Image_2_Extract From Plectranthus amboinicus Inhibit Maturation and Release of Interleukin 1β Through Inhibition of NF-κB Nuclear Translocation and NLRP3 Inflammasome Activation.TIF

Uncontrolled inflammation may produce massive inflammatory cytokines, in which interleukin 1β (IL-1β) plays a key role, resulting in tissue damage and serious disorders. The activation of NLRP3 inflammasome is one of the major mechanisms in maturation and release of IL-1β. Plectranthus amboinicus is a perennial herb. Several pharmacological activities of natural components and crude extracts from P. amboinicus have been reported including anti-inflammation; however, the underlying mechanism is not clear. Phorbol-12-myristate 13-acetate-differentiated THP-1 monocytic leukemia cells were used as a reliable model in this study to examine the effect on inflammasome signaling pathway by PA-F4, an extract from Plectranthus amboinicus. PA-F4 inhibited ATP-induced release of caspase-1, IL-1β, and IL-18 from lipopolysaccharides (LPS)-primed cells. PA-F4 induced a concentration-dependent inhibition of both ASC dimerization and oligomerization in cells under LPS priming plus ATP stimulation. Co-immunoprecipitation of NLRP3 and ASC demonstrated that PA-F4 significantly blunted the interaction between NLRP3 and ASC. Furthermore, PA-F4 completely abolished ATP-induced K+ efflux reaction in LPS-primed cells. Taken together, PA-F4 displayed an inhibitory activity on NLRP3 inflammasome activation. Moreover, PA-F4 also inhibited LPS-induced p65 NF-κB activation, suggesting an inhibitory activity on LPS priming step. Further identification showed that rosmarinic acid, cirsimaritin, salvigenin, and carvacrol, four constituents in PA-F4, inhibited LPS-induced IL-6 release. In contrast, rosmarinic acid, cirsimaritin and carvacrol but not salvigenin inhibited ATP-induced caspase-1 release from LPS-primed cells. In conclusion, PA-F4 displayed an inhibitory activity on activation of NLRP3 inflammasome. PA-F4 inhibited LPS priming step through block of p65 NF-κB activation. It also inhibited ATP-induced signaling pathways in LPS-primed cells including the inhibition of both ASC dimerization and oligomerization, K+ efflux reaction, and the release reaction of caspase-1, IL-1β, and IL-18. Rosmarinic acid, cirsimaritin, salvigenin, and carvacrol could partly explain PA-F4-mediated inhibitory activity on blocking the activation of NLRP3 inflammasome.</p

Image_1_Extract From Plectranthus amboinicus Inhibit Maturation and Release of Interleukin 1β Through Inhibition of NF-κB Nuclear Translocation and NLRP3 Inflammasome Activation.TIF

Uncontrolled inflammation may produce massive inflammatory cytokines, in which interleukin 1β (IL-1β) plays a key role, resulting in tissue damage and serious disorders. The activation of NLRP3 inflammasome is one of the major mechanisms in maturation and release of IL-1β. Plectranthus amboinicus is a perennial herb. Several pharmacological activities of natural components and crude extracts from P. amboinicus have been reported including anti-inflammation; however, the underlying mechanism is not clear. Phorbol-12-myristate 13-acetate-differentiated THP-1 monocytic leukemia cells were used as a reliable model in this study to examine the effect on inflammasome signaling pathway by PA-F4, an extract from Plectranthus amboinicus. PA-F4 inhibited ATP-induced release of caspase-1, IL-1β, and IL-18 from lipopolysaccharides (LPS)-primed cells. PA-F4 induced a concentration-dependent inhibition of both ASC dimerization and oligomerization in cells under LPS priming plus ATP stimulation. Co-immunoprecipitation of NLRP3 and ASC demonstrated that PA-F4 significantly blunted the interaction between NLRP3 and ASC. Furthermore, PA-F4 completely abolished ATP-induced K+ efflux reaction in LPS-primed cells. Taken together, PA-F4 displayed an inhibitory activity on NLRP3 inflammasome activation. Moreover, PA-F4 also inhibited LPS-induced p65 NF-κB activation, suggesting an inhibitory activity on LPS priming step. Further identification showed that rosmarinic acid, cirsimaritin, salvigenin, and carvacrol, four constituents in PA-F4, inhibited LPS-induced IL-6 release. In contrast, rosmarinic acid, cirsimaritin and carvacrol but not salvigenin inhibited ATP-induced caspase-1 release from LPS-primed cells. In conclusion, PA-F4 displayed an inhibitory activity on activation of NLRP3 inflammasome. PA-F4 inhibited LPS priming step through block of p65 NF-κB activation. It also inhibited ATP-induced signaling pathways in LPS-primed cells including the inhibition of both ASC dimerization and oligomerization, K+ efflux reaction, and the release reaction of caspase-1, IL-1β, and IL-18. Rosmarinic acid, cirsimaritin, salvigenin, and carvacrol could partly explain PA-F4-mediated inhibitory activity on blocking the activation of NLRP3 inflammasome.</p

Image_2_Para-Toluenesulfonamide Induces Anti-tumor Activity Through Akt-Dependent and -Independent mTOR/p70S6K Pathway: Roles of Lipid Raft and Cholesterol Contents.TIF

Castration-resistant prostate cancer (CRPC) cells can resist many cellular stresses to ensure survival. There is an unmet medical need to fight against the multiple adaptive mechanisms in cells to achieve optimal treatment in patients. Para-toluenesulfonamide (PTS) is a small molecule that inhibited cell proliferation of PC-3 and DU-145, two CRPC cell lines, through p21- and p27-independent G1 arrest of cell cycle in which cyclin D1 was down-regulated and Rb phosphorylation was inhibited. PTS also induced a significant loss of mitochondrial membrane potential that was attributed to up-regulation of both Bak and PUMA, two pro-apoptotic Bcl-2 family members, leading to apoptosis. PTS inhibited the phosphorylation of m-TOR, 4E-BP1, and p70S6K in both cell lines. Overexpression of constitutively active Akt rescued the inhibition of mTOR/p70S6K signaling in PC-3 cells indicating an Akt-dependent pathway. In contrast, Akt-independent effect was observed in DU-145 cells. Lipid rafts serve as functional platforms for multiple cellular signaling and trafficking processes. Both cell lines expressed raft-associated Akt, mTOR, and p70S6K. PTS induced decreases of expressions in both raft-associated total and phosphorylated forms of these kinases. PTS-induced inhibitory effects were rescued by supplement of cholesterol, an essential constituent in lipid raft, indicating a key role of cholesterol contents. Moreover, the tumor xenograft model showed that PTS inhibited tumor growth with a T/C (treatment/control) of 0.44 and a 56% inhibition of growth rate indicating the in vivo efficacy. In conclusion, the data suggest that PTS is an effective anti-tumor agent with in vitro and in vivo efficacies through inhibition of both Akt-dependent and -independent mTOR/p70S6K pathways. Moreover, disturbance of lipid raft and cholesterol contents may at least partly explain PTS-mediated anti-tumor mechanism.</p

Image_1_Para-Toluenesulfonamide Induces Anti-tumor Activity Through Akt-Dependent and -Independent mTOR/p70S6K Pathway: Roles of Lipid Raft and Cholesterol Contents.TIF

Castration-resistant prostate cancer (CRPC) cells can resist many cellular stresses to ensure survival. There is an unmet medical need to fight against the multiple adaptive mechanisms in cells to achieve optimal treatment in patients. Para-toluenesulfonamide (PTS) is a small molecule that inhibited cell proliferation of PC-3 and DU-145, two CRPC cell lines, through p21- and p27-independent G1 arrest of cell cycle in which cyclin D1 was down-regulated and Rb phosphorylation was inhibited. PTS also induced a significant loss of mitochondrial membrane potential that was attributed to up-regulation of both Bak and PUMA, two pro-apoptotic Bcl-2 family members, leading to apoptosis. PTS inhibited the phosphorylation of m-TOR, 4E-BP1, and p70S6K in both cell lines. Overexpression of constitutively active Akt rescued the inhibition of mTOR/p70S6K signaling in PC-3 cells indicating an Akt-dependent pathway. In contrast, Akt-independent effect was observed in DU-145 cells. Lipid rafts serve as functional platforms for multiple cellular signaling and trafficking processes. Both cell lines expressed raft-associated Akt, mTOR, and p70S6K. PTS induced decreases of expressions in both raft-associated total and phosphorylated forms of these kinases. PTS-induced inhibitory effects were rescued by supplement of cholesterol, an essential constituent in lipid raft, indicating a key role of cholesterol contents. Moreover, the tumor xenograft model showed that PTS inhibited tumor growth with a T/C (treatment/control) of 0.44 and a 56% inhibition of growth rate indicating the in vivo efficacy. In conclusion, the data suggest that PTS is an effective anti-tumor agent with in vitro and in vivo efficacies through inhibition of both Akt-dependent and -independent mTOR/p70S6K pathways. Moreover, disturbance of lipid raft and cholesterol contents may at least partly explain PTS-mediated anti-tumor mechanism.</p

Presentation_1_The Combination of MK-2206 and WZB117 Exerts a Synergistic Cytotoxic Effect Against Breast Cancer Cells.pptx

Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death in women. Hormone receptor-positive breast cancer is usually subjected to hormone therapy, while triple-negative breast cancer is more formidable and poses a therapeutic challenge. Glucose transporters are potential targets for the development of anticancer drugs. In search of anticancer agents whose effect could be enhanced by a GLUT1 inhibitor WZB117, we found that MK-2206, a potent allosteric Akt inhibitor, when combined with WZB117, showed a synergistic effect on growth inhibition and apoptosis induction in breast cancer cells, including ER(+) MCF-7 cells and triple-negative MDA-MB-231 cells. The combination index values at 50% growth inhibition were 0.45 and 0.21, respectively. Mechanism studies revealed that MK-2206 and WZB117 exert a synergistic cytotoxic effect in both MCF-7 and MDA-MB-231 breast cancer cells by inhibiting Akt phosphorylation and inducing DNA damage. The combination may also compromise DNA damage repair and ultimately lead to apoptosis. Our findings suggest that the combination of Akt inhibitors and GLUT1 inhibitors could be a novel strategy to combat breast cancer.</p

1-Benzyl-3-(5‘-hydroxymethyl-2‘-furyl)indazole (YC-1) Derivatives as Novel Inhibitors Against Sodium Nitroprusside-Induced Apoptosis

Antiapoptotic agents based on 1-benzyl-3-(5‘-hydroxymethyl-2‘-furyl)indazole (22, YC-1) derivatives were explored for effective treatment of sepsis and septic shock. We found that compound 22, 1-benzyl-3-(5‘-methoxymethyl-2‘-furyl)indazole (27), and 1-phenyl-3-(5‘-hydroxymethyl-2‘-furyl)indazole (23) were the most effective inhibitors of sodium nitroprusside-induced vascular smooth muscle cell apoptosis. These three compounds are proposed as potential therapeutic agents for the treatment of sepsis

Presentation1_The Combination of a Novel GLUT1 Inhibitor and Cisplatin Synergistically Inhibits Breast Cancer Cell Growth By Enhancing the DNA Damaging Effect and Modulating the Akt/mTOR and MAPK Signaling Pathways.PPT

Breast cancer is the most prevalent cancer and the second leading cause of cancer death in women. Cisplatin is a commonly used chemotherapeutic drug for breast cancer treatment. Owing to serious side effects, the combination of cisplatin with other drugs is an effective strategy to simultaneously reduce side effects and increase the anticancer efficacy. GLUT1 is an emerging target for cancer treatment since cancer cells usually consume more glucose, a phenomenon called the Warburg effect. In this study, we found that the combination of cisplatin and a novel GLUT1 inhibitor #43 identified from our previous high-throughput screening exerted a synergistic anticancer effect in MCF-7 and MDA-MB-231 breast cancer cells. Mechanism studies in MCF-7 cells revealed that combination of cisplatin and #43 significantly induced apoptosis, intracellular reactive oxygen species, and loss of mitochondrial membrane potential. Furthermore, #43 enhanced the DNA damaging effect of cisplatin. Akt/mTOR downstream signaling and the ERK signaling pathway usually involved in cell growth and survival were inhibited by the combination treatment. On the other hand, phosphorylation of p38 and JNK, which may be associated with apoptosis, was induced by the combination treatment. Altogether, our data indicate that oxidative stress, DNA damage, the Akt/mTOR and MAPK signaling pathways, and apoptosis may be involved in the synergism of cisplatin and #43 in breast cancer cells.</p

Effect of reevesioside A on the expression of several cell cycle regulators.

<p>(A) PC-3 cells were incubated in the absence or presence of reevesioside A (50 nM) for various times. Cells were harvested and lysed for the detection of the indicated protein expression by Western blot analysis. The expression was quantified using the computerized image analysis system ImageQuant (Amersham Biosciences). The data are expressed as mean±SEM of three to five independent experiments. * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001 compared with 100% control. (B) After the treatment, the cells were harvested for immunoprecipitation assay. The protein expression was detected by Western blot analysis. Data are representative of three independent experiments.</p

Determination of functional involvement of c-myc.

<p>(A) PC-3 cells were incubated in the absence or presence of reevesioside A (50 nM) for various times. The cells were harvested and lysed for the detection of the indicated protein by Western blot analysis. The expression was quantified using the computerized image analysis system ImageQuant (Amersham Biosciences). The data are expressed as mean±SEM of three independent experiments. *** <i>P</i><0.001 compared with 100% control. (B) PC-3 cells were incubated in the absence or presence of reevesioside A (50 nM) for the indicated times. The cells were harvested for the determination of mRNA expression by RT-PCR. (C) PC-3 cells were transfected with the indicated plasmid. The cells were treated without or with reevesioside A (50 nM) for 6 hours. After treatment, the cells were harvested and lysed for the detection of the indicated protein by Western blot analysis.</p

Correlation between Na⁺/K⁺-ATPase α₃ subunit and anti-proliferative activity.

<p>(A) The expression of Na⁺/K⁺-ATPase α₃ subunit was detected using Western blot analysis. The anti-proliferative IC₅₀ values were determined using SRB assays except for HL-60 cells by MTT assays. (B) PC-3 cells were incubated in the absence or presence of reevesioside A (50 nM) for the indicated times. After the treatment, the cells were harvested for the detection of intracellular Ca²⁺ levels using flow cytometric analysis of the staining with fluo-3 AM. Data are expressed as mean±SEM of three independent experiments. * <i>P</i><0.05 compared with the control.</p