nglin Li

Bioactive tanshinones in Salvia miltiorrhiza inhibit the growth of prostate cancer cells in vitro and in mice

Searching for efficacious and safe agents for the chemoprevention and therapy of prostate cancer has become the top priority of research. The objective of this study was to determine the effects of a group of tanshinones from a Chinese herb Salvia Miltiorrhiza, cryptotanshinone (CT), tanshinone IIA (T2A) and tanshinone I (T1) on prostate cancer. The in vitro studies showed that these tanshinones inhibited the growth of human prostate cancer cell lines in a dose‐dependent manner via cell cycle arrest and apoptosis induction. Among three compounds, T1 had the most potent activity with IC50s around 3–6 μM. On the other hand, tanshinones had much less adverse effects on the growth of normal prostate epithelial cells. The epigenetic pathway focused array assay identified Aurora A kinase as a possible target of tanshinone actions. The expression of Aurora A was overexpressed in prostate cancer cell lines. Moreover, knockdown of Aurora A in prostate cancer cells significantly decreased cell growth. Tanshinones significantly downregulated the Aurora A expression, suggesting Aurora A may be a functional target of tanshinones. Tanshinones, especially T1, also showed potent anti‐angiogenesis activity in vitro and in vivo. Furthermore, T1 inhibited the growth of DU145 prostate tumor in mice associated with induction of apoptosis, decrease of proliferation, inhibition of angiogenesis and downregulation of Aurora A, whereas it did not alter food intake or body weight. Our results support that T1 may be an efficacious and safe chemopreventive or therapeutic agent against prostate cancer progression.

Daidzein-rich isoflavone aglycones are potentially effective in reducing hot flashes in menopausal women.

Objective: The aim of this study was to determine the effect of DRIs on hot flash symptoms in menopausal women. Design: This was a randomized, double-blind, placebo-controlled trial of menopausal women, aged 38 to 60 years, who experienced 4 to 14 hot flashes per day. After a 1-week run-in period, a total of 190 menopausal women were randomized to receive a placebo or 40 or 60 mg/day of a DRI for 12 weeks. The primary outcome was the mean changes from baseline to week 12 in the frequency of hot flashes recorded in the participant diary. The secondary outcomes included changes in quality of life and hormonal profiles. Results: A total of 147 women (77%) completed the study. It was found that 40 and 60 mg of DRI improved hot flash frequency and severity equally. At 8 weeks hot flash frequency was reduced by 43% in the 40-mg DRI group and by 41% in the 60-mg DRI group, compared with 32% in the placebo group (P = not significant vs placebo). The corresponding numbers for 12 weeks were 52%, 51%, and 39%, respectively (P = 0.07 and 0.09 vs placebo). When comparing the two treatment groups with the placebo group, there were significant reductions in mean daily hot flash frequency. The supplement (either 40 or 60 mg) reduced hot flash frequency by 43% at 8 weeks (P = 0.1) and 52% at 12 weeks (P = 0.048) but did not cause any significant changes in endogenous sex hormones or thyroid hormones. Menopausal quality of life improved in all three groups, although there were no statistically significant differences between groups. Conclusions: DRI supplementation may be an effective and acceptable alternative to hormone treatment for menopausal hot flashes.

Pannexin 1 Channels Link Chemoattractant Receptor Signaling to Local Excitation and Global Inhibition Responses at the Front and Back of Polarized Neutrophils

Background: Chemotaxis requires excitatory and inhibitory signals at the front and back of cells. PANX1 and P2Y2 receptors provide local excitation at the front. Results: We found that PANX1 triggers A2A receptor activation and inhibits chemotactic signaling at the back. Conclusion: PANX1 thus integrates excitatory and inhibitory signals that regulate chemotaxis at the front and back of cells. Significance: These findings suggest new strategies to modulate neutrophil chemotaxis. Neutrophil chemotaxis requires excitatory signals at the front and inhibitory signals at the back of cells, which regulate cell migration in a chemotactic gradient field. We have previously shown that ATP release via pannexin 1 (PANX1) channels and autocrine stimulation of P2Y2 receptors contribute to the excitatory signals at the front. Here we show that PANX1 also contributes to the inhibitory signals at the back, namely by providing the ligand for A2A adenosine receptors. In resting neutrophils, we found that A2A receptors are uniformly distributed across the cell surface. In polarized cells, A2A receptors redistributed to the back where their stimulation triggered intracellular cAMP accumulation and protein kinase A (PKA) activation, which blocked chemoattractant receptor signaling. Inhibition of PANX1 blocked A2A receptor stimulation and cAMP accumulation in response to formyl peptide receptor stimulation. Treatments that blocked endogenous A2A receptor signaling impaired the polarization and migration of neutrophils in a chemotactic gradient field and resulted in enhanced ERK and p38 MAPK signaling in response to formyl peptide receptor stimulation. These findings suggest that chemoattractant receptors require PANX1 to trigger excitatory and inhibitory signals that synergize to fine-tune chemotactic responses at the front and back of neutrophils. PANX1 channels thus link local excitatory signals to the global inhibitory signals that orchestrate chemotaxis of neutrophils in gradient fields.

A novel pathway involving melanoma differentiation associated gene-7/interleukin-24 mediates nonsteroidal anti-inflammatory drug-induced apoptosis and growth arrest of cancer cells.

Numerous studies show that nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in chemoprevention or treatment of cancer. Nevertheless, the mechanisms underlying these antineoplastic effects remain poorly understood. Here, we report that induction of the cancer-specific proapoptotic cytokine melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24) by several NSAIDs is an essential step for induction of apoptosis and G(2)-M growth arrest in cancer cells in vitro and inhibition of tumor growth in vivo. We also show that MDA-7/IL-24-dependent up-regulation of growth arrest and DNA damage inducible 45 alpha (GADD45alpha) and GADD45gamma gene expression is sufficient for cancer cell apoptosis via c-Jun NH(2)-terminal kinase (JNK) activation and growth arrest induction through inhibition of Cdc2-cyclin B checkpoint kinase. Knockdown of GADD45alpha and GADD45gamma transcription by small interfering RNA abrogates apoptosis and growth arrest induction by the NSAID treatment, blocks JNK activation, and restores Cdc2-cyclin B kinase activity. Our results establish MDA-7/IL-24 and GADD45alpha and GADD45gamma as critical mediators of apoptosis and growth arrest in response to NSAIDs in cancer cells.

Resuscitation of traumatic hemorrhagic shock patients with hypertonic saline - without dextran - inhibits neutrophil and endothelial cell activation

ABSTRACT Posttraumatic inflammation and excessive neutrophil activation cause multiple organ dysfunction syndrome (MODS), a major cause of death among hemorrhagic shock patients. Traditional resuscitation strategies may exacerbate inflammation; thus, novel fluid treatments are needed to reduce such posttraumatic complications. Hypertonic resuscitation fluids inhibit inflammation and reduce MODS in animal models. Here we studied the anti-inflammatory efficacy of hypertonic fluids in a controlled clinical trial. Trauma patients in hypovolemic shock were resuscitated in a prehospital setting with 250 mL of either 7.5% hypertonic saline (HS; n = 9), 7.5% hypertonic saline + 6% dextran 70 (HSD; n = 8), or 0.9% normal saline (NS; n = 17). Blood samples were collected on hospital admission and 12 and 24 h after resuscitation. Multicolor flow cytometry was used to quantify neutrophil expression of cell-surface activation/adhesion (CD11b, CD62L, CD64) and degranulation (CD63, CD66b, CD35) markers as well as oxidative burst activity. Circulating concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVACM-1), P- and E-selectins, myeloperoxidase (MPO), and matrix metalloproteinase 9 (MMP-9) were assessed by immunoassay. Multiple organ dysfunction syndrome, leukocytosis, and mortality were lower in the HS and HSD groups than in the NS group. However, these differences were not statistically significant. Hypertonic saline prevented priming and activation and neutrophil oxidative burst and CD11b and CD66b expression. Hypertonic saline also reduced circulating markers of neutrophil degranulation (MPO and MMP-9) and endothelial cell activation (sICAM-1, sVCAM-1, soluble E-selectin, and soluble P-selectin). Hypertonic saline + 6% dextran 70 was less capable than HS of suppressing the upregulation of most of these activation markers. This study demonstrates that initial resuscitation with HS, but neither NS nor HSD, can attenuate posttraumatic neutrophil and endothelial cell activation in hemorrhagic shock patients. These data suggest that hypertonic resuscitation without dextran may inhibit posttraumatic inflammation. However, despite this effect, neither HS nor HSD reduced MODS in trauma patients with hemorrhagic shock.

Mitochondria Are Gate-keepers of T Cell Function by Producing the ATP That Drives Purinergic Signaling

Background: Autocrine purinergic signaling regulates T cell function. Results: We found that mitochondria are the source of ATP that drives these signaling mechanisms. Conclusion: Mitochondria are the gate-keepers of T cell function that control purinergic signaling and T cell activation. Significance: These findings suggest that mitochondria could be therapeutic targets to modulate T cell responses. T cells play a central role in host defense. ATP release and autocrine feedback via purinergic receptors has been shown to regulate T cell function. However, the sources of the ATP that drives this process are not known. We found that stimulation of T cells triggers a spike in cellular ATP production that doubles intracellular ATP levels in <30 s and causes prolonged ATP release into the extracellular space. Cell stimulation triggered rapid mitochondrial Ca2+ uptake, increased oxidative phosphorylation, a drop in mitochondrial membrane potential (Δψm), and the accumulation of active mitochondria at the immune synapse of stimulated T cells. Inhibition of mitochondria with CCCP, KCN, or rotenone blocked intracellular ATP production, ATP release, intracellular Ca2+ signaling, induction of the early activation marker CD69, and IL-2 transcription in response to cell stimulation. These findings demonstrate that rapid activation of mitochondrial ATP production fuels the purinergic signaling mechanisms that regulate T cells and define their role in host defense.

P2X7 Integrates PI3K/AKT and AMPK-PRAS40-mTOR Signaling Pathways to Mediate Tumor Cell Death

Background Extracellular adenosine triphosphate (ATP) functions as a novel danger signal that boosts antitumor immunity and can also directly kill tumor cells. We have previously reported that chronic exposure of tumor cells to ATP provokes P2X7-mediated tumor cell death, by as yet incompletely defined molecular mechanisms. Methodology/Principal Findings Here, we show that acute exposure of tumor cells to ATP results in rapid cytotoxic effects impacting several aspects of cell growth/survival, leading to inhibition of tumor growth in vitro and in vivo. Using agonist and antagonist studies together with generation of P2X7 deficient tumor cell lines by lentiviral shRNA delivery system, we confirm P2X7 to be the central control node transmitting extracellular ATP signals. We identify that downstream intracellular signaling regulatory networks implicate two signaling pathways: the known P2X7-PI3K/AKT axis and remarkably a novel P2X7-AMPK-PRAS40-mTOR axis. When exposed to high levels of extracellular ATP, these two signaling axes perturb the balance between growth and autophagy, thereby promoting tumor cell death. Conclusions Our study defines novel molecular mechanisms underpinning the antitumor actions of P2X7 and provides a further rationale for purine-based drugs in targeted cancer therapy.

Tanshinones Inhibit the Growth of Breast Cancer Cells through Epigenetic Modification of Aurora A Expression and Function

The objectives of this study were to evaluate the effects of tanshinones from a Chinese herb Salvia Miltiorrhiza on the growth of breast cancer cells, and to elucidate cellular and molecular mechanisms of action. Tanshinones showed the dose-dependent effect on the growth inhibition of breast cancer cells in vitro, with tanshinone I (T1) the most potent agent. T1 was also the only tanshinone to have potent activity in inhibiting the growth of the triple-negative breast cancer cell line MDA-MB231. T1 caused cell cycle arrests of both estrogen-dependent and estrogen-independent cell lines associated with alterations of cyclinD, CDK4 and cyclinB, and induced breast cancer cell apoptosis associated with upregulation of c-PARP and downregulation of survivin and Aurora A. Among these associated biomarkers, Aurora A showed the most consistent pattern with the anti-growth activity of tanshinones. Overexpression of Aurora A was also verified in breast tumors. The gene function assay showed that knockdown of Aurora A by siRNA dramatically reduced the growth-inhibition and apoptosis-induction activities of T1, suggesting Aurora A as an important functional target of T1 action. On the other hand, tanshinones had much less adverse effects on normal mammary epithelial cells. Epigenetic mechanism studies showed that overexpression of Aurora A gene in breast cancer cells was not regulated by gene promoter DNA methylation, but by histone acetylation. T1 treatment significantly reduced acetylation levels of histone H3 associated with Aurora A gene. Our results supported the potent activity of T1 in inhibiting the growth of breast cancer cells in vitro in part by downregulation of Aurora A gene function. Our previous studies also demonstrated that T1 had potent anti-angiogenesis activity and minimal side effects in vivo. Altogether, this study warrants further investigation to develop T1 as an effective and safe agent for the therapy and prevention of breast cancer.

Combinatorial Effect of Non-Steroidal Anti-inflammatory Drugs and NF-κB Inhibitors in Ovarian Cancer Therapy

Several epidemiological studies have correlated the use of non-steroidal anti-inflammatory drugs (NSAID) with reduced risk of ovarian cancer, the most lethal gynecological cancer, diagnosed usually in late stages of the disease. We have previously established that the pro-apoptotic cytokine melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24) is a crucial mediator of NSAID-induced apoptosis in prostate, breast, renal and stomach cancer cells. In this report we evaluated various structurally different NSAIDs for their efficacies to induce apoptosis and mda-7/IL-24 expression in ovarian cancer cells. While several NSAIDs induced apoptosis, Sulindac Sulfide and Diclofenac most potently induced apoptosis and reduced tumor growth. A combination of these agents results in a synergistic effect. Furthermore, mda-7/IL-24 induction by NSAIDs is essential for programmed cell death, since inhibition of mda-7/IL-24 by small interfering RNA abrogates apoptosis. mda-7/IL-24 activation leads to upregulation of growth arrest and DNA damage inducible (GADD) 45 α and γ and JNK activation. The NF-κB family of transcription factors has been implicated in ovarian cancer development. We previously established NF-κB/IκB signaling as an essential step for cell survival in cancer cells and hypothesized that targeting NF-κB could potentiate NSAID-mediated apoptosis induction in ovarian cancer cells. Indeed, combining NSAID treatment with NF-κB inhibitors led to enhanced apoptosis induction. Our results indicate that inhibition of NF-κB in combination with activation of mda-7/IL-24 expression may lead to a new combinatorial therapy for ovarian cancer.

Bioactive tanshinone I inhibits the growth of lung cancer in part via downregulation of Aurora A function

Lung cancer is the leading cause of cancer death in the world, and the searching for novel efficacious and safe agents for lung cancer prevention remains the top priority of lung cancer research. In the present study, we evaluated the effect of bioactive tanshinones from a Chinese herb Salvia miltiorrhiza, cryptotanshinone (CT), tanshinone I (T1) and tanshinone IIA (T2A), on the proliferation inhibition of lung cancer cell lines. Tanshinones inhibited the lung cancer cell proliferation in vitro, with T1 the most potent, via cell cycle arrest and apoptosis induction. Gene function assay showed that Aurora A knockdown by siRNA dramatically eliminated the T1 activity in vitro, suggesting that Aurora A is an important functional target for T1. We further evaluated the effectiveness of T1 on the growth of H1299 nonsmall lung cancer cell in a mouse model. Tanshinone I inhibited the growth of H1299 lung tumor in a dose‐dependent manner. Tanshinone I at 200 mg/kg body weight significantly reduced final tumor weight by 34% (P < 0.05) associated with inhibiting proliferation and inducing apoptosis of lung cancer cells by 54% (P < 0.001) and 193% (P < 0.001), respectively, inhibiting lung tumor angiogenesis by 72% (P < 0.001), and reducing Aurora A expression by 67% (P < 0.001). On the other hand, T1 did not significantly alter food intake or body weight. Our results provided experimental evidence to suggest that T1 may be an efficacious and safe agent for the prevention of lung cancer progression and Aurora A may be an important molecular target for T1 action against lung cancer.