Journal: bioRxiv

Article Title: WNT inhibition creates a BRCA-like state in Wnt-addicted cancer

doi: 10.1101/2020.06.17.157024

Figure Lengend Snippet: A-B . PORCN inhibitor ETC-159 synergizes with PARP inhibitor olaparib in suppressing proliferation of HPAF-II pancreatic cancer cells in soft agar. The ED 50 (dose that reduced colony formation by 50% of the maximal inhibition) was determined (Table S1) and cells were treated with ETC-159, olaparib or the combination at the indicated dose (for example 0.25 × ED 50 of Olaparib or ETC-159, or 0.25 × ED 50 of Olaparib + 0.25 × ED 50 of ETC-159, respectively). (A) The data is representative of two independent experiments with each point representing an average colony count ± SD of duplicates. (B) The Combination Index (CI) values of olaparib and ETC-159 calculated for the two independent experiments using the Chou-Talalay CompuSyn software. CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, respectively. The lower the CI, the stronger the synergism. C-D . Olaparib and ETC-159 synergize to prevent the growth of HPAF-II xenografts in mice. NSG mice with established HPAF-II subcutaneous xenografts were randomized into four groups. Mice were gavaged daily with ETC-159 (10 mg/kg), Olaparib (50 mg/kg) or a combination of ETC-159 (10 mg/kg) and Olaparib (50 mg/kg). Treatment was initiated after HPAF-II tumors were established. (C) Tumor volumes were measured starting from day 0 and during the course of treatment as shown. Data points represent the mean ± SD. n = 7-8 tumors/group. (D) Tumor weights in the respective groups at the end of the treatment are shown, each dot represents an individual tumor. p-values were calculated with Mann-Whitney test. E. Olaparib and ETC-159 synergize in multiple Wnt-addicted cancer cells. Soft agar colony formation assays were performed as in with the indicated cell lines treated with varying concentrations of ETC-159, olaparib or a combination of both as indicated and the combination index was calculated. PARP inhibitor olaparib and ETC-159 synergistically prevent colony formation of EGI-1, MCAS and CFPAC-1 cells in soft agar. F. Representative image of soft agar colonies of EGI-1 cells is shown.

Article Snippet: Averages of the total number of colonies obtained as a fraction of the control were then used to determine the Combination Index values using the Chou-Talalay CompuSyn Software ( http://www.combosyn.com/ )( ).

Techniques: Inhibition, Software, MANN-WHITNEY

Journal: bioRxiv

Article Title: WNT inhibition creates a BRCA-like state in Wnt-addicted cancer

doi: 10.1101/2020.06.17.157024

Figure Lengend Snippet: A. Flow cytometric analysis of endogenous cell surface FZD levels in PaTu8988T cells with inactivating RNF43 mutation (F69C) shows high cell surface abundance of FZDs. PaTu8988T cells were stained with pan-FZD antibody clone F2.A followed by anti-human Fc fragment APC-conjugated secondary antibody. MFI = median fluorescence intensity. B. Wnt inhibition using siRNA against β-catenin or ETC-159 treatment reduces the expression of HR and FA pathway genes in PaTu8988T cells. PaTu8988T cells were transfected with indicated siRNAs or treated with ETC-159 (1 μ M) for 48 hours, total RNA was isolated and expression of β-catenin ( CTNNB1 ), AXIN2 and DNA repair genes was measured by qRT-PCR. C-D. ETC-159 and olaparib synergistically inhibit the growth of ETC-159 resistant pancreatic cancer cell line PaTu8988T. (C) PaTu8988T cells were seeded in soft agar and treated with ETC-159, olaparib or the combination of the two inhibitors at an equivalent dose as described in . Representative image of soft agar colonies from PaTu8988T cells combination study in a 48-well plate is shown. (D) The Combination Index (CI) values of olaparib and ETC-159 calculated from two independent experiments using the Chou-Talalay CompuSyn software. E. Flow cytometric analysis of endogenous cell surface FZD levels in Panc 08.13 cells with wild-type RNF43 shows low cell surface abundance of FZDs. Panc 08.13 cells were stained as described in followed by flow cytometric analysis. MFI = median fluorescence intensity. F. Wnt inhibition does not synergize with olaparib in Wnt-low Panc 08.13 cells . Panc 08.13 cells were seeded in soft agar and treated with ETC-159, olaparib or the combination of the two inhibitors at the indicated doses. The data is representative of two independent experiments with each point representing an average colony count ± SD of duplicates. G. Wnt inhibition through ETC-159 does not change the expression of HR and FA pathway genes in Panc 08.13 cells. Panc 08.13 cells were cultured in low adherence plates and treated with ETC-159 for 72 hours, total RNA was isolated and expression of indicated genes was measured by qRT-PCR.

Article Snippet: Averages of the total number of colonies obtained as a fraction of the control were then used to determine the Combination Index values using the Chou-Talalay CompuSyn Software ( http://www.combosyn.com/ )( ).

Techniques: Mutagenesis, Staining, Fluorescence, Inhibition, Expressing, Transfection, Isolation, Quantitative RT-PCR, Software, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Synergistic Effect of Simultaneous versus Sequential Combined Treatment of Histone Deacetylase Inhibitor Valproic Acid with Etoposide on Melanoma Cells

doi: 10.3390/ijms221810029

Figure Lengend Snippet: Fa-CI plot analysis of the synergistic and antagonistic effects of different VPA and ETO treatment schemes on inhibiting melanoma cells. The combination index (CI) values for simultaneous versus sequential combined treatments of VPA and ETO treatment schemes on ( a ) B16-F10 and ( b ) SK-MEL-2-Luc cells were calculated based on Chou and Talalay’s method via CompuSyn analysis software, 2005 Edition (ComboSyn, Paramus, NJ, USA). This analysis evaluates the effectiveness of combination treatment under the specified order of drug administration. In the Fa-CI plot, fractions of affected cells (Fa) represent the dead cells after drug treatments. Circle, square, and triangle symbols represent the CI values of each Fa under the specified treatment scheme with different orders of drug administration. The range of Fa is from 0 (no inhibition) to 1 (complete inhibition). The CI values are quantitative grading of synergism (CI < 1), additive (CI = 1), and antagonism (CI > 1). The IC 12.5 , IC 25, and IC 50 values are listed in . IC 50 values are summarized in .

Article Snippet: Next, CompuSyn calculation software, 2005 Edition (ComboSyn, Paramus, NJ, USA) calculated the combination index (CI) values based on the Fa values uploaded.

Techniques: Software, Inhibition

Journal: Scientific Reports

Article Title: Concerted cell and in vivo screen for pancreatic ductal adenocarcinoma (PDA) chemotherapeutics

doi: 10.1038/s41598-020-77373-8

Figure Lengend Snippet: HDAC inhibitor (TSA) and BRD4 inhibitor (JQ1) are potent suppressors of early passage PDA primary cells. Primary mouse PDA cells of increasing passage number (10, 50, 100) were treated with increasing concentrations (1, 25, 50, 100, 200, 400, 800, 1600 nM) of ( A ) TSA or ( B ) JQ1 for 72 h. Cell viability was measured by Resazurin sodium salt assay. Data are expressed as Mean ± SEM from three independent experiments. ( C ) Primary mouse PDA cells were treated with increasing concentrations of JQ1 at three concentrations of TSA (25, 50, or 100 nM) for 72 h. Cell viability at sub uM concentrations of JQ1 significantly decreased with increasing [TSA]. Data are shown as Mean ± SEM from two independent experiments. ( D ) Combination index (CI) analysis showed synergistic effects for combined TSA + JQ1. The line at CI = 1 indicates additivity; data points below indicate synergy. Synergy with JQ1 increased at higher concentrations of TSA. ( E ) EC 50 of JQ1 at three concentrations of TSA (25, 50, or 100 nM). Graphpad Prism v7 software [ https://www.graphpad.com/ (current version); licensed] and CompuSyn ( https://www.combosyn.com/ ; publicly available) were used for data analysis and visualization.

Article Snippet: CompuSyn synergy software ( https://www.combosyn.com/ ; publicly available) based on the drug combination principles of Chou-Martin was used for combination index (CI) values quantification.

Techniques: Software

Journal: Scientific Reports

Article Title: Concerted cell and in vivo screen for pancreatic ductal adenocarcinoma (PDA) chemotherapeutics

doi: 10.1038/s41598-020-77373-8

Figure Lengend Snippet: Gemcitibine (Gem) cytotoxicity in PDA primary cells is potentiated by combined TSA and JQ1. ( A ) Primary mouse PDA cells were treated with Gem alone or increasing concentrations of Gem and two concentrations (50 or 100 nM) of TSA or JQ1 or ( B ) combination of Gem + TSA + JQ1 for 72 h. Cell viability was measured by Resazurin sodium salt assay. Data are shown as Mean ± SEM from three independent experiments. ( C ) Combination index (CI) analysis was performed to identify synergistic effects for the drug combinations in ( C ). The dotted line at CI = 1 indicates additivity; data points below indicate synergy. ( D ) EC 50 of Gem at different concentrations of TSA + JQ1. Graphpad Prism v7 software ( https://www.graphpad.com/ (current version); licensed) and CompuSyn ( https://www.combosyn.com/ ; publicly available) were used for data analysis and visualization.

Article Snippet: CompuSyn synergy software ( https://www.combosyn.com/ ; publicly available) based on the drug combination principles of Chou-Martin was used for combination index (CI) values quantification.

Techniques: Software