Journal: Breast cancer research : BCR

Article Title: Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-κB inhibition.

doi: 10.1186/bcr3477

Figure Lengend Snippet: Figure 1 Parthenolide and costunolide decrease detyrosinated tubulin in human breast carcinoma cells. (A) Bt‐549 (N = 3) and MDA‐MB‐ 157 (N = 6) cells treated for six hours with DMSO (Veh; 0.1%), parthenolide (Parth; 5 μM, 10 μM, 25 μM), costunolide (Cost; 5 μM, 10 μM, 25 μM), resveratrol (ResV; 50 μg/ml), colchicine (Col; 50 μM), and Taxol (Tax; 0.5 μg/ml). Parthenolide (10 μM, 25 μM), costunolide (10 μM, 25 μM), and colchicine (50 μM) significantly reduced detyrosinated tubulin (Detyr) levels compared to vehicle (*P <0.05; ** P <0.001, t‐test). Resveratrol, a non‐ sesquiterpene lactone NF‐κB inhibitor, did not significantly affect detyrosinated tubulin (P >0.5, t‐test). Taxol significantly increased detyrosinated tubulin (‡Bt‐549 Tax value is × 2.5; MDA‐MB‐157 Tax value is × 10). None of the compounds induced apoptosis in these cells, as gauged by PARP cleavage. Columns, mean densitometry for N = 3 (Bt‐549) or N = 6 (MDA‐MB‐157) experiments; bars, SD. (B) Comparison of the compounds used show parthenolide and costunolide, two sesquiterpene lactones, are structurally similar while the other compounds are structurally dissimilar. DMSO, dimethyl sulfoxide; NF-κB, nuclear factor-kappaB; PARP, poly(ADP-ribose) polymerase.

Article Snippet: Costunolide (Cost) was purchased from Chromadex (Santa Ana, CA, USA).

Techniques: Comparison

Journal: Breast cancer research : BCR

Article Title: Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-κB inhibition.

doi: 10.1186/bcr3477

Figure Lengend Snippet: Figure 2 Tubulin detyrosination is independent of NF-κB activation. (A) NF-κB -luciferase reporter adenovirus infected MDA-MB-157 and Bt-549 show that a four-hour treatment of parthenolide (Parth; 10 μM, 25 μM) and costunolide (Cost; 10 μM, 25 μM) inhibit TNF-α-induced (100 ng/ml) NF-κB activation at concentrations that reduced detyrosinated tubulin. Resveratrol (ResV; 50 μg/ml, 100 μg/ml) inhibits NF-κB but does not affect detyrosination. Colchicine (Col; 50 μM) and Taxol (Tax; 0.5 μg/ml) do not inhibit NF-κB at concentrations that affect detyrosinated tubulin. (B) Cell viability assay shows that non-toxic drug concentrations are used. All compounds are expressed as a % of vehicle (set at 100%; horizontal dotted line). NF-κB, nuclear factor-kappaB.

Article Snippet: Costunolide (Cost) was purchased from Chromadex (Santa Ana, CA, USA).

Techniques: Activation Assay, Luciferase, Infection, Viability Assay

Journal: Breast cancer research : BCR

Article Title: Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-κB inhibition.

doi: 10.1186/bcr3477

Figure Lengend Snippet: Figure 3 Parthenolide and costunolide decrease detyrosinated tubulin without compromising the overall microtubule network. Immunofluorescence of six-hour drug treated (A) Bt-549 and (B) MDA-MB-157 stained for detyrosinated tubulin and α-tubulin show that parthenolide (Parth; 10 μM) and costunolide (Cost; 25 μM) decrease and disrupt filamentous detyrosinated tubulin (Detyr) while leaving the overall microtubule network intact (α-tub). Treatment with colchicine (Col; 50 μM) disrupts both detyrosinated and α-tubulin filaments. Taxol (Tax; 0.5 μg/ml) increases bundling and disrupts organization of detyrosinated and α-tubulin filaments. Resveratrol (ResV; 50 μg/ml) did not significantly affect detyrosinated or α-tubulin.

Article Snippet: Costunolide (Cost) was purchased from Chromadex (Santa Ana, CA, USA).

Techniques: Immunofluorescence, Staining

Journal: Breast cancer research : BCR

Article Title: Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-κB inhibition.

doi: 10.1186/bcr3477

Figure Lengend Snippet: Figure 4 Parthenolide and costunolide decrease microtentacle (McTN) frequency and attachment. (A) Detached Bt-549 and MDA-MB-157 were pretreated for six hours and suspended in drug-containing media for blind McTN scoring. Parthenolide (Parth; 10 μM), costunolide (Cost; 25 μM), and colchicine (Col; 50 μM) show a significant decrease compared to vehicle treated (*P <0.05; ** P <0.001, t-test). Resveratrol (ResV; 50 μg/ ml) and Taxol (Tax; 0.5 μg/ml) did not have a significant effect on McTN frequency. Columns, mean %McTNs for six independent experiments in which at least 100 cells were scored blindly; bars, SD. (B) Live population images of suspended Bt-549 and MDA-MB-157. McTNs are observed in vehicle, resveratrol, and Taxol (black arrows).

Article Snippet: Costunolide (Cost) was purchased from Chromadex (Santa Ana, CA, USA).

Techniques:

Journal: Breast cancer research : BCR

Article Title: Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-κB inhibition.

doi: 10.1186/bcr3477

Figure Lengend Snippet: Figure 5 Parthenolide and costunolide reduce reattachment efficiency of human breast carcinoma cells. Real-time electrical impedance monitoring shows that parthenolide (10 μM) and costunolide (25 μM) significantly reduce attachment when compared to vehicle while Taxol (0.5 μg/ml) and resveratrol (50 μg/ml) did not. Colchicine (50 μM) reduced attachment to a greater extent in Bt-549 than in MDA-MB-157. Lines, mean for three triplicate wells; bars, SD; representative graph is shown. Three independent experiments were performed [see Additional file 4: Figure S4].

Article Snippet: Costunolide (Cost) was purchased from Chromadex (Santa Ana, CA, USA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Lettuce Costunolide Synthase ( CYP71BL2 ) and Its Homolog ( CYP71BL1 ) from Sunflower Catalyze Distinct Regio- and Stereoselective Hydroxylations in Sesquiterpene Lactone Metabolism *

doi: 10.1074/jbc.M110.216804

Figure Lengend Snippet: Costunolide biosynthetic pathway in Asteraceae. COS, costunolide synthase.

Article Snippet: The authentic costunolide standard was purchased from AvaChem Scientific (San Antonio, TX).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Lettuce Costunolide Synthase ( CYP71BL2 ) and Its Homolog ( CYP71BL1 ) from Sunflower Catalyze Distinct Regio- and Stereoselective Hydroxylations in Sesquiterpene Lactone Metabolism *

doi: 10.1074/jbc.M110.216804

Figure Lengend Snippet: Biochemical and chemical characterizations of germacrene A acid 8β-hydroxylase. A and B, (±)LC-MS analyses of C12 (HaG8H) enzymatic products are shown. Microsomes from the yeast expressing C12 and CPR catalyzes the synthesis of a new compound (labeled as 3) with [M-H2O+H]+ ion at m/z 233 in A and with [M-H]− ion at m/z 249 in B. The 6-hydroxy-GAA (compound 2) was prepared by alkaline hydrolysis of authentic costunolide standard (1). The identity of 2 was confirmed by reverting it to costunolide. The chemical identity of the peak marked by asterisk is unknown. The compound marked by the star in A (inset) is a minor compound displaying m/z 233, but it showed different retention time from the costunolide (8.03 min versus 7.66 min). C, structures of the new compound (3) purified from the in vivo feeding assay (8β-hydroxygermacrene A acid) and its rearranged product in an acidic condition (8β-hydroxyilicic acid). In the 8β-hydroxyilicic acid, the stereochemistry of a C15 methyl and a hydroxyl group attached to C4 could not be determined due to NMR signal overlapping.

Article Snippet: The authentic costunolide standard was purchased from AvaChem Scientific (San Antonio, TX).

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Labeling, Purification, In Vivo, Feeding Assay

Journal: The Journal of Biological Chemistry

Article Title: Lettuce Costunolide Synthase ( CYP71BL2 ) and Its Homolog ( CYP71BL1 ) from Sunflower Catalyze Distinct Regio- and Stereoselective Hydroxylations in Sesquiterpene Lactone Metabolism *

doi: 10.1074/jbc.M110.216804

Figure Lengend Snippet: Biochemical and chemical characterization of costunolide synthase. A, (+)LC-MS scan at m/z 233 demonstrated that compounds 1 and 2 showed identical retention times with costunolide and 6-hydroxygermacrene A acid, respectively. Asterisks indicate unknown compounds, which are likely due to the unspecific activities of LsCOS. B, the structures of the standards are depicted. C, metabolite profile of the culture extraction from the EPY300 strain expressing GAS, LsGAO, CPR, and with or without LsCOS by (+)LC-MS scan at m/z 233. D, product ion scans of the costunolide standard and compound 1 by (+)LC-MS-MS showed identical fragmenting patterns. Diamonds indicate the parental ion at m/z 233.

Article Snippet: The authentic costunolide standard was purchased from AvaChem Scientific (San Antonio, TX).

Techniques: Liquid Chromatography with Mass Spectroscopy, Extraction, Expressing