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

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. 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. 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. The present results suggest that the combination of MTE and gefitinib may be a promising therapeutic approach to enhance gefitinib efficacy in resistant NSCLC.

Han SY ，Zhao W ，Sun H ，Zhou N ，Zhou F ，An G ，Li PP ... -  《-》

Marsdenia tenacissima extract restored gefitinib sensitivity in resistant non-small cell lung cancer cells.

Most non-small cell lung cancer (NSCLC) patients responding to gefitinib harbor activating mutations in the epidermal growth factor receptor (EGFR). However, the responsive cases eventually develop the resistance to gefitinib. Besides, K-ras mutations were identified as the primary resistance to gefitinib. We investigated whether Marsdenia tenacissima extract (MTE, trade name: Xiao-Ai-Ping injection) combined with gefitinib could overcome the resistance of NSCLC cells to gefitinib. NSCLC cell lines with different sensitivities to gefitinib were studied. Cell growth and apoptosis were evaluated by MTT assay and flow cytometry, respectively. The EGFR-related signaling molecule phosphorylation was assessed by Western blotting. We found that MTE inhibited cell growth in gefitinib-sensitive and -resistant cells. In gefitinib-resistant cells, the MTE→MTE+gefitinib (M→M+G) treatment was most potent over the concurrent administration of MTE and gefitinib (M+G) or gefitinib→gefitinib+MTE (G→G+M) treatment in cell growth inhibition and apoptosis induction. The M→M+G treatment significantly reduced the phosphorylation of EGFR downstream signaling molecules PI3K/Akt/mTOR and ERK, on which MTE and gefitinib alone had no obvious effects on the resistant cells. The M→M+G treatment attenuated c-Met phosphorylation in H460 and H1975 as well. Thus, we found that the M→M+G treatment improved the sensitivity of resistant NSCLC cells carrying T790M or K-ras mutations to gefitinib, suggesting that the M→M+G treatment may be a promising therapeutic strategy to overcome gefitinib resistance in NSCLC.

Han SY ，Zhao MB ，Zhuang GB ，Li PP ... -  《-》

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

Han SY ，Ding HR ，Zhao W ，Teng F ，Li PP ... -  《BMC Complementary and Alternative Medicine》

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

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. 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. 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. 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.

Han SY ，Zhao HY ，Zhou N ，Zhou F ，Li PP ... -  《-》

Marsdenia tenacissima extract promotes gefitinib accumulation in tumor tissues of lung cancer xenograft mice via inhibiting ABCG2 activity.

Marsdenia tenacissima extract (MTE) is the water-soluble part of a traditional Chinese medicine Marsdenia tenacissima (Roxb.) Wight & Arn, and is commercially available in China for treating cancers. MTE has been revealed to be effective in improving gefitinib efficacy in treating non-small cell lung cancer (NSCLC). However, the mechanisms remain to be defined. To determine the effects of MTE on gefitinib metabolism and accumulation in vivo, and to explore the underlying mechanisms. MTE or vehicle were intraperitoneally administrated to the H1975 xenograft model, followed by intragastric administration of gefitinib 12 h later. Mice plasma, tumors and liver tissues were harvested for further analysis. Hoechst 33342, a specific substrate of ATP Binding Cassette Subfamily G Member 2 (ABCG2), was used to determine the effects of MTE on activities of ABCG2 in tumor cells. A higher concentration of plasma gefitinib was detected in MTE-treated mice at 24 h after delivery of gefitinib, however, it became insignificant in another 24 h. By contrast, gefitinib levels were continuously higher in MTE-pretreated mice tumor tissues at 12-48 h post gefitinib administration. MTE suppressed plasma levels of gefitinib metabolites (M523595, M608236 and M537194) in the first 24 h after gefitinib delivery, and inhibited activities of liver CYP2D6 and CYP3A4 at early stage (within 6 h) after gefitinib treatment. Strikingly, the activities of ABCG2, the primary drug transporter for gefitinib, were significantly inhibited by MTE in H1975 lung cancer cells. Further, it was identified that tenacissoside H, but not tenacissoside I, may contribute to the ABCG2-suppressive effects of MTE. MTE pretreatment temporarily elevated plasma concentrations of gefitinib via inhibiting CYP450 enzymes. Most importantly, MTE promoted gefitinib accumulation in tumor tissues in a long-lasting manner via decreasing activities of ABCG2, a drug transporter responsible for gefitinib efflux.

Zhao C ，Hao H ，Zhao H ，Ren W ，Jiao Y ，An G ，Sun H ，Han S ，Li P ... -  《-》