ophiopogonin-b and Carcinoma--Non-Small-Cell-Lung

Lung adenocarcinoma is the most common metastatic cancer, and is associated with high patient mortality. Therefore, investigation of anti‑metastatic treatments for lung adenocarcinoma is crucial. Ophiopogonin B (OP‑B) is a bioactive component of Radix Ophiopogon Japonicus, which is often used in Chinese traditional medicine to treat pulmonary disease. Screening of transcriptome and digital gene expression (DGE) profiling data in NSCLC cell lines showed that OP‑B regulated the epithelial‑mesenchymal transition (EMT) pathway in A549 cells. Further results showed that 10 µmol/l OP‑B downregulated EphA2 expression and phosphorylation (Ser897) in A549 cells but upregulated them in NCI‑H460 cells. Meanwhile, the Ras/ERK pathway was unaffected in A549 cells and stimulated in NCI‑H460 cells. More importantly, detection of the EMT pathway showed that OP‑B treatment increased the epithelial markers ZO‑1 and E‑cadherin and decreased the expression of the mesenchymal marker N‑cadherin and the transcriptional repressors Snail, Slug and ZEB1. Furthermore, through Transwell migration and scratch wound healing assays, we found that 10 µmol/l OP‑B significantly reduced the invasion and migration of A549 cells. In vivo, we found that 75 mg/kg OP‑B inhibited A549 cell metastasis in a pulmonary metastasis nude mouse model. In addition, we also found that 10 µmol/l OP‑B significantly inhibited tube formation in EA.hy926 cells. The expression of VEGFR2 and Tie‑2, the phosphorylation of Akt (S473) and PLC (S1248), and the levels of EphA2 and phosphorylated EphA2 (S897) were all inhibited by OP‑B in this cell line. In vivo, using a Matrigel plug assay, we found that OP‑B inhibited angiogenesis and the hemoglobin content of A549 transplanted tumors. Taken together, OP‑B inhibited the metastasis and angiogenesis of A549 cells by inhibiting EphA2/Akt and the corresponding pathway. The investigation gives new recognition to the anticancer mechanism of OP‑B in NSCLC and this compound is a promising inhibitor of metastasis and angiogenesis of lung adenocarcinoma cells.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Proto-Oncogene Proteins c-akt; Receptor, EphA2; Saponins; Signal Transduction; Spirostans; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Ophiopogonin B (OP-B), a saponin compound isolated from Radix Ophiopogon japonicus, was verified to inhibit cell proliferation in numerous non-small cell lung cancer (NSCLC) cells in our previous study. However, the precise mechanisms of action have remained unclear. In the present study, we mainly investigated the effects of OP-B on adenocarcinoma A549 cells to further elaborate the underlying mechanisms of OP-B in different NSCLC cell lines. Detection by high content screening (HCS) and TUNEL assay verified that OP-B induced apoptosis in this cell line, while detection of Caspase-3, Bcl-2 and Bax showed that OP-B induced cell death was caspase and mitochondrial independent. Further experiments showed that OP-B induced cell cycle arrest in the S and G2/M phases by inhibiting the expression of Myt1 and phosphorylation of Histone H3 (Ser10), which resulted in mitotic catastrophe in the cells. Transmission electron microscopy (TEM) observation of cell micro-morphology combined with detection of Atgs by western blot analysis showed that OP-B induced autophagy in this cell line. Autophagy inhibition by the lysosome inhibitor CQ or Beclin1-siRNA knockdown both attenuated cell viability, demonstrated that autophagy also being the vital reason resulted in cell death. More importantly, the xenograft model using A549 cells provided further evidence of the inhibition of OP-B on tumor proliferation. Immunohistochemistry detection of LC3 and Tunel assay both verified that high dose of OP-B (75 mg/kg) induced autophagy and apoptosis in vivo, and western blot detection of p-Histone H3 (Ser10), Survivin and XIAP further indicated the molecular mechanism of OP-B in vivo. As our findings revealed, multiple types of cell death overlapped in OP-B treated A549 cells, it displayed multitarget characteristics of the compounds extracted from the Chinese herbal, which may be used as candidate anticancer medicine in clinic.

Topics: A549 Cells; Apoptosis; Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Proliferation; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Humans; Mitosis; RNA, Small Interfering; Saponins; Spirostans; Xenograft Model Antitumor Assays

Ophiopogonin B (OP-B) is a bioactive component of Radix Ophiopogon Japonicus, which is often used in Chinese traditional medicine to treat pulmonary disease. However, whether or not OP-B has any potential antitumor activity has not been reported. Here, we show that the non-small cell lung cancer (NSCLC) cell lines NCI-H157 and NCI-H460 treated with OP-B grow more slowly and accumulate vacuoles in their cytoplasm compared to untreated control cells. Flow cytometric analysis showed that the cells were arrested in G0/G1 phase. Nuclear morphology, Annexin-V/PI staining, and expression of cleaved caspase-3 all confirm that OP-B does not induce apoptosis. Instead, based on results from both transmission electron microscopy (TEM) and the expression of microtubule-associated protein 1 light chain 3-II (LC3-II), we determined that OP-B treatment induced autophagy in both cell lines. Next, we examined the PI3K/Akt/mTOR signaling pathway and found that OP-B inhibited phosphorylation of Akt (Ser473, Thr308) in NCI-H157 cells and also inhibited several key components of the pathway in NCI-H460 cells, such as p-Akt(Ser473, Thr308), p-p70S6K (Thr389). Additionally, insulin-mediated activation of the PI3K/Akt/mTOR pathway provides evidence that activation of this pathway may correlate with induction of autophagy in H460 cells. Therefore, OP-B is a prospective inhibitor of PI3K/Akt and may be used as an alternative compound to treat NSCLC.

Topics: Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cytoplasm; Humans; Lung Neoplasms; Microtubule-Associated Proteins; Phosphatidylinositol 3-Kinases; Plant Extracts; Proto-Oncogene Proteins c-akt; Saponins; Signal Transduction; Spirostans; Vacuoles