Mistletoe Extract Targets the STAT3-FOXM1 Pathway to Induce Apoptosis and Inhibits Metastasis in Breast Cancer Cells.

Mistletoe extracts (Viscum album L.) have been widely used as complementary and alternative medicines for the treatment of cancer, and their cytotoxic effects have been reported on various types of cancer. However, the molecular targets of mistletoe extracts have not been well studied. Herein, we investigated molecules associated with the in vitro and in vivo anticancer effects of mistletoe extract using 4T1 murine breast cancer cells. Mistletoe extract induced apoptosis and inhibited the signal transducer and activator of transcription3 (STAT3) phosphorylation. This inhibition was accompanied by the downregulations of forkhead box M1 (FOXM1) and the DNA repair proteins, RAD51 and survivin. Mistletoe extract simultaneously increased the expression of the DNA damage marker proteins, phosphorylated H2A histone family member X (H2A.X), and phosphorylated p38. Furthermore, mistletoe extract effectively suppressed tumor growth in 4T1 tumor-bearing BALB/c mice. In addition to tumor growth inhibition, mistletoe extract inhibited lung metastasis in the tumor-bearing mice and cell invasiveness by downregulating the expressions of matrix metalloproteinases (MMPs), urokinase-type plasminogen activator (uPA), uPA receptor, and markers of epithelial-mesenchymal transition (snail and fibronectin). Taken together, our results suggest that mistletoe extract targets the STAT3-FOXM1 pathway for its cytotoxic effects, and that mistletoe extracts might be useful for the treatment of patients with cancers highly expressing the STAT3-FOXM1 pathway.

Lotus (Nelumbo nucifera) seedpod extract inhibits cell proliferation and induces apoptosis in non-small cell lung cancer cells via downregulation of Axl.

Non-small-cell lung cancer (NSCLC) is the most frequent cause of cancer-related death. In this study, we found the anticancer activity of lotus seedpod extract (LSPE) in NSCLC cells, since LSPE treatment inhibited cell proliferation of A549 and H460 cells in a dose-dependent manner and the clonogenic activities of LSPE-treated cells were also reduced. In LSPE-treated cells, the cleavage of poly (ADP-ribose) polymerase (PARP) and phosphorylation of H2X, were also observed, indicating the pro-apoptotic effect of LSPE. Next, we found that LPSE treatment diminished the levels of protein and mRNA of Axl, a receptor tyrosine kinase (RTK) that transduces critical signals for cell proliferation and inhibition of apoptosis. The promoter activity of Axl was found to be dose-dependently decreased in response to LSPE treatment, implying that LSPE inhibited Axl gene expression at transcriptional level. In addition, Axl overexpression was found to decrease the effects of LSPE on inhibition of cell proliferation and colony formation as well as induction of PARP cleavage and phosphorylation of H2AX, while the same activities of LPSE were increased by knockdown of Axl gene expression, indicating that the antiproliferative and pro-apoptotic effect of LSPE is inversely proportional to the protein level of Axl. Taken together, we found that the LSPE suppressed cell proliferation and induced apoptosis of NSCLC cells, which is attenuated or augmented by overexpression or RNA interference of Axl expression, respectively. Our data suggest that Axl is a novel therapeutic target of LSPE to inhibit cell proliferation and promote apoptosis in NSCLC cells. PRACTICAL APPLICATIONS: In this study, lotus seedpod extract (LSPE) was found to have the cytotoxic and apoptosis-inducing potentials in non-small-cell lung cancer (NSCLC) cells. LSPE downregulated the Axl expression at transcriptional level and the effects of LSPE on cell proliferation as well as apoptosis were affected by Axl protein level. Therefore, the inference of Axl-mediated intracellular signals by LSPE must be a novel approach to control NSCLC. Since our data imply that LSPE contains bioactive compounds targeting Axl, further studies to elucidate these compounds might discover a potent therapeutic agent.

Bufalin down-regulates Axl expression to inhibit cell proliferation and induce apoptosis in non-small-cell lung cancer cells.

Axl, a member of the TAM (Tyro3, AXL, Mer) receptor tyrosine kinase family, plays critical roles in cell growth, proliferation, apoptosis, and migration. In the present study, we demonstrated that the anti-cancer activity of bufalin, a major bioactive component of the Chinese traditional medicine Chan Su, is mediated by the down-regulation of Axl in non-small-cell lung cancer (NSCLC) cells. We observed the inhibitory effect of bufalin on the proliferation of A549 and H460 NSCLC cells and the clonogenicity of these cells was reduced by bufalin treatment in a dose-dependent manner. Next, we found that the protein level of Axl was decreased in proportion to the concentration of bufalin in both A549 and H460 cells. Moreover, the promoter activity of the Axl gene was decreased by bufalin in a dose- and time-dependent manner, indicating that bufalin down-regulates Axl gene expression at the transcriptional level. We further examined if the anti-proliferative property of bufalin is influenced by Axl at the protein level. Axl overexpression attenuated the effect of bufalin in inhibiting cell proliferation and colony formation and inducing apoptosis in H460 cells, while knockdown of Axl gene expression induced the opposite effect. Taken together, our data indicate that the anti-proliferative and pro-apoptotic effects of bufalin were associated with the protein level of Axl, suggesting that Axl is a potent therapeutic target of bufalin in suppressing proliferation and inducing apoptosis in NSCLC cells.

Mistletoe (Viscum album) extract targets Axl to suppress cell proliferation and overcome cisplatin- and erlotinib-resistance in non-small cell lung cancer cells.

BACKGROUND: Mistletoe extract of Visucm album extract (VAE) contains many biologically active components and has been reported to be not only a complementary and alternative medicine, but also a potent therapeutic agent for many types of cancer. PURPOSE: In this study, we examined the effect of VAE on expression and activation of Axl and scrutinized the involvement of Axl in the anti-cancer activity of VAE in parental and chemo-resistant non-small cell lung cancer (NSCLC) cells. METHODS: The levels of Axl protein and mRNA were determined by Western blot analysis and RT-PCR, respectively. Phosphorylation of Axl upon Gas6 stimulation was observed by Western blot analysis. For ectopic expression or gene silencing of Axl, the recombinant plasmid, pcDNA3-Axl, or specific siRNA targeting Axl were transfected into A549 and H460 cells using Lipofectamine 2000, respectively. The anti-cancer activity of mistletoe extract was examined against the parental cells and each of their cisplatin- or erlotinib-resistant cells using trypan blue exclusion assays and colony formation assay. RESULTS: The levels of Axl mRNA were also reduced by VAE treatment, implying the transcriptional downregulation of Axl expression by VAE. In addition, the phosphorylation of Axl protein upon its ligand, Gas6, stimulation was found to be abrogated by VAE. We next found cytotoxic effect of VAE on both the parental NSCLC cells and their variants which are resistant to cisplatin (A549/CisR and H460/CisR) or erlotinib (H460/ER and H1975/ER). Treatment of these cells with VAE caused a dose-dependent decrease of cell viability and clonogenicity. This anti-proliferative effect of VAE was attenuated in Axl-overexpressing cells, while it was augmented in cells transfected Axl specific siRNA. Next, we also found that in cisplatin-resistant cells and erlotinib-resistant cells, VAE treatment decreased Axl protein level, colonogenicity. The levels of several cell cycle regulator, p21 and apoptosis related protein, X-linked inhibitor of apoptosis, was found to be induced and reduced by VAE treatment, respectively. CONCLUSION: Taken together, our data provide that VAE targets Axl to suppress cell proliferation and to circumvent cisplatin- and erlotinib-resistance in NSCLC cells.

Cortical effect and functional recovery by the electromyography-triggered neuromuscular stimulation in chronic stroke patients.

We investigated the effect of electromyography (EMG)-triggered neuromuscular electrical stimulation (NMES; EMG-stim) on functional recovery of the hemiparetic hand and the related cortical activation pattern in chronic stroke patients. We enrolled 14 stroke patients, who were randomly assigned to the EMG-stim (n=7) or the control groups (n=7). The EMG-stim was applied to the wrist extensor of the EMG-stim group for two sessions (30 min/session) a day, five times per week for 10 weeks. Four functional tests (box and block, strength, the accuracy index, and the on/offset time of muscle contraction) and functional MRI (fMRI) were performed before and after treatment. fMRI was measured at 1.5 T in parallel with timed finger flexion-extension movements at a fixed rate. Following treatment, the EMG-stim group showed a significant improvement in all functional tests. The main cortical activation change with such functional improvement was shifted from the ipsilateral sensorimotor cortex (SMC) to the contralateral SMC. We demonstrated that 10-week EMG-stim can induce functional recovery and change of cortical activation pattern in the hemiparetic hand of chronic stroke patients.

Primary motor cortex activation by transcranial direct current stimulation in the human brain.

Transcranial direct current stimulation (tDCS) can modulate motor cortex excitability in the human brain. We attempted to demonstrate the cortical stimulation effect of tDCS on the primary motor cortex (M1) using functional MRI (fMRI). An fMRI study was performed for 11 right-handed healthy subjects at 1.5 T. Anodal tDCS was applied to the scalp over the central knob of the M1 in the left hemisphere. A constant current with an intensity of 1.0 mA was applied. The total fMRI paradigm consisted of three sessions with a 5-min resting period between each session. Each session consisted of five successive phases (resting-tDCS-tDCS-tDCS-tDCS), and each of the phases was performed for 21s. Our findings revealed that no cortical activation was detected in any of the stimulation phases except the fourth tDCS phase. In the result of group analysis for the fourth tDCS phase, the average map indicated that the central knob of the left primary motor cortex was activated. In addition, there were activations on the left supplementary motor cortex and the right posterior parietal cortex. We demonstrated that tDCS has a direct stimulation effect on the underlying cortex. It seems that tDCS is a useful modality for stimulating a target cortical region.

Cortical activation changes induced by visual biofeedback tracking training in chronic stroke patients.

OBJECTIVES: We tried to examine whether visual biofeedback tracking training (VBTT) can improve both the gait performance and cortical activation pattern in chronic stroke patients. DESIGN: We enrolled 10 chronic hemiparetic patients with stroke(mean age 46.3 +/- 5.19 years). The patients were randomly assigned to the training group (5 patients) or the control group (5 patients). VBTT was to follow the PC-generated sine waves with the knee joint electrogoniometer, and the two sine waves should appear as close to overlapping as possible on the PC monitor. The training was performed for 39 minutes/day, 5 days/week, for 4 weeks. Pre-training and post-training accuracy of tracking, functional status of gait, and functional MRI (fMRI) were measured. fMRI was performed at 1.5 T in parallel with timed knee flexion-extension movements at a fixed rate. RESULTS: The accuracy of the tracking performance, walking speed, and motor scale for gait improved in the training group. Primary sensorimotor cortex (SM1) cortical activation shifted significantly from the unaffected to the affected hemisphere in the training group. CONCLUSIONS: We demonstrated that cortical activation changes occurred with gait function improvement in chronic stroke patients throughout the 4-week VBTT program. It seems that the cortical reorganization was induced by VBTT.