally advanced or metastatic non-small cell lung cancer. A number of studies have been reported on its pharmacokinetics profiles, especially on the metabolism. In this review, we have comprehensively summarized the pharmacokinetic characteristics of gefitinib: absorption, distribution, metabolism and excretion (ADME). Overall, gefitinib reached the maximum plasma level relatively fast and distributed extensively. It underwent extensive biotransformation and predominantly excreted in feces, with less than 7% in the urine. CYP450 enzymes played critical roles in the process of gefitinib metabolism. The major enzyme involved in the metabolism was CYP3A4, with other CYP450 enzymes playing a secondary role. A high clearance of gefitinib might result in drug resistance by lowering drug concentration. The enhanced efflux and decreased uptake by transporters were important resistance mechanisms. The transporters involved in pharmacokinetics of gefitinib consist of the ATP-binding cassette and the solute carrier superfamily. Understanding the pharmacokinetics property of gefitinib may provide valuable and new information for dealing with drug resistance and making personalized therapy regarding their interindividual variability.

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