Crude extract of Rheum palmatum L. Induces cell cycle arrest S phase and apoptosis through mitochondrial-dependent pathways in U-2 OS human osteosarcoma cells.

Cancer is the second cause of death in children. Osteosarcoma is the most common primary malignancy of solid bone cancer primarily affecting adolescents and young adults. In the Chinese population, the crude extract of Rheum palmatum L. (CERP) has been used for treating different diseases, including SARS, rheumatoid arthritis, coxsackievirus B3, and human colon cancer cell, pancreatic cancer. There are no reports on CERP and human osteosarcoma cells. The present study examined effects of CERP on cytotoxicity including cell cycle distribution and cell death (apoptosis) in U-2 OS human osteosarcoma cells. CERP significantly induced S phase arrest in U-2 OS cells in a dose-dependent. CERP produced DNA damage and DNA condensation. Other effects of CERP were stimulation of ROS and Ca(2+) , mitochondria impairment, and activation of caspase-3, -8, and -9. CERP increased the levels of Bax, Bak, Bad, cyclin B, Fas, PARP, GRP78, GADD153, AIF, Endo G, Calpain-2, p21, and p27, but decreased the levels of Bcl-2, BCL-X, XIAP, Akt, CDC25A, CDK2, Cyclin A, and Cyclin E of U-2 OS cells. It was also observed that CERP promoted the expression of AIF, Endo G, GADD153, and cytochrome c. These results indicate that CERP has anticancer effects in vitro and provide the foundation for in vivo studies of animal models of osteosarcoma. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 957-969, 2016.

Crude extract of Polygonum cuspidatum promotes immune responses in leukemic mice through enhancing phagocytosis of macrophage and natural killer cell activities in vivo.

Polygonum cuspidatum is a traditional Chinese herbal medicine used in the treatment of various diseases. In the present study, we investigated whether the crude extract of Polygonum cuspidatum (CEPC) could affect immune responses of murine leukemia cells in vivo. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemic mice and then were treated orally with CEPC at 0, 50, 100 and 200 mg/kg for three weeks. Animals were weighed and blood, liver, spleen samples were collected for further analyses. Results indicated that CEPC did not significantly affect the body and liver weight of animals, but reduced the weight of spleen when compared to control groups. Flow cytometric assay demonstrated that CEPC increased the percentage of CD3- (T-cell marker) and CD19- (B-cell marker) positive cells, but reduced that of CD11b-positive ones (monocytes). However, it did not significantly affect the proportion of Mac-3-positive cells (macrophages), compared to control groups. Results indicated that CEPC promoted phagocytosis by macrophages from blood samples at all examined doses but did not affect that of macrophages from the peritoneal cavity. CEPC also promoted natural killer cell activity of splenocytes at 200 mg/kg of CEPC. CEPC promoted B-cell proliferation at 200 mg/kg treatment when cells were stimulated with lipopolysaccharides but did not promote T-cell proliferation at three doses of CEPC treatment on concanavalin A stimulation.

Crude extract of Polygonum cuspidatum stimulates immune responses in normal mice by increasing the percentage of Mac-3-positive cells and enhancing macrophage phagocytic activity and natural killer cell cytotoxicity.

Polygonum cuspidatum is a natural plant that is used in traditional Chinese herbal medicine. The crude extract of Polygonum cuspidatum (CEPC) has numerous biological effects; however, there is a lack of studies on the effects of CEPC on immune responses in normal mice. The aim of the present study was to determine the in vivo effects of CEPC on immune responses in normal mice. CEPC (0, 50, 100, 150 and 200 mg/kg) was orally administered to BALB/c mice for three weeks, following which blood, liver, and spleen samples were collected. CEPC did not significantly affect the total body weight, or tissue weights of the liver or spleen, as compared with the control mice. CEPC increased the percentages of CD3 (T-cell marker), 11b (monocytes) and Mac-3 (macrophages) positive-cells, and reduced the percentage of CD19-positive cells (B-cell marker), as compared with the control mice. CEPC (100 mg/kg) stimulated macrophage phagocytosis of blood samples but did not affect macrophage phagocytosis in the peritoneum. Activity of the splenic natural killer cells was increased in response to CEPC (50 mg/kg) treatment. Furthermore, CEPC inhibited T- and B-cell proliferation when the cells were stimulated with concanavalin A and lipopolysaccharide, respectively.

Crude extract of Rheum palmatum L inhibits migration and invasion of LS1034 human colon cancer cells acts through the inhibition of matrix metalloproteinase-2/-9 by MAPK signaling.

Crude extract of Rheum palmatum L. (CERP) has been used to treat different diseases in the Chinese population for decades. In this study, we investigated the anti-metastasis effects of CERP on LS1034 human colorectal cancer cells in vitro and examined potential mechanisms of its effects. CERP significantly inhibited cell migration and invasion of LS1034 cells. We also found that CERP inhibited protein levels of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9), and cytosolic NF-kB p65, RHO A, ROCK 1. Furthermore, we found CERP inhibited protein levels of GRB2, SOS1, MKK7, FAK, Rho A, ROCK 1, VEGF, PKC, AKT, phosphor-AKT (Thr308), Cyclin D, iNOS, COX2, NF-kB p65, p-ERK1/2, p-JNK1/2, p-p38, p-c-jun, MMP-2, MMP-9, MMP-1, MMP-7, MMP-10, UPA and increased the protein level of Ras in LS1034 cells. In conclusion, our results suggest that CERP may be used as a novel anti-metastasis agent for the treatment of human colon cancer cells.

The crude extract of Corni Fructus inhibits the migration and invasion of U-2 OS human osteosarcoma cells through the inhibition of matrix metalloproteinase-2/-9 by MAPK signaling.

Osteosarcoma is the most common primary malignancy of the bone cancers. In the Chinese population, the crude extract of Corni Fructus (CECF) has been used as Traditional Chinese medicine to treat several different diseases for hundreds of years. In the present study, effects of CECF on inhibition of migration and invasion in U-2 OS human osteosarcoma cells were examined. CECF significantly inhibited migration and invasion of U-2 OS human osteosarcoma cells. We also found that CECF inhibited activities of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9). CECF decreased protein levels of FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF-κB p65, COX-2, HIF-1α, PI3K, Rho A, ROCK-1, IRE-1α, p-JNK1/2, p-ERK1/2, p-p38, Ras, p-PERK, MMP-2, MMP-9, and VEGF in U-2 OS cells. Results of this study indicate that CECF may have potential as a novel anticancer agent for the treatment of osteosarcoma by inhibiting migration and invasion of cancer cells.

Bufalin induces cell death in human lung cancer cells through disruption of DNA damage response pathways.

Bufalin is a key component of a Chinese medicine (Chan Su) and has been proved effective in killing various cancer cells. Its role in inducing DNA damage and the inhibition of the DNA damage response (DDR) has been reported, but none have studied such action in lung cancer in detail. In this study, we demonstrated bufalin-induced DNA damage and condensation in NCI-H460 cells through a comet assay and DAPI staining, respectively. Western blotting indicated that bufalin suppressed the protein levels associated with DNA damage and repair, such as a DNA dependent serine/threonine protein kinase (DNA-PK), DNA repair proteins breast cancer 1, early onset (BRCA1), 14-3-3 σ (an important checkpoint keeper of DDR), mediator of DNA damage checkpoint 1 (MDC1), O6-methylguanine-DNA methyltransferase (MGMT) and p53 (tumor suppressor protein). Bufalin could activate phosphorylated p53 in NCI-H460 cells. DNA damage in NCI-H460 cells after treatment with bufalin up-regulated its ATM and ATR genes, which encode proteins functioning as sensors in DDR, and also up-regulated the gene expression (mRNA) of BRCA1 and DNA-PK. But bufalin suppressed the gene expression (mRNA) of p53 and 14-3-3 σ, however, bufalin did not significantly affect the mRNA of MGMT. In conclusion, bufalin induced DNA damage in NCI-H460 cells and also inhibited its DNA repair and checkpoint function.

Establishment of reporter platforms capable of detecting NF-κB mediated immuno-modulatory activity.

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are transcriptional targets of nuclear factor kappa B (NF-κB) that are involved in inflammatory responses. The aim of this study is to develop a method for efficiently detecting inflammation modulatory activities. Here we established RAW264.7 macrophage cells stably expressing a luciferase reporter gene directed by iNOS or COX-2 promoter. Lipopolysaccharide (LPS) treatment stimulated the luciferase activity which paralleled with increased iNOS and COX-2 mRNA levels determined by RT-q-PCR. The LPS-stimulated luciferase activity was blocked by NF-κB inhibitor CAPE and by nobiletin, an anti-inflammatory natural product from citrus peels. We have applied the platforms to screen various mushroom species; analysis by scatter plot revealed a strong correlation to the results obtained by ELISA-based detection of TNF-α. Together we have established luciferase reporter systems sensitive to NF-κB-dependent iNOS and COX-2 activation, which provides an alternative screening method for identifying food components with immune-modulatory activities.

Factors associated with newly diagnosed tic disorders among children in Taiwan: a 10-year nationwide longitudinal study.

Increased attention has been paid to tic disorders clinically, yet relatively few studies have probed potential factors that account for the occurrence of tic disorders in the general population. In this study, we used data derived from the Taiwan's National Health Insurance Research Database to examine an array of factors related to the diagnosis of tic disorders and to further probe gender heterogeneity in clinical manifestation. Poisson regression analyses were applied to model the effects of birth cohort, period, and age, separately, on tic disorders. A total of 880 newly diagnosed tic disorders were identified from 2002 to 2009 among 100,516 youngsters in the study dataset who were born between 1997 and 2005. The results showed that a significant increase in the adjusted incidence rate ratio (IRR) was observed when age increased, with the highest adjusted IRR found at age 8-9 years. Compared to the time period from 2002 to 2005, an elevated IRR was found in the time period from 2006 to 2009 (adjusted IRR: 1.37; 95% CI: 1.05-1.80). Boys tended to be more likely to receive their initial diagnosis from psychiatrists and have higher comorbid attention-deficit/hyperactivity disorder (ADHD), as compared with their girl counterparts. In conclusion, the findings indicate that the effects of age and period, respectively, influence the occurrence of newly diagnosed tic disorders. Gender difference and higher frequent comorbid ADHD in boys than in girls were observed in this study.

Subamolide B Isolated from Medicinal Plant Cinnamomum subavenium Induces Cytotoxicity in Human Cutaneous Squamous Cell Carcinoma Cells through Mitochondrial and CHOP-Dependent Cell Death Pathways.

Subamolide B is a butanolide isolated from Cinnamomum subavenium, a medicinal plant traditionally used to treat various ailments including carcinomatous swelling. We herein reported for the first time that subamolide B potently induced cytotoxicity against diverse human skin cancer cell lines while sparing nonmalignant cells. Mechanistic studies on human cutaneous squamous cell carcinoma (SCC) cell line SCC12 highlighted the involvement of apoptosis in subamolide B-induced cytotoxicity, as evidenced by the activation of caspases-8, -9, -4, and -3, the increase in annexin V-positive population, and the partial restoration of cell viability by cotreatment with the pan-caspase inhibitor z-VAD-fmk. Additionally, subamolide B evoked cell death pathways mediated by FasL/Fas, mitochondria, and endoplasmic reticulum (ER) stress, as supported by subamolide B-induced FasL upregulation, BCL-2 suppression/cytosolic release of cytochrome c, and UPR activation/CHOP upregulation, respectively. Noteworthy, ectopic expression of c-FLIPL or dominant-negative mutant of FADD failed to impair subamolide B-induced cytotoxicity, whereas BCL-2 overexpression or CHOP depletion greatly rescued subamolide B-stimulated cells. Collectively, these results underscored the central role of mitochondrial and CHOP-mediated cell death pathways in subamolide B-induced cytotoxicity. Our findings further implicate the potential of subamolide B for cutaneous SCC therapy or as a lead compound for developing novel chemotherapeutic agents.

Crude extract of Euphorbia formosana inhibits the migration and invasion of DU145 human prostate cancer cells: The role of matrix metalloproteinase-2/9 inhibition via the MAPK signaling pathway.

Prostate cancer is a common worldwide health problem in males with a poor prognosis due in part to tumor invasion and migration. The crude extract of Euphorbia formosana (CEEF) has been used for the treatment of numerous diseases, however, its effects on the migration and invasion of prostate cancer cells have yet to be examined. In the present study, we investigated the effects of CEEF on the migration and invasion of DU145 human prostate cancer cells in vitro. The wound healing assay and the Matrigel-uncoated migration assay were used to examine the migration of cancer cells. Western blotting was used to examine the levels of proteins associated with migration and invasion, and gelatin zymography was used to examine the secretion levels of matrix metalloproteinases-2 and -9 (MMP­2/9) from DU145 cells following exposure to CEEF. The results indicated that CEEF suppressed the migration and invasion of DU145 prostate cancer cells and that these effects are exerted in a concentration- and time-dependent manner. CEEF inhibited the ERK1/2, p38, JNK, SOS1, PKC, PI3K and MMP-2/9 protein expression in DU145 cells. The results demonstrated that CEEF suppressed the migration and invasion of DU145 cells through inhibition of the mitogen-activated protein kinase (MAPK) signaling pathway resulting in the inhibition of MMP-2/9 in DU145 human prostate cancer cells.