Biphasic Dose-Response of Components From Coptis chinensis on Feeding and Detoxification Enzymes of Spodoptera litura Larvae.

Due to long-term coevolution, secondary metabolites present in plants apparently function as chemical defense against insect feeding, while various detoxification enzymes in insects are adaptively induced as a prosurvival mechanism. Coptis chinensis, a medicinal plant used in traditional Chinese medicine for a thousand years, was found to be less prey to insects in our earlier field observations. Herein, 4 crude extracts obtained from sequential partition of aqueous extract of Rhizoma coptidis with petroleum ether, ethyl acetate, and n-butanol exhibited antifeedant activity against Spodoptera litura (Fabricius) larvae at high doses and inducing activity at low doses. Furthermore, a similar biphasic dose-response of the antifeedant activity against S litura larvae was also observed for jateorhizine, palmatine, and obakunone in Coptis chinensis. Notably, the enzyme activities of glutathione-S-transferase and carboxyl esterase in S litura larvae affected by the different components (jateorhizine, palmatine, obakunone, berberine, and coptisine) of C chinensis also showed a biphasic dose-response with an increasing trend at low doses and a decreasing trend at high doses. Together, our study suggests that the components of C chinensis may play a chemical defensive role against S litura larvae in a hormetic manner.

Cantharidin Induces Apoptosis and Promotes Differentiation of AML Cells Through Nuclear Receptor Nur77-Mediated Signaling Pathway.

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by uncontrolled proliferation and accumulation of myeloblasts in the bone marrow (BM), blood, and other organs. The nuclear receptors Nur77 is a common feature in leukemic blasts and has emerged as a key therapeutic target for AML. Cantharidin (CTD), a main medicinal component of Mylabris (blister beetle), exerts an anticancer effect in multiple types of cancer cells. PURPOSE: This study aims to characterize the anti-AML activity of CTD in vitro and in vivo and explore the potential role of Nur77 signaling pathway. STUDY DESIGN/METHODS: The inhibition of CTD on cell viability was performed in different AML cells, and then the inhibition of CTD on proliferation and colony formation was detected in HL-60 cells. Induction of apoptosis and promotion of differentiation by CTD were further determined. Then, the potential role of Nur77 signaling pathway was assessed. Finally, anti-AML activity was evaluated in NOD/SCID mice. RESULTS: In our study, CTD exhibited potent inhibition on cell viability and colony formation ability of AML cells. Moreover, CTD significantly induced the apoptosis, which was partially reversed by Z-VAD-FMK. Meanwhile, CTD promoted the cleavage of caspases 8, 3 and PARP in HL-60 cells. Furthermore, CTD obviously suppressed the proliferation and induced the cell cycle arrest of HL-60 cells at G2/M phase. Meanwhile, CTD effectively promoted the differentiation of HL-60 cells. Notably, CTD transiently induced the expression of Nur77 protein. Interestingly, CTD promoted Nur77 translocation from the nucleus to the mitochondria and enhanced the interaction between Nur77 and Bcl-2, resulting in the exposure of the BH3 domain of Bcl-2, which is critical for the conversion of Bcl-2 from an antiapoptotic to a proapoptotic protein. Importantly, silencing of Nur77 attenuated CTD-induced apoptosis, reversed CTD-mediated cell cycle arrest and differentiation of HL-60 cells. Additionally, CTD also exhibited an antileukemic effect in NOD/SCID mice with the injection of HL-60 cells into the tail vein. CONCLUSIONS: Our studies suggest that Nur77-mediated signaling pathway may play a critical role in the induction of apoptosis and promotion of differentiation by CTD on AML cells.

Ethyl acetate extract of Caesalpinia sappan L. inhibited acute myeloid leukemia via ROS-mediated apoptosis and differentiation.

BACKGROUND: The dried heartwood of Caesalpinia sappan L. is traditionally prescribed in the formula of traditional Chinese medicine (TCM) for the treatment of acute myeloid leukemia (AML), while nothing is yet known of the active fractions and the underlying mechanisms. PURPOSE: This study aims to investigate the effect of the ethyl acetate extract of the dried heartwood of Caesalpinia sappan L. (C-A-E) on induction of apoptosis and promotion of differentiation in vitro and anti-AML activity in vivo. STUDY DESIGN/METHODS: The aqueous extract was sequentially separated with solvents of increasing polarity and the active fraction was determined through the inhibition potency. The inhibition of the active fraction on cell viability, proliferation and colony formation was performed in different AML cells. Induction of apoptosis and the promotion of differentiation were further determined. Then, the level of the reactive oxygen species (ROS) and its potential role were assessed. Finally, anti-AML activity was evaluated in NOD/SCID mice. RESULTS: C-A-E exhibited the highest inhibition on the cell viability of HL-60 cells. Meanwhile, C-A-E significantly suppressed the proliferation and the colony formation ability of HL-60 and Kasumi-1 cells. Moreover, C-A-E significantly induced the apoptosis, which was partially reversed by Z-VAD-FMK. C-A-E also reduced the level of mitochondrial membrane potential, promoted the release of cytochrome C, decreased the Bcl-2/Bax ratio, and promoted the cleavage of caspase-9 and -3. In addition, Mdivi-1 (mitochondrial fission blocker) remarkably reduced the apoptosis caused by C-A-E. Meanwhile, C-A-E also induced the expression of Mff and Fis1 and increased the location of Drp1 in mitochondria. Furthermore, C-A-E obviously promoted the differentiation of AML cells characterized by the typic morphological changes, the increased NBT positive cells, as well as the increased CD11b and CD14 levels. Notably, C-A-E significantly enhanced the intracellular ROS level. Moreimportantly, C-A-E-mediated apoptosis and differentiation of HL-60 cells was significantly mitigated by NAC. Additionally, C-A-E also exhibited an obvious anti-AML effect in NOD/SCID mice with the injection of HL-60 cells. CONCLUSIONS: C-A-E exhibited an inhibitory effect on AML cells by inducing mitochondrial apoptosis and promoting differentiation, both of which were highly correlated to the activation of ROS.

Z-Ligustilide Exerted Hormetic Effect on Growth and Detoxification Enzymes of Spodoptera litura Larvae.

Plants have evolved a variety of phytochemicals to defense insect feeding, whereas insects have also evolved diverse detoxification enzymes, which are adaptively induced as a prosurvival mechanism. Herein, Z-ligustilide in Ligusticum chuanxiong Hort. was found to exhibit a similar trend in the accumulation from December to May as the occurrence of Spodoptera litura (Fabricius) larvae. Importantly, S. litura larvae feeding enhanced Z-ligustilide level in the stem and leaf (p < 0.01). Moreover, Z-ligustilide ranging from 1 to 5 mg·g-1 exhibited remarkable larvicidal activity, antifeedant activity, and growth inhibition against S. litura larvae. The LC50 values of larvicidal activity for phthalides in L. chuanxiong were compared as follows: Z-ligustilide > levistilide A > senkyunolide A > 3-butylidenephthalide > senkyunolide I, implicating the critical role of conjugated structure. Notably, there was a biphasic dose response for glutathione S-transferase (GST), cytochrome P450 (CYP) 450, Acetylcholinesterase (AChE), and Carboxylesterase (CarE) activities and GSTs1, cytochrome P450 (CYP) 4S9, and CYP4M14 mRNA expression. Particularly, low dose (0.1 mg·g-1) of Z-ligustilide conferred the resistance of S. litura larvae against chlorpyrifos (p < 0.05). Together, our data suggest that Z-ligustilide may function in a hormetic way in the chemical defense of L. chuanxiong against S. litura larvae.

ß-Asarone Induces Apoptosis and Cell Cycle Arrest of Human Glioma U251 Cells via Suppression of HnRNP A2/B1-Mediated Pathway In Vitro and In Vivo.

HnRNP A2/B1 has been found to be an oncogenic protein strongly related to the growth of human glioma cells. Herein, ß-asarone, the main component in the volatile oil of Acori tatarinowii Rhizoma, inhibited the cell viability, proliferation, and colony formation ability of U251 cells. Moreover, ß-asarone induced apoptosis and cell cycle arrest at the G1 phase. Notably, ß-asarone suppressed the expression of hnRNP A2/B1 and hnRNPA2/B1 overexpression remarkably reversed ß-asarone-mediated apoptosis and cell cycle arrest. Importantly, ß-asarone promoted the alternative splicing of Bcl-x by enhancing the ratio of Bcl-xS/Bcl-xL. Meanwhile, hnRNPA2/B1 overexpression mitigated the promoting effect of ß-asarone on the alternative splicing of Bcl-x. ß-asarone also regulated the level of the key proteins involved in the death receptor pathway and mitochondrial apoptosis pathway. Additionally, ß-asarone modulated the cell cycle-related proteins p21, p27, Cdc25A, cyclin D, cyclin E, and CDK2. Finally, ß-asarone inhibited tumor growth and induced apoptosis in nude mice bearing U251 tumor xenografts. ß-asarone also suppressed the hnRNP A2/B1 expression, enhanced the expression of cleaved-caspase 3 and p27 and the ratio of Bcl-xS/Bcl-xL, and reduced the expression of CDK2 in U251 xenografts. Together, ß-asarone-induced apoptosis and cell cycle arrest of U251 cells may be related to the suppression of hnRNPA2/B1-mediated signaling pathway.

ß-Asarone Inhibits Invasion and EMT in Human Glioma U251 Cells by Suppressing Splicing Factor HnRNP A2/B1.

ß-asarone, the main component in the volatile oil of Acori tatarinowii Rhizoma, has been found to possess antitumor activity. However, its effect and mechanisms against tumor invasion and epithelial-mesenchymal transition (EMT) are still unclear. In this study, no or less cytotoxicity was caused by ß-asarone within 0-120 µM in human glioma U251 cells for 48 h. ß-asarone (30 and 60 µM) inhibited the migration of U251 cells in the wound healing assay, suppressed the invasion of U251 cells in the Boyden chamber invasion assay, and inhibited the adhesion of U251 cells onto the Matrigel. Moreover, ß-asarone suppressed EMT with the up-regulation of E-cadherin and the down-regulation of vimentin. HnRNP A2/B1, a well-characterized oncogenic protein, was shown at a high basal level in U251 cells and ß-asarone reduced hnRNP A2/B1 expression in a concentration and time-dependent way. Importantly, hnRNP A2/B1 overexpression significantly counteracted the inhibition of ß-asarone on the migration, invasion, and adhesion of U251 cells and reversed the modulation of EMT markers by ß-asarone. Additionally, ß-asarone decreased the MMP-9 and p-STAT3 in U251 cells, which was also reversed by hnRNP A2/B1 overexpression. Together, our results suggest that hnRNP A2/B1 may be a potential molecular target underlying the inhibitory effect of ß-asarone on invasion and EMT in glioma cells.

Sensitization of tamoxifen-resistant breast cancer cells by Z-ligustilide through inhibiting autophagy and accumulating DNA damages.

Autophagy plays a pro-survival role in the tamoxifen-resistant breast cancer cells. Herein we found that autophagy was concomitantly induced in tamoxifen-resistant MCF-7 (MCF-7TR5) cells through the dissociation of Bcl-2 from Beclin 1 and subsequent enhancement of interaction among the ATG14-Beclin1-PI3KC3 complex. Moreover, higher level of DNA damage was observed in MCF-7TR5 cells with the decreased BRCA1 and RAD51 level and the increased Ku80 level. Interestingly, Nur77 was selectively degraded by autophagy, which causes the release of Ku80 from the Nur77-Ku80 complex, resulting in the increase of the DNA binding of Ku80 and DNA-PKcs. Meanwhile, Z-ligustilide, a phthalide compound from Radix Angelica sinensis, was shown to inhibit the autophagic flux by blocking the autophagosome-lysosome fusion. Importantly, Z-ligustilide-mediated autophagy inhibition restored Nur77 expression in MCF-7TR5 cells. Furthermore, Z-ligustilide promoted the interaction of Nur77 with Ku80 and thereby abolished the association of DNA-PKcs with DNA ends. Moreover, Z-ligustilide sensitized MCF-7TR5 cells in a caspase-independent cell death and enhanced the DNA damage caused by tamoxifen, which was found to be attenuated by shNur77. Together, these findings not only provide important insights into the formation of tamoxifen resistance in breast cancer cells, but also suggest Z-ligustilide may function as a novel autophagy inhibitor to overcome chemoresistance.

Proteomic Identification of eEF1A1 as a Molecular Target of Curcumol for Suppressing Metastasis of MDA-MB-231 Cells.

Curcumol, a major volatile component in Rhizoma Curcumae, exhibits a potent antimetastatic effect on breast cancer cells. However, its molecular mechanism remains poorly understood. In this study, we employed two-dimensional gel electrophoresis-based proteomics to investigate the cellular targets of curcumol in MDA-MB-231 cells and identified 10 differentially expressed proteins. Moreover, Gene Ontology analysis revealed that these proteins are mainly involved in nine types of cellular components, seven different biological processes, and nine kinds of molecular functions, and 35 pathways (p < 0.05) were enriched by KEGG pathway analysis. Specially, eEF1A1, a well-characterized actin binding protein, draws our attention. Curcumol decreased eEF1A1 expression at both mRNA and protein levels. EEF1A1 expression was shown to be correlated with the invasiveness of cancer cells. Importantly, overexpression of eEF1A1 significantly reversed the inhibition of curcumol regarding the invasion and adhesion of MDA-MB-231 cells (p < 0.05). Together, our data suggest that eEF1A1 may be a potential molecular target underlying the antimetastatic effect of curcumol.