Crosstalk with cancer-associated fibroblasts induces resistance of non-small cell lung cancer cells to epidermal growth factor receptor tyrosine kinase inhibition.

Although lung cancers with activating mutations in the epidermal growth factor receptor (EGFR) are highly sensitive to selective EGFR tyrosine kinase inhibitors (TKIs), these tumors invariably develop acquired drug resistance. Host stromal cells have been found to have a considerable effect on the sensitivity of cancer cells to EGFR TKIs. Little is known, however, about the signaling mechanisms through which stromal cells contribute to the response to EGFR TKI in non-small cell lung cancer. This work examined the role of hedgehog signaling in cancer-associated fibroblast (CAF)-mediated resistance of lung cancer cells to the EGFR TKI erlotinib. PC9 cells, non-small cell lung cancer cells with EGFR-activating mutations, became resistant to the EGFR TKI erlotinib when cocultured in vitro with CAFs. Polymerase chain reaction and immunocytochemical assays showed that CAFs induced epithelial to mesenchymal transition phenotype in PC9 cells, with an associated change in the expression of epithelial to mesenchymal transition marker proteins including vimentin. Importantly, CAFs induce upregulation of the 7-transmembrane protein smoothened, the central signal transducer of hedgehog, suggesting that the hedgehog signaling pathway is active in CAF-mediated drug resistance. Indeed, downregulation of smoothened activity with the smoothened antagonist cyclopamine induces remodeling of the actin cytoskeleton independently of Gli-mediated transcriptional activity in PC9 cells. These findings indicate that crosstalk with CAFs plays a critical role in resistance of lung cancer to EGFR TKIs through induction of the epithelial to mesenchymal transition and may be an ideal therapeutic target in lung cancer.

Aucubin promotes neurite outgrowth in neural stem cells and axonal regeneration in sciatic nerves.

Aucubin is an iridoid glycoside with a wide range of biological activities, including anti-inflammatory, anti-microbial, anti-algesic as well as anti-tumor activities. Recently, it has been shown that aucubin prevents neuronal death in the hippocampal CA1 region in rats with diabetic encephalopathy. In addition, it has protective effects on H2O2-induced apoptosis in PC12 cells. We have shown here that aucubin promotes neuronal differentiation and neurite outgrowth in neural stem cells cultured primarily from the rat embryonic hippocampus. We also investigated whether aucubin facilitates axonal elongation in the injured peripheral nervous system. Aucubin promoted lengthening and thickness of axons and re-myelination at 3 weeks after sciatic nerve injury. These results indicate that administration of aucubin improved nerve regeneration in the rat model of sciatic nerve injury, suggesting that aucubin may be a useful therapeutic compound for the human peripheral nervous system after various nerve injuries.

Establishment and characterization of a lung cancer cell line, SMC-L001, from a lung adenocarcinoma.

Lung cancer cell lines are a valuable tool for elucidating lung tumorigenesis and developing novel therapies. However, the majority of cell lines currently available were established from tumors in patients of Caucasian origin, limiting our ability to investigate how cancers in patients of different ethnicities differ from one another in terms of tumor biology and drug responses. In this study, we established a human non-small cell lung carcinoma cell line, SMC-L001, and characterized its genome and tumorigenic potential. SMC-L001 cells were isolated from a Korean lung adenocarcinoma patient (male, pStage IIb) and were propagated in culture. SMC-L001 cells were adherent. DNA fingerprinting analysis indicated that the SMC-L001 cell line originated from parental tumor tissue. Comparison of the genomic profile of the SMC-L001 cell line and the original tumor revealed an identical profile with 739 mutations in 46 cancer-related genes, including mutations in TP53 and KRAS. Furthermore, SMC-L001 cells were highly tumorigenic, as evidenced by the induction of solid tumors in immunodeficient mice. In summary, we established a new lung cancer cell line with point mutations in TP53 and KRAS from a Korean lung adenocarcinoma patient that will be useful for investigating ethnic differences in lung cancer biology and drug response.

Tumor-stromal interactions with direct cell contacts enhance motility of non-small cell lung cancer cells through the hedgehog signaling pathway.

The metastatic potential of non-small cell lung cancer (NSCLC) has been shown to be associated with interactions with the tumor microenvironment, which primarily comprises of cancer-associated fibroblasts (CAFs). Heterotypic cell-cell interactions occur via released signaling molecules and direct physical contact. To investigate the differential contribution of direct cell-cell contact and paracrine signaling factors to NSCLC metastasis, we performed two types of co-cultures: direct co-cultures of the NSCLC cell line H358 with primary cultures of CAFs from patients with resected NSCLC; and indirect co-cultures across a separable membrane. We showed that CAFs more potently induce epithelial-to-mesenchymal transition (EMT) in NSCLC H358 cells through direct contacts than through indirect interactions, as indicated by an elongated and disseminated appearance. Immunocytochemical experiments show that EMT accompanies the expression of mesenchymal cytoskeletal proteins, including vimentin. However, H358 cells proliferate more slowly in direct co-culture than in indirect co-culture. Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed that H358 cells in direct contact with CAFs up-regulate the expression of the pan-mesenchymal markers α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), transforming growth factor-ß (TGFß) signaling effector SMAD family number-3 (SMAD3), and hedgehog signaling effector GLI family zinc finger-1 (GLI1), compared with the indirect co-culture system. Furthermore, we found that the direct GLI1 transcription targets snail family zinc finger-1 (SNAI1) and SNAI2 are up-regulated, suggesting that the hedgehog signaling pathway is active in direct co-culture. A scratch wound assay showed that direct contact co-culture increases the motility of H358 cells. In conclusion, these findings provide evidence that paracrine factors and direct physical contact between NSCLC cells and CAFs might control the metastatic potential of NSCLC through the hedgehog signaling pathway.

Human lung cancer-associated fibroblasts enhance motility of non-small cell lung cancer cells in co-culture.

The metastatic potential of non-small cell lung cancer (NSCLC) cells has been shown to be associated with the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment, regulating tumor cell function by secreting growth factors, chemokines, and extracellular matrix (ECM). In this study, we examined the role of CAFs in the tumor progression of NSCLC. Firstly, we established primary cultures of CAFs and matched normal fibroblasts (NFs) from patients with resected NSCLC. CAFs exhibited greater expression of the pan-mesenchymal marker α-smooth muscle actin (α-SMA) than did NFs, although they displayed similar morphology. Furthermore, we employed a direct co-culture assay with human NSCLC A549 and H358 cells, and found that CAFs were more potent in inducing the epithelial-to-mesenchymal transition (EMT) phenotype than NFs, as indicated by an elongated and disseminated appearance. CAF-induced EMT led to an increase in motility and a decrease in proliferation of NSCLC cells through SMAD family number-3 (SMAD3)-dependent up-regulation of the growth inhibitory gene p21(CIP1) [cyclin-dependent kinase inhibitor-1A (CDKN1A)] and α-SMA. Taken together, these findings provide evidence that lung CAFs have tumor-promoting capacity distinct from NFs and might play a significant role in the metastatic potential of NSCLC.

Memory improvement in ibotenic acid induced model rats by extracts of Scutellaria baicalensis.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria baicalensis Georgi (Labiatae) extracts have been used as traditional Korean medicine, to treat cerebral ischemia in addition to bacterial infection and inflammatory diseases. AIM OF THE STUDY: The improvement effect on learning and memory by the administration of Scutellaria baicalensis extracts was evaluated and the underlying mechanisms were investigated. MATERIALS AND METHODS: Memory behavior was tested by the passive avoidance test and Y-maze test. We also investigated the cells expressing neuronal markers related to memory processes by immunofluorescence staining analysis in memory deficient animal model (Ibo model) rats and in hippocampal progenitor cells. RESULTS: We found neuronal cells immunoreactive to choline acetyltransferase (ChAT), a marker for cholinergic neurons were increased in the hippocampus, while cells producing GABA and glutamate were not after 30 mg/kg Scutellaria baicalensis administration. Futhermore, Scutellaria baicalensis extracts enhanced the survival of a hippocampal progenitor cell line, HiB5 and its differentiation to ChAT immunoreactive cells. The increased expression of memory related neurotransmitter, NMDA receptor and a reduction of activated microglia in the hippocampus were also observed in the Ibo model when administrated Scutellaria baicalensis extracts. CONCLUSIONS: These results imply that Scutellaria baicalensis has significant neuroprotective effects in the Ibo model.