Root Extract of Trichosanthes kirilowii Suppresses Metastatic Activity of EGFR TKI-Resistant Human Lung Cancer Cells by Inhibiting Src-Mediated EMT.

The roots of Trichosanthes kirilowii (TK) have been used in traditional oriental medicine for the treatment of respiratory diseases. In this study, we investigated whether an ethanolic root extract of TK (ETK) can regulate the metastatic potency of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant human lung cancer cells. The relative migration and invasion abilities of erlotinib-resistant PC9 (PC9/ER) and gefitinib-resistant PC9 (PC9/GR) cells were higher than those of parental PC9 cells. Mesenchymal markers were overexpressed, whereas epithelial markers were downregulated in resistant cells, suggesting that resistant cells acquired the EMT phenotype. ETK reduced migration and invasion of resistant cells. The expression levels of N-cadherin and Twist were downregulated, whereas Claudin-1 was upregulated by ETK, demonstrating that ETK suppresses EMT. As a molecular mechanism, Src was dephosphorylated by ETK. The anti-metastatic effect of ETK was reduced by transfecting PC9/ER cells with a constitutively active form of c-Src. Dasatinib downregulated N-cadherin, Twist, and vimentin, suggesting that Src regulates EMT in resistant cells. Notably, CuB played a key role in mediating the anti-metastatic activity of ETK. Collectively, our results demonstrate that ETK can attenuate the metastatic ability of EGFR-TKI-resistant lung cancer cells by inhibiting Src-mediated EMT.

The Ethanolic Extract of Trichosanthes Kirilowii Root Exerts anti-Cancer Effects in Human Non-Small Cell Lung Cancer Cells Resistant to EGFR TKI.

The aim of this study was to investigate whether the ethanol extract of the Trichosanthes kirilowii root (ETK), traditionally used to treat lung diseases, exhibits anticancer activity in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant non-small cell lung cancer (NSCLC) cells. ETK treatment suppressed the growth of EGFR TKI-resistant NSCLC cells, including H1299, H1975, PC9/ER (erlotinib-resistant PC9) and PC9/GR (gefitinib-resistant PC9) cells, in a concentration- and time-dependent manner. Dose-dependent decline in anchorage-dependent and -independent colony formation was also detected following ETK treatment. We demonstrate that the growth-inhibitory effect of ETK was related to apoptosis induction, based on flow cytometry results showing ETK-induced increase in the percentage of cells with sub-G1 DNA and the population of annexin V-positive cells. Consistently, ETK induced chromatin condensation and cleavage of poly(ADP-ribose) polymerase (PARP). As a molecular mechanism, the phosphorylation level of signal transducer and activator of transcription 3 (STAT3) and Src was decreased by ETK. ETK-induced apoptosis was partially reversed by transfection of constitutively activated STAT3, indicating that STAT3 inactivation mediated ETK-induced apoptosis in EGFR TKI-resistant NSCLC cells. Our results provide basic evidence supporting the role of ETK as a novel therapeutic in EGFR TKI-resistant NSCLC.

The Root Extract of Peucedanum praeruptorum Dunn Exerts Anticancer Effects in Human Non-Small-Cell Lung Cancer Cells with Different EGFR Mutation Statuses by Suppressing MET Activity.

The aim of this study was to investigate the anticancer effects of the root extract of Peucedanum praeruptorum Dunn (EPP) in human non-small-cell lung cancer (NSCLC) cells and explore the mechanisms of action. We used four types of human lung cancer cell lines, including H1299 (epidermal growth factor receptor (EGFR) wild-type), PC9 (EGFR Glu746-Ala750 deletion mutation in exon 19; EGFR tyrosine kinase inhibitor (TKI)-sensitive), H1975 (EGFR L858R/T790M double-mutant; EGFR TKI-resistant), and PC9/ER (erlotinib-resistant) cells. EPP suppressed cell growth and the colony formation of NSCLC cells in a concentration-dependent manner. EPP stimulated chromatin condensation, increased the percentage of sub-G1 phase cells, and enhanced the proportion of annexin V-positive cells, demonstrating that EPP triggered apoptosis in NSCLC cells regardless of the EGFR mutation and EGFR TKI resistance status. The phosphorylation level of the signal transducer and activator of transcription 3 (STAT3) and AKT was decreased by EPP. The expression of STAT3 target genes was also downregulated by EPP. EPP reversed hepatocyte growth factor (HGF)-induced MET phosphorylation and gefitinib resistance. Taken together, our results demonstrate that EPP exerted anticancer effects not only in EGFR TKI-sensitive NSCLC cells, but also in EGFR TKI-resistant NSCLC cells, by suppressing MET activity.

Root Bark of Morus Alba L. Induced p53-Independent Apoptosis in Human Colorectal Cancer Cells by Suppression of STAT3 Activity.

The root bark of Morus alba L. (MA) used in traditional oriental medicine exerts various bioactivities including anticancer effects. In this study, we investigated the molecular mechanism underlying the methylene chloride extract of MA (MEMA)-induced apoptosis in colorectal cancer (CRC) cells. We observed that MEMA decreased cell viability and colony formation in both HCT116 p53+/+ cells and HCT116 p53-/- cells. In addition, MEMA increased the sub-G1 phase DNA content, the annexin V-positive cell population, and the expression of apoptosis marker proteins in both cell lines, indicating that MEMA induced apoptosis regardless of the p53 status. Interestingly, the phosphorylation level, transcriptional activity, and target genes expression of signal transducer and activator of transcription 3 (STAT3) were commonly decreased by MEMA. The overexpression of constitutively active STAT3 in HCT116 cells reversed MEMA-induced apoptosis, demonstrating that MEMA-triggered apoptosis was mediated by the inactivation of STAT3. Taken together, we suggest that MEMA can be applied not only to p53 wild-type CRC in the early stages but also to p53-mutant advanced CRC with hyperactivated STAT3. Even though a wide range of studies are required to validate the anticancer effects of MEMA, we propose MEMA as a novel material for the treatment of CRC.

The Root Extract of Scutellaria baicalensis Induces Apoptosis in EGFR TKI-Resistant Human Lung Cancer Cells by Inactivation of STAT3.

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) is a major obstacle in managing lung cancer. The root of Scutellaria baicalensis (SB) traditionally used for fever clearance and detoxification possesses various bioactivities including anticancer effects. The purpose of this study was to investigate whether SB exhibited anticancer activity in EGFR TKI-resistant lung cancer cells and to explore the underlying mechanism. We used four types of human lung cancer cell lines, including H1299 (EGFR wildtype; EGFR TKI-resistant), H1975 (acquired TKI-resistant), PC9/ER (acquired erlotinib-resistant), and PC9/GR (acquired gefitinib-resistant) cells. The ethanol extract of SB (ESB) decreased cell viability and suppressed colony formation in the four cell lines. ESB stimulated nuclear fragmentation and the cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3. Consistently, the proportion of sub-G1 phase cells and annexin V+ cells were significantly elevated by ESB, indicating that ESB induced apoptotic cell death in EGFR TKI-resistant cells. ESB dephosphorylated signal transducer and activator of transcription 3 (STAT3) and downregulated the target gene expression. The overexpression of constitutively active STAT3 reversed ESB-induced apoptosis, suggesting that ESB triggered apoptosis in EGFR TKI-resistant cells by inactivating STAT3. Taken together, we propose the potential use of SB as a novel therapeutic for lung cancer patients with EGFR TKI resistance.

Blue laser-induced selective vasorelaxation by the activation of NOSs.

Phototherapy has been tried for treating cardiovascular diseases. In particular, ultraviolet and blue visible lights were suggested to be useful due to their nitric oxide (NO)-production ability in the skin. However, the effects of blue light on the arterial contractility are controversial. Here, we hypothesized that appropriate protocol of blue laser can induce selective vasorelaxation by activating vasodilating signaling molecules in arteries. Using organ chamber arterial mechanics, NO assay, Matrigel assay, and microarray, we showed that a 200-Hz, 300-µs, 445-nm pulsed-laser (total energy of 600 mJ; spot size 4 mm) induced selective vasorelaxation, without vasocontraction in rat mesenteric arteries. The laser stimulation increased NO production in the cord blood-endothelial progenitor cells (CB-EPCs). Both the laser-induced vasorelaxation and NO production were inhibited by a non-selective, pan-NO synthase inhibitor, L-NG-Nitro arginine methyl ester. Microarray study in CB-EPCs suggested up-regulation of cryptochrome (CRY)2 as well as NO synthase (NOS)1 and NOSTRIN (NOS trafficking) by the laser. In conclusion, this study suggests that the 445-nm blue puled-laser can induce vasorelaxation possibly via the CRY photoreceptors and NOSs activation. The blue laser-therapy would be useful for treating systemic hypertension as well as improving local blood flow depending on the area of irradiation.

The root bark of Morus alba L. regulates tumor-associated macrophages by blocking recruitment and M2 polarization of macrophages.

Tumor-associated macrophages (TAMs) promote tumor growth and metastasis, and are closely related with poor prognosis of cancers. Therefore, TAMs have been an attractive target in cancer therapy. This study investigated whether the root bark of Morus alba L. (MA) regulates TAMs. Methylene chloride extract of MA (MEMA) decreased the migration of RAW264.7 cells and THP-1 macrophages toward cancer cells via inhibition of focal adhesion kinase and Src activity. In addition, MEMA inhibited the phorbol myristate acetate-stimulated secretion of plasminogen activator inhibitor-1 from cancer cells, leading to the decreased chemotaxis of macrophages. Finally, MEMA-suppressed M2 macrophage polarization induced by interleukin (IL)-4/IL-13 or IL-6. MEMA downregulated the mRNA expression of M2 macrophage markers and decreased the phosphorylation of signal transducer and activator of transcription (STAT) 6 and STAT3 in RAW264.7 cells. Suppression of M2 polarization of macrophages by MEMA resulted in the reduced migration of Lewis lung carcinoma cells when the conditioned media from RAW264.7 cells was used as a chemoattractant. Taken together, our results demonstrate that MEMA regulates TAMs by blocking the recruitment of macrophages into tumor microenvironments and by inhibiting M2 polarization of macrophages.

The Root Bark of Morus alba L. Suppressed the Migration of Human Non-Small-Cell Lung Cancer Cells through Inhibition of Epithelial⁻Mesenchymal Transition Mediated by STAT3 and Src.

The root bark of Morus alba L. (MA) has been traditionally used for the treatment of various lung diseases in Korea. Although recent research has demonstrated its anticancer effects in several cancer cells, it is still unclear whether MA inhibits the migratory ability of lung cancer cells. The present study investigated the effects of MA on the migration of lung cancer cells and explored the underlying mechanism. Results from a transwell assay and wound-healing assay demonstrated that methylene chloride extracts of MA (MEMA) suppressed the migration and invasion of H1299, H460, and A549 human non-small-cell lung cancer (NSCLC) cells in a concentration-dependent manner. Results from Western blot analyses showed that MEMA reduced the phosphorylation of STAT3 and Src. In addition, MEMA downregulated the expression of epithelial-mesenchymal transition (EMT) marker proteins including Slug, Snail, Vimentin, and N-cadherin, while upregulating the expression of Occludin-a tight-junction protein. The regulation of EMT markers and the decrease of migration by MEMA treatment were reversed once phospho-mimetic STAT3 (Y705D) or Src (Y527F) was transfected into H1299 cells. In conclusions, MEMA inhibited the migratory activity of human NSCLC cells through blocking Src/STAT3-mediated EMT.

Preclinical studies of ropivacaine extended-release from a temperature responsive hydrogel for prolonged relief of pain at the surgical wound.

Postoperative pain is a common form of acute pain that has been treated commonly by local anesthetics through regional nerve blocking. In this study, a series of experiments were conducted using rats to investigate the pharmacokinetic, distribution, and efficacy of a temperature responsive hydrogel-based drug delivery device (PF-72) containing ropivacaine (0.75%) for extended relief of postoperative pain by allowing the prolonged release of ropivacaine. When the ropivacaine was administered using PF-72, its concentration-time curve (AUClast) and peak concentration (Cmax) were 577.0 h*ng/mL and 271.9 ng/mL, respectively. In contrast when the ropivacaine solution was administered using saline solution, its AUClast and Cmax were 982.8 h*ng/mL and 423.6 ng/mL, respectively. In the tissue distribution study, the peak concentration and mean area under the curve of the ropivacaine in injection area (target tissue) were found about 2-fold higher in the case of PF-72 compared with the case of conventional ropivacaine solution. These results clearly demonstrate the capability of PF-72 hydrogel to retain the ropivacaine at the injection site for an extended period. Effective extended (at least 24 h) pain relief of ropivacaine administered using PF-72 was found in the pharmacodynamic study of prolonged analgesic effect. The results of this study indicated that local drug delivery by PF-72 hydrogel formulation may be an effective method to achieve extended relief of pain. Other advantages of ropivacaine administration using PF-72 include reduced systemic side effects and high localization of a drug in target tissues.

Effects of an ethanolic extract of mulberry fruit on blood pressure and vascular remodeling in spontaneous hypertensive rats.

Mulberry (Morus alba) has been used in traditional oriental medicine since ages. Recently, it has been reported that mulberry produces hypotensive effects through the eNOS signaling pathway. However, the mechanism underlying the hypotensive effects of mulberry is not entirely clear. Moreover, the effects of mulberry on vascular remodeling events such as hyperplasia, an important etiology in the pathogenesis of hypertension and arteriosclerosis, are also ambiguous. Here, we hypothesized that an ethanolic extract of mulberry fruit (EMF) has beneficial effects on vascular remodeling and produces hypotensive effects. The effects of a 6-week oral administration of EMF were examined in spontaneously hypertensive rats (SHRs). The animals were divided into four groups: normotensive control (Wistar Kyoto rats), non-treated SHR, low-dose (100 mg/kg) EMF-treated SHR, and high-dose (300 mg/kg) EMF-treated SHR. Our results showed that the EMF-diet normalizes hypertension in SHRs in a dose-dependent manner, by preventing smooth muscle proliferation, thickening of the tunica media, and vascular hyper-reactivity. The endothelial functions were not substantially affected by the EMF diet in our experimental setting. In conclusion, we suggest that the mulberry fruit could act as a food supplement for reducing blood pressure in hypertensive subjects through its effects on smooth muscle proliferation and vascular contractility.

Nonviral delivery of genetic medicine for therapeutic angiogenesis.

Genetic medicines that induce angiogenesis represent a promising strategy for the treatment of ischemic diseases. Many types of nonviral delivery systems have been tested as therapeutic angiogenesis agents. However, their delivery efficiency, and consequently therapeutic efficacy, remains to be further improved, as few of these technologies are being used in clinical applications. This article reviews the diverse nonviral gene delivery approaches that have been applied to the field of therapeutic angiogenesis, including plasmids, cationic polymers/lipids, scaffolds, and stem cells. This article also reviews clinical trials employing nonviral gene therapy and discusses the limitations of current technologies. Finally, this article proposes a future strategy to efficiently develop delivery vehicles that might be feasible for clinically relevant nonviral gene therapy, such as high-throughput screening of combinatorial libraries of biomaterials.