Inhibition of Polyinosinic-Polycytidylic Acid-Induced Acute Pulmonary Inflammation and NF-κB Activation in Mice by a Banana Plant Extract.

NF-κB activation is pivotal for the excess inflammation causing the critical condition and mortality of respiratory viral infection patients. This study was aimed to evaluate the effect of a banana plant extract (BPE) on suppressing NF-κB activity and acute lung inflammatory responses in mice induced by a synthetic double-stranded RNA viral mimetic, polyinosinic-polycytidylic acid (poly (I:C)). The inflammatory responses were analyzed by immunohistochemistry and HE stains and ELISA. The NF-κB activities were detected by immunohistochemistry in vivo and immunofluorescence and Western blot in vitro. Results showed that BPE significantly decreased influx of immune cells (neutrophils, lymphocytes, and total WBC), markedly suppressed the elevation of pro-inflammatory cytokines and chemokines (IL-6, RANTES, IFN-γ, MCP-1, keratinocyte-derived chemokine, and IL-17), and restored the diminished anti-inflammatory IL-10 in the bronchoalveolar lavage fluid (BALF) of poly (I:C)-stimulated mice. Accordingly, HE staining revealed that BPE treatment alleviated poly (I:C)-induced inflammatory cell infiltration and histopathologic changes in mice lungs. Moreover, immunohistochemical analysis showed that BPE reduced the pulmonary IL-6, CD11b (macrophage marker), and nuclear NF-κB p65 staining intensities, whilst restored that of IL-10 in poly (I:C)-stimulated mice. In vitro, BPE antagonized poly(I:C)-induced elevation of IL-6, nitric oxide, reactive oxygen species, NF-κB p65 signaling, and transient activation of p38 MAPK in human lung epithelial-like A549 cells. Taken together, BPE ameliorated viral mimic poly(I:C)-induced acute pulmonary inflammation in mice, evidenced by reduced inflammatory cell infiltration and regulation of both pro- and anti-inflammatory cytokines. The mechanism of action might closely associate with NF-κB signaling inhibition.

Corrigendum to "Honokiol Eliminates Human Oral Cancer Stem-Like Cells Accompanied with Suppression of Wnt/ß-Catenin Signaling and Apoptosis Induction".

[This corrects the article DOI: 10.1155/2013/146136.].

Changes in urinary Cu, Zn, and Se levels in cancer patients after treatment with Sha Shen Mai Men Dong Tang.

Sha Shen Mai Men Dong Tang (SMD-2; sha shen mài dong tang) is a Chinese medicinal herb (CMH; zhong cǎo yào) used to treat symptoms associated with cancer therapy. The objective of this study was to assess the effect of SMD-2 on the levels of urinary copper (Cu), zinc (Zn), and selenium (Se) in lung cancer patients and head and neck cancer patients receiving chemoradiotherapy. Forty-two head and neck cancer patients and 10 lung cancer patients participated in our clinical trial. Each patient received chemoradiotherapy for 4 weeks. In addition, each patient was treated with SMD-2 for 8 weeks, including 2 weeks prior to and after the chemoradiotherapy treatment. Comparison of urinary Cu, Zn, and Se levels and the ratios of Zn to Cu and Se to Cu at three time points in the two types of cancer were assessed using the generalized estimating equations (GEEs). After the patients received chemoradiotherapy for 4 weeks, SMD-2 treatment was found to be associated with a significant decrease in urinary Cu levels, whereas urinary Zn and Se levels increased significantly. In addition, the ratios of Zn to Cu and Se to Cu in the urine samples of these patients also increased significantly. Both the urinary Zn levels and the ratio of Zn to Cu in head and neck cancer patients were significantly higher than in lung cancer patients. Urinary Zn and Se levels and the ratios of Zn to Cu and Se to Cu, but not urinary Cu levels, increased significantly during and after treatment when assessed using the GEE model. The SMD-2 treatments significantly increased Zn and Se levels in the urine of head and neck cancer patients. Increased Zn and Se levels in urine strengthened immune system.

Elimination of cancer stem-like cells and potentiation of temozolomide sensitivity by Honokiol in glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) is the most common adult malignant glioma with poor prognosis due to the resistance to radiotherapy and chemotherapy, which might be critically involved in the repopulation of cancer stem cells (CSCs) after treatment. We had investigated the characteristics of cancer stem-like side population (SP) cells sorted from GBM cells, and studied the effect of Honokiol targeting on CSCs. GBM8401 SP cells possessed the stem cell markers, such as nestin, CD133 and Oct4, and the expressions of self-renewal related stemness genes, such as SMO, Notch3 and IHH (Indian Hedgehog). Honokiol inhibited the proliferation of both GBM8401 parental cells and SP cells in a dose-dependent manner, the IC50 were 5.3±0.72 and 11±1.1 µM, respectively. The proportions of SP in GBM8401 cells were diminished by Honokiol from 1.5±0.22% down to 0.3±0.02% and 0.2±0.01% at doses of 2.5 µM and 5 µM, respectively. The SP cells appeared to have higher expression of O6-methylguanine-DNA methyltransferase (MGMT) and be more resistant to Temozolomide (TMZ). The resistance to TMZ could be only slightly reversed by MGMT inhibitor O6-benzylguanine (O6-BG), but markedly further enhanced by Honokiol addition. Such significant enhancement was accompanied with the higher induction of apoptosis, greater down-regulation of Notch3 as well as its downstream Hes1 expressions in SP cells. Our data indicate that Honokiol might have clinical benefits for the GBM patients who are refractory to TMZ treatment.

Elimination of cancer stem-like side population in human glioblastoma cells accompanied with stemness gene suppression by Korean herbal recipe MSC500.

BACKGROUND: High-grade gliomas are the most common and invasive malignant brain tumors in adults, and they are almost universally fatal because of drug resistance and recurrence. In spite of the progress in adjuvant therapy (like temozolomide) and irradiation after surgery, no effective salvage therapy is currently available for relapsed patients. A Korean herbal recipe MSC500 has been reported to have beneficial therapeutic effects in patients with high-grade gliomas who are relapsed or refractory to conventional treatments. But the underlying molecular mechanisms remain unclear. METHODS: As Cancer stem cell (CSC) plays a pivotal role in the resistance to conventional cancer therapy, we explored the effects of MSC500 on the CSC-like side population (SP) in GBM8401 human glioblastoma multiforme cells. RESULTS: Compared with the parental cells, the SP cells were more resistant to temozolomide but sensitive to MSC500. The mRNA levels of stemness genes such as Nanog, CD133, and ABCG2 were much higher in the SP cells, and so was E-cadherin, which was reported to correlate with the aggressiveness of glioblastoma multiforme. Treatment with MSC500 decreased the proportion of SP cells and high ALDH activity cells from 1.6% to 0.3% and from 0.9% to 0.1%, respectively, accompanied with suppression of the aforementioned stemness genes and E-cadherin, as well as other CSC markers such as ABCB5, Oct-4, Sox-2, ß-catenin, Gli-1, and Notch-1. CONCLUSION: Our results suggest the potential role of MSC500 as an integrative and complementary therapeutic for advanced or refractory high-grade glioma patients.

Activation of estrogen receptors with E2 downregulates peroxisome proliferator-activated receptor γ in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality and occurs more often in men than in women; however, little is known about its underlying molecular mechanisms. The present study investigated the effect of estrogen receptor (ER)α and ERß on peroxisome proliferator-activated receptor γ (PPARγ) expression in Hep3B cells. We examined PPARγ, ERα and ERß mRNA and protein expression by RT-PCR and western blotting. In order to determine whether PPARγ plays a central role in HCC, we screened for PPARγ expression in liver cancer patient tissues and differentially differentiated HCC cell lines (HA22T, Huh-7, Hep3B and HepG2). We found that PPARγ expression was highly expressed in liver cancer tissues and in Hep3B cells. Furthermore, overexpression of ERα and ERß was found to decrease PPARγ expression at the transcriptional as well as at the translational level in a ligand-dependent manner. In summary, the present study demonstrated that both ERα and ß were sufficient to inhibit PPARγ and provide a valuable therapeutic option for the treatment of HCC patients.

Tetrandrine induces cell death in SAS human oral cancer cells through caspase activation-dependent apoptosis and LC3-I and LC3-II activation-dependent autophagy.

Numerous studies have demonstrated that autophagy is associated with cancer development. Thus, agents to induce autophagy could be employed in some cases for the treatment of cancer. Our results showed that tetrandrine significantly decreased the viability of SAS cells in a concentration- and time-dependent manner. Tetrandrine induced nuclear condensation, demonstrated by DAPI staining. The early events in apoptosis analysed by Annexin V/PI staining indicated that the percentage of cells staining positive for Annexin V was slightly increased in SAS cells with tetrandrine treatment but was much lower following bafilomycin A1 pre-treatment. Tetrandrine caused AVO and MDC induction in SAS cells in a concentration-dependent manner by fluorescence microscopy. Tetrandrine also caused LC-3 expression in SAS cells in a time-dependent manner. Our results show that tetrandrine treatment induced the levels of cleaved caspase-3 in a concentration- and time-dependent manner. Tetrandrine treatment induced the levels of LC-3 II, Atg-5, beclin-1, p-S6, p-ULK, p-mTOR, p-Akt (S473) and raptor. Tetrandrine decreased cell viability, but bafilomycin A1, 3-MA, chloroquine and NAC protected tetrandrine-treated SAS cells against decrease of cell viability. Atg-5, beclin-1 siRNA decreased tetrandrine-induced cleaved caspase-3 and cleaved PARP in SAS cells and protected tetrandrine-treated SAS cells against decrease in cell viability. Chloroquine, NAC and bafilomycin A1 also decreased tetrandrine-induced cleaved caspase-3 and cleaved PARP in SAS cells. Our results indicate the tetrandrine induces apoptosis and autophagy of SAS human cancer cells via caspase-dependent and LC3-I and LC3-II­dependent pathways.

Honokiol Eliminates Human Oral Cancer Stem-Like Cells Accompanied with Suppression of Wnt/ ß -Catenin Signaling and Apoptosis Induction.

Honokiol, an active compound of Magnolia officinalis, exerted many anticancer effects on various types of cancer cells. We explored its effects on the elimination of cancer stem-like side population (SP) cells in human oral squamous cell carcinoma SAS cells. The sorted SP cells possessed much higher expression of stemness genes, such as ABCG2, ABCC5, EpCAM, OCT-4, CD133, CD44, and ß -catenin, and more clonogenicity as compared with the Non-SP cells. After 48 h of treatment, honokiol dose dependently reduced the proportion of SP from 2.53% to 0.09%. Apoptosis of honokiol-treated SP cells was evidenced by increased annexin V staining and cleaved caspase-3 as well as decreased Survivin and Bcl-2. Mechanistically, honokiol inhibited the CD44 and Wnt/ ß -catenin signaling of SP cells. The Wnt signaling transducers such as ß -catenin and TCF-4 were decreased in honokiol-treated SP cells, while the ß -catenin degradation promoting kinase GSK-3 α / ß was increased. Consistently, the protein levels of ß -catenin downstream targets such as c-Myc and Cyclin D1 were also downregulated. Furthermore, the ß -catenin-related EMT markers such as Slug and Snail were markedly suppressed by honokiol. Our findings indicate honokiol may be able to eliminate oral cancer stem cells through apoptosis induction, suppression of Wnt/ ß -catenin signaling, and inhibition of EMT.

Antioxidant effects of diallyl trisulfide on high glucose-induced apoptosis are mediated by the PI3K/Akt-dependent activation of Nrf2 in cardiomyocytes.

BACKGROUND: Hyperglycemia-induced reactive oxygen species (ROS) generation contributes to development of diabetic cardiomyopathy. Nuclear factor E2-related factor 2 (Nrf2), a redox-sensing transcription factor, induces the antioxidant enzyme expressions. Diallyl trisulfide (DATS) is the most powerful antioxidant among the sulfur-containing compounds in garlic oil. We investigated whether DATS inhibits hyperglycemia-induced ROS production via Nrf2-mediated activation of antioxidant enzymes in cardiac cells exposed to high glucose (HG). METHODS AND RESULTS: Treatment of H9c2 cells with HG resulted in an increase in intracellular ROS level and caspase-3 activity, which were markedly reduced by the administration of DATS (10 µM). DATS treatment significantly increased Nrf2 protein stability and nuclear translocation, upregulated downstream gene HO-1, and suppressed its repressor Keap1. However, apoptosis was not inhibited by DATS in cells transfected with Nrf2-specific siRNA. Inhibition of PI3K/Akt signaling by LY294002 (PI3K inhibitor) or PI3K-specific siRNA not only decreased the level of DATS-induced Nrf2-mediated HO-1 expression, but also diminished the protective effects of DATS. Similar results were also observed in high glucose-exposed neonatal primary cardiomyocytes and streptozotocin-treated diabetic rats fed DATS at a dose of 40 mg/kg BW. CONCLUSIONS: Our findings indicate that DATS protects against hyperglycemia-induced ROS-mediated apoptosis by upregulating the PI3K/Akt/Nrf2 pathway, which further activates Nrf2-regulated antioxidant enzymes in cardiomyocytes exposed to HG.

Elimination of cancer stem-like "side population" cells in hepatoma cell lines by chinese herbal mixture "tien-hsien liquid".

There are increasing pieces of evidence suggesting that the recurrence of cancer may result from a small subpopulation of cancer stem cells, which are resistant to the conventional chemotherapy and radiotherapy. We investigated the effects of Chinese herbal mixture Tien-Hsien Liquid (THL) on the cancer stem-like side population (SP) cells isolated from human hepatoma cells. After sorting and subsequent culture, the SP cells from Huh7 hepatoma cells appear to have higher clonogenicity and mRNA expressions of stemness genes such as SMO, ABCG2, CD133, ß-catenin, and Oct-4 than those of non-SP cells. At dose of 2 mg/mL, THL reduced the proportion of SP cells in HepG2, Hep3B, and Huh7 cells from 1.33% to 0.49%, 1.55% to 0.43%, and 1.69% to 0.27%, respectively. The viability and colony formation of Huh7 SP cells were effectively suppressed by THL dose-dependently, accompanied with the inhibition of stemness genes, e.g., ABCG2, CD133, and SMO. The tumorigenicity of THL-treated Huh7 SP cells in NOD/SCID mice was also diminished. Moreover, combination with THL could synergize the effect of doxorubicin against Huh7 SP cells. Our data indicate that THL may act as a cancer stem cell targeting therapeutics and be regarded as complementary and integrative medicine in the treatment of hepatoma.

The role of the ERK1/2 pathway as an alternative to the aging-diminished cyclic AMP pathway in calcitonin-mediated chondrogenesis in human nucleus pulposus.

Human disc degeneration initiated by aging in the central nucleus pulposus (hNP) is an irreversible process and the recovery has become seriously emerging. In this study, the related mechanisms of calcitonin on the regeneration of hNP and the effects of calcitonin on the age-related alterations were examined. The harvested hNP population was designated as YhNP (from young donor, age <50) and OhNP (from old donor, age >50). Primary OhNP cells showed more hypertrophic phenotypes than YhNP. However, calcitonin (10(-8)-10(-6) M) was able to induce the same chondrogenesis in both YhNP and OhNP by elevating chondrogenic specific-mRNA and protein expressions. Their cell viabilities were increased with calcitonin treatment. No significant differences of calcitonin receptor (CTR) were expressed between YhNP and OhNP cells. Interestingly, in calcitonin-induced pathways for chondrogenesis, highly increased cyclic AMP (cAMP) was detected in YhNP but was strongly diminished by aging in OhNP after calcitonin treatment. However, to maintain the chondrogenesis, calcitonin-induced an alterative phosphorylated ERK1/2 (p-ERK) in both cells. After inhibiting ERK1/2 by PD98059, calcitonin-induced chondrogenesis in OhNP was almost restrained while YhNP cells were not affected. Our results demonstrated that the regeneration of calcitonin on hNP was maintained with aging which was satisfied by an alternative signaling pathway. Therefore, calcitonin shows great potential for clinical therapy for disc regeneration without aging considerations.

Glycyrrhizic acid induces apoptosis in WEHI-3 mouse leukemia cells through the caspase- and mitochondria-dependent pathways.

Leukemia, one of the causes of cancer-related death in humans, is an aggressive malignancy via the rapid growth of abnormal white blood cells. The aim of this study was to determine the anti-leukemia effect of glycyrrhizic acid (GA) on a mouse leukemia cell line, WEHI-3. GA, an active compound in Glycyrrhiza glabra, has been proven to induce cytotoxic effects in many cancer cell lines. In the current study, we investigated the effects of GA in mouse leukemia cells in vitro. The results indicated that GA induced morphological changes, G0/G1 phase arrest, apoptosis and DNA damage in WEHI-3 cells as determined by phase contrast microscopy, DAPI-staining, flow cytometry and comet assay. The results from the flow cytometric assay showed that GA increased ROS levels, reduced the mitochondrial membrane potential (ΔΨm) and stimulated caspase-3 activity in WEHI-3 cells. GA regulated the intrinsic and extrinsic apoptosis-associated protein expression which was determined by western blotting. In addition, endoplasmic reticulum (ER) stress responses were observed in GA-treated WEHI-3 cells. GA promoted the trafficking of apoptosis-inducing factor (AIF), cytochrome c and endonuclease G (Endo G) in WEHI-3 cells. Based on this evidence, GA-triggered apoptosis occurs through the death receptor, mitochondria-mediated and ER stress multiple signaling pathways.

Induction of DNA damage by deguelin is mediated through reducing DNA repair genes in human non-small cell lung cancer NCI-H460 cells.

It has been shown that deguelin, one of the compounds of rotenoids from flavonoid family, induced cytotoxic effects through induction of cell cycle arrest and apoptosis in many types of human cancer cell lines, but deguelin-affected DNA damage and repair gene expression (mRNA) are not clarified yet. We investigated the effects of deguelin on DNA damage and associated gene expression in human lung cancer NCI-H460 cells in vitro. DNA damage was assayed by using the comet assay and DNA gel electrophoresis and the results indicated that NCI-H460 cells treated with 0, 50, 250 and 500 nM deguelin led to a longer DNA migration smear based on the single cell electrophoresis and DNA fragmentation occurred based on the examination of DNA gel electrophoresis. DNA damage and repair gene expression (mRNA) were evaluated by using real-time PCR assay and the results indicated that 50 and 250 nM deguelin for a 24-h exposure in NCI-H460 cells, decreased the gene levels of breast cancer 1, early onset (BRCA1), DNA-dependent serine/threonine protein kinase (DNA-PK), O6-methylguanine-DNA methyltransferase (MGMT), p53, ataxia telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad3-related (ATR) mRNA expressions. Collectively, the present study showed that deguelin caused DNA damage and inhibited DNA damage and repair gene expressions, which might be due to deguelin-inhibited cell growth in vitro.

NADPH oxidase-derived superoxide anion-induced apoptosis is mediated via the JNK-dependent activation of NF-κB in cardiomyocytes exposed to high glucose.

Hyperglycemia-induced generation of reactive oxygen species (ROS) can lead to cardiomyocyte apoptosis and cardiac dysfunction. However, the mechanism by which high glucose causes cardiomyocyte apoptosis is not clear. In this study, we investigated the signaling pathways involved in NADPH oxidase-derived ROS-induced apoptosis in cardiomyocytes under hyperglycemic conditions. H9c2 cells were treated with 5.5 or 33 mM glucose for 36 h. We found that 33 mM glucose resulted in a time-dependent increase in ROS generation as well as a time-dependent increase in protein expression of p22(phox), p47(phox), gp91(phox), phosphorylated IκB, c-Jun N-terminal kinase (JNK) and p38, as well as the nuclear translocation of NF-kB. Treatment with apocynin or diphenylene iodonium (DPI), NADPH oxidase inhibitors, resulted in reduced expression of p22(phox), p47(phox), gp91(phox), phosphorylated IκB, c-Jun N-terminal kinase (JNK) and p38. In addition, treatment with JNK and NF-kB siRNAs blocked the activity of caspase-3. Furthermore, treatment with JNK, but not p38, siRNA inhibited the glucose-induced activation of NF-κB. Similar results were obtained in neonatal cardiomyocytes exposed to high glucose concentrations. Therefore, we propose that NADPH oxidase-derived ROS-induced apoptosis is mediated via the JNK-dependent activation of NF-κB in cardiomyocytes exposed to high glucose.

Arsenic trioxide (As2O3) inhibits murine WEHI-3 leukemia in BALB/c mice in vivo.

Arsenic trioxide (As2O3) is used clinically to treat acute promyelocytic leukemia (APL) and has activity in vitro for induction of apoptosis in several solid tumor cell lines. To investigate the potential therapeutic application of As2O3 for leukemia, we analyzed the effects of As2O3 on the WEHI-3 cells-induced orthotopic leukemia animal model in vivo in this study. We established the WEHI-3 cells leukemia mice through the injection of murine WEHI-3 cells into BALB/c mice, and they were then treated with As2O3 (0.9 and 4.5 mg kg⁻¹ ; p.o.) and/or combined with all-trans-retinoic acid (ATRA), (30 mg kg⁻¹ ; i.p.). The results indicated that (1) As2O3 alone or As2O3 combined with ATRA promoted the total survival rate of leukemia mice and these effects are dose-dependent; (2) As2O3 did not affect the body weight but decreased the spleen weight; however, it did not affect liver weight; (3) As2O3 alone or As2O3 combined with ATRA increased the levels of CD3 and CD19, indicating that the differentiation of T and B cells were promoted; and (4) As2O3 alone or As2O3 combined with ATRA did not change the levels of Mac-3 and CD11b markers, indicating that the differentiation of the precursor of macrophage were not inhibited. Based on these observations, As2O3 alone or As2O3 combined with ATRA have efficacious antileukemia activity in WEHI-3 cells leukemia in vivo.

Gypenosides suppress growth of human oral cancer SAS cells in vitro and in a murine xenograft model: the role of apoptosis mediated by caspase-dependent and caspase-independent pathways.

PURPOSE: Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino. The authors investigated the effects of Gyp on cell morphology, viability, cell cycle distribution, and induction of apoptosis in human oral cancer SAS cells and the determination of murine SAS xenograft model in vivo. EXPERIMENTAL DESIGN: Flow cytometry was used to quantify the percentage of viable cells; cell cycle distribution; sub-G1 phase (apoptosis); caspase-3, -8, and -9 activity; reactive oxygen species (ROS) production, intracellular Ca(2+) determination; and the level of mitochondrial membrane potential (ΔΨ(m)). Western blotting was used to examine levels of apoptosis-associated proteins, and confocal laser microscopy was used to examine the translocation of proteins in cells. RESULTS: Gyp induced morphological changes, decreased the percentage of viable cells, caused G0/G1 phase arrest, and triggered apoptotic cell death in SAS cells. Cell cycle arrest induced by Gyp was associated with apoptosis. The production of ROS, increased intracellular Ca(2+) levels, and the depolarization of ΔΨ(m) were observed. Gyp increased levels of the proapoptotic protein Bax but inhibited the levels of the antiapoptotic proteins Bcl-2 and Bcl-xl. Gyp also stimulated the release of cytochrome c and Endo G. Translocation of GADD153 to the nucleus was stimulated by Gyp. Gyp in vivo attenuated the size and volume of solid tumors in a murine xenograft model of oral cancer. CONCLUSIONS: Gyp-induced cell death occurs through caspase-dependent and caspase-independent apoptotic signaling pathways, and the compound reduced tumor size in a xenograft nu/nu mouse model of oral cancer.

Bee venom induces apoptosis through intracellular Ca2+ -modulated intrinsic death pathway in human bladder cancer cells.

OBJECTIVES: To focus on bee venom-induced apoptosis in human bladder cancer TSGH-8301 cells and to investigate its signaling pathway to ascertain whether intracellular calcium iron (Ca(2+)) is involved in this effect. METHODS: Bee venom-induced cytotoxic effects, productions of reactive oxygen species and Ca(2+) and the level of mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry. Apoptosis-associated proteins were examined by Western blot analysis and confocal laser microscopy. RESULTS: Bee venom-induced cell morphological changes and decreased cell viability through the induction of apoptosis in TSGH-8301 cell were found. Bee venom promoted the protein levels of Bax, caspase-9, caspase-3 and endonuclease G. The enhancements of endoplasmic reticulum stress-related protein levels were shown in bee venom-provoked apoptosis of TSGH-8301 cells. Bee venom promoted the activities of caspase-3, caspase-8, and caspase-9, increased Ca(2+) release and decreased the level of ΔΨm. Co-localization of immunofluorescence analysis showed the releases of endonuclease G and apoptosis-inducing factor trafficking to nuclei for bee venom-mediated apoptosis. The images revealed evidence of nuclear condensation and formation of apoptotic bodies by 4',6-diamidino-2-phenylindole staining and DNA gel electrophoresis showed the DNA fragmentation in TSGH-8301 cells. CONCLUSIONS: Bee venom treatment induces both caspase-dependent and caspase-independent apoptotic death through intracellular Ca(2+) -modulated intrinsic death pathway in TSGH-8301 cells.

Gallic acid induces G0/G1 phase arrest and apoptosis in human leukemia HL-60 cells through inhibiting cyclin D and E, and activating mitochondria-dependent pathway.

Gallic acid (GA) induces apoptosis in different types of cancer cell lines. In this study, we investigate the apoptotic effects induced by GA in human promyelocytic leukemia HL-60 cells, and clarify the underlying mechanism. Our results showed that GA reduced the viability of HL-60 cells in a dose- and time-dependent manner. GA led to G(0)/G(1) phase arrest in HL-60 cells through promoting p21 and p27 and inhibiting the levels of cyclin D and cyclin E. GA caused DNA damage and fragmentation in HL-60 cells as assayed using DAPI staining and Comet assay. Flow cytometric analysis revealed that GA increased Ca(2+) levels and reduced the mitochondrial membrane potential (ΔΨ(m)) in HL-60 cells. Apoptotic protein expressions were determined by Western blotting. The results indicated that GA-mediated apoptosis of HL-60 cells mainly depended on mitochondrial pathway, by promoting the release of cytochrome c, apoptosis-inducing factor (AIF) and endonuclease G (Endo G) and by up-regulating the protein expression of Bcl-2-associated X protein (BAX), caspase-4, caspase-9 and caspase-3. In addition, GA also activated the death receptor-dependent pathway by enhancing the protein expressions of fatty acid synthase (FAS), FAS ligand (FASL), caspase-8 and BCL-2 interacting domain (BID). We determined the mRNA expression of the gene levels of these proteins by real-time PCR. The results showed that GA-mediated apoptosis of HL-60 cells mainly depended on up-regulation of the mRNA of caspase-8, caspase-9, caspase-3, AIF and Endo G. In conclusion, GA-induced apoptosis occurs through the death receptor and mitochondria-mediated pathways. The evaluation of GA as a potential therapeutic agent for treatment of leukemia seems warranted.

Gallic acid inhibits the migration and invasion of A375.S2 human melanoma cells through the inhibition of matrix metalloproteinase-2 and Ras.

Melanoma is one of the most common cancers worldwide and its incidence has been increasing over the past few decades. Gallic acid (GA) can inhibit the growth of human cancer cells in vitro and in vivo. However, there is no available information to address the effects of GA on migration and invasion of human skin cancer cells. Matrix metalloproteinases (MMPs), zinc-dependent proteolytic enzymes, play an important role in the invasion, metastasis, and angiogenesis of cancer cells. Therefore, MMPs are one of the targets for agents to suppress and that could inhibit the migration and invasion of cancer cells. GA affected the viable A375.S2 cells by propidium iodide exclusion and flow cytometric analysis. Cell migration and invasion were investigated by Boyden chamber assay and we also determined the levels of protein and mRNA expression cell migration and invasion by gelatin zymography, western blotting, and real-time PCR assays. In this study, we examined the influence of GA on the protein levels and gene expression of MMP-2 and MMP-9 and in-vitro migration and invasiveness of human melanoma cells. GA decreases the MMPs and associated signal pathway protein and MMPs mRNA levels in A375.S2 human melanoma cells. Our findings suggest that GA has antimetastatic potential by decreasing invasiveness of cancer cells. Moreover, this action of GA was involved in the Ras, p-ERK signaling pathways resulting in inhibition of MMP-2 in A375.S2 human melanoma cells. These data, therefore, provide evidence for the role of GA as a potential cancer chemotherapeutic agent, which can markedly inhibit the invasive capacity of melanoma cells.

Dilong: role in peripheral nerve regeneration.

Dilong, also known as earthworm, has been widely used in traditional Chinese medicine (TCM) for thousands of years. Schwann cell migration and proliferation are critical for the regeneration of injured nerves and Schwann cells provide an essentially supportive role for neuron regeneration. However, the molecular mechanisms of migration and proliferation induced by dilongs in Schwann cells remain unclear. Here, we discuss the molecular mechanisms that includes (i) migration signaling, MAPKs (mitogen-activated protein kinases), mediated PAs and MMP2/9 pathway; (ii) survival and proliferative signaling, IGF-I (insulin-like growth factor-I)-mediated PI3K/Akt pathways and (iii) cell cycle regulation. Dilong stimulate RSC96 cell proliferation and migration. It can induce phosphorylation of ERK1/2 and p38, but not JNK, and activate the downstream signaling expression of PAs (plasminogen activators) and MMPs (matrix metalloproteinases) in a time-dependent manner. In addition, Dilong stimulated ERK1/2 and p38 phosphorylation was attenuated by pretreatment with chemical inhibitors (U0126 and SB203580), and small interfering ERK1/2 and p38 RNA, resulting in migration and uPA-related signal pathway inhibition. Dilong also induces the phosphorylation of IGF-I-mediated PI3K/Akt pathway, activates protein expression of PCNA (proliferating cell nuclear antigen) and cell cycle regulatory proteins (cyclin D1, cyclin E and cyclin A) in a time-dependent manner. In addition, it accelerates G(1)-phase progression with earlier S-phase entry and significant numbers of cells entered the S-phase. The siRNA-mediated knockdown of PI3K that significantly reduces PI3K protein expression levels, resulting in Bcl(2) survival factor reduction, revealing a marked blockage of G(1) to S transition in proliferating cells. These results reveal the unknown RSC96 cell migration and proliferation mechanism induced by dilong, which find use as a new medicine for nerve regeneration.