Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synergistic targeting of the regulatory and catalytic subunits of PI3Kδ in mature B cell malignancies

doi: 10.1158/1078-0432.CCR-17-2218

Figure Lengend Snippet: (A) Cell proliferation data for six independent DLBCL lines were accrued following 48h (HBL-1, WSU-NHL, OCI-Ly7,) or 72h (OCI-Ly3, OCI-Ly10, OCI-Ly18) exposure to the indicated compounds at progressively increasing doses. The combination of roflumilast and idelalisib synergistically suppressed cell proliferation relative to single-agent treatments as determined by the combination index (CI) analysis; CI <0.1 very strong synergism; CI=0.1–0.3 strong synergism; CI=0.3–0.85 synergism. (B) The combination of roflumilast and idelalisib significantly enhanced the suppression of PI3K activity relative to single agent treatments (*** p<0.001 ** p<0.01, ANOVA; Bonferroni’s multiple comparisons post-test, single agents relative to combination). Data shown are mean ± SD of experiments completed in triplicate. At least three biologic replicates were completed to all assays.

Article Snippet: Proliferation of DLBCL cell lines in response to increasing doses of the PDE4 inhibitor roflumilast (1.25 to 10μM) and the PI3Kδ inhibitor idelalisib (0.03 to 0.6μM), used as single agents or in combination, was measured using the CellTiter Proliferation assay (MTS; Promega, Madison, WI).

Techniques: Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synergistic targeting of the regulatory and catalytic subunits of PI3Kδ in mature B cell malignancies

doi: 10.1158/1078-0432.CCR-17-2218

Figure Lengend Snippet: (A) Tumor volume in mice inoculated with OCI-Ly7 and randomized into four treatment arms: 1) vehicle control, 2) roflumilast, 3) idelalisib 4) idelalisib and roflumilast. The cohort treated with the combination of idelalisib and roflumilast showed significantly reduced tumor volume relative to the single agent and vehicle groups at days 10 and 14 of dosing (*p<0.05, two tailed Student’s t-test). (B) All mice were sacrificed and tumors harvest on treatment day 14. PI3K activity was quantified in all tumors, and those from mice treated with the combination of PDE4 and PI3Kδ inhibitors showed significantly more pronounced suppression of PI3K activity relative to single-agent treatments (p<0.001, two-tailed Student’s t-test). Tumor volume data are mean ± SEM (6 mice/group), and PI3K activity data are mean ± SD of 24 tumors (6/group), each quantified in triplicate.

Article Snippet: Proliferation of DLBCL cell lines in response to increasing doses of the PDE4 inhibitor roflumilast (1.25 to 10μM) and the PI3Kδ inhibitor idelalisib (0.03 to 0.6μM), used as single agents or in combination, was measured using the CellTiter Proliferation assay (MTS; Promega, Madison, WI).

Techniques: Control, Two Tailed Test, Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synergistic targeting of the regulatory and catalytic subunits of PI3Kδ in mature B cell malignancies

doi: 10.1158/1078-0432.CCR-17-2218

Figure Lengend Snippet: (A) Annexin V data from 10 independent primary CLL samples was obtained by FACS following 96h of exposure to the indicated compounds either as single agents (10 μM roflumilast, 0.5μM idelalisib) or in combination. The combination of roflumilast and idelalisib significantly enhanced apoptosis relative to single-agent treatments (*** p<0.001, ** p<0.01, * P<0.05 – Bonferroni’s post-tests, single agents relative to combination; Two-way ANOVA P<0.0001). Data shown are mean ± SD of measurement performed in triplicate. (B) The combination of roflumilast and idelalisib is significantly more effective in suppression of PI3K activity in primary CLL samples than each agent used alone (p<0.05, two-sided Student’s t-test). Data are mean ± SD of primary CLL exposed the indicated agents, each sample quantified in triplicate.

Article Snippet: Proliferation of DLBCL cell lines in response to increasing doses of the PDE4 inhibitor roflumilast (1.25 to 10μM) and the PI3Kδ inhibitor idelalisib (0.03 to 0.6μM), used as single agents or in combination, was measured using the CellTiter Proliferation assay (MTS; Promega, Madison, WI).

Techniques: Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synergistic targeting of the regulatory and catalytic subunits of PI3Kδ in mature B cell malignancies

doi: 10.1158/1078-0432.CCR-17-2218

Figure Lengend Snippet: (A) Western blot analysis shows that elevation of intracellular cyclic-AMP levels with forskolin is associated with a decrease in phosphorylation of the P85/P55 subunit of PI3K, with a consequent reduction in phospholipid PI(3,4,5)P3 production by PI3K in the PDE4-null/low DLBCL cell lines SU-DHL4, SU-DHL6, and SU-DHL10; left and right panels, respectively (*** p<0.001 ** p<0.01, two-tailed Student’s t-test). (B) Western blots show that roflumilast suppresses P85 phosphorylation, and consequently PI3K activity, in the PDE4-high cell lines HBL-1, OCI-Ly3, and OCI-Ly10; left and right panels, respectively (***p<0.001, two tailed Student’s t-test). (C) Western blot analysis shows that expression of wild-type (WT) PDE4B but not of a phosphodiesterase-inactive (PI) mutant enzyme abrogates cyclic-AMP inhibitory effects on P85 phosphorylation and PI3K activity, left and right panels, respectively (*** p<0.001, two-tailed Student’s t-test). (D) Western blot analysis shows that stable expression of a constitutively active (CA) SYK variant blunts the cyclic-AMP inhibitory effects on P85 phosphorylation and PI3K activity, left and right panels, respectively. (**p<0.01, two- tailed Student’s t-test). Cell expressing PDE4B-WT and –PI are included as controls. (E) Western blot analysis shows that roflumilast but not idelalisib suppresses P85 phosphorylation in PDE4B-high DLBCL cell lines. All data shown are mean ± SD of assays performed in triplicate. A minimum of three biologic replicates were completed for each assay. Densitometric quantification of pP85/P55 suppression is shown at the bottom of the western blots.

Article Snippet: Proliferation of DLBCL cell lines in response to increasing doses of the PDE4 inhibitor roflumilast (1.25 to 10μM) and the PI3Kδ inhibitor idelalisib (0.03 to 0.6μM), used as single agents or in combination, was measured using the CellTiter Proliferation assay (MTS; Promega, Madison, WI).

Techniques: Western Blot, Phospho-proteomics, Two Tailed Test, Activity Assay, Expressing, Mutagenesis, Variant Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synergistic targeting of the regulatory and catalytic subunits of PI3Kδ in mature B cell malignancies

doi: 10.1158/1078-0432.CCR-17-2218

Figure Lengend Snippet: (A) Western blot analysis shows that roflumilast suppresses BTK phosphorylation in multiple PDE4-high DLBCL cell lines (B) Western blots show that elevation of intracellular cyclic-AMP levels with forskolin suppresses BTK phosphorylation in multiple PDE4-null/low DLBCL cell lines (C) Western blot analysis shows that idelalisib does not suppress BTK phosphorylation at Y223. (D) Western blot shows that expression of PDE4B-WT or SYK-CA, but not PDE4B-PI, blunts the suppressive cyclic-AMP effects towards BTK phosphorylation. At least three biologic replicates were completed for each assay. Densitometry with quantification of pBTK suppression is shown at the bottom of the western blots.

Article Snippet: Proliferation of DLBCL cell lines in response to increasing doses of the PDE4 inhibitor roflumilast (1.25 to 10μM) and the PI3Kδ inhibitor idelalisib (0.03 to 0.6μM), used as single agents or in combination, was measured using the CellTiter Proliferation assay (MTS; Promega, Madison, WI).

Techniques: Western Blot, Phospho-proteomics, Expressing

Journal: Nutrients

Article Title: Nobiletin Stimulates Adrenal Hormones and Modulates the Circadian Clock in Mice

doi: 10.3390/nu16101491

Figure Lengend Snippet: PDE kinase assays and the acute clock gene expression changes by PDE4 inhibitor or ROR α/γ agonist. ( A ) Inhibitory effect of nobiletin on PDE4A1, 4B1, and 10A2. Various concentrations of nobiletin (1–100 μM) were used for IC50 determination . Values represent the mean of two experiments. Positive controls were rolipram for PDE4A1 and 4B1 and papaverine for PDE10A2. ( B ) Mice received control (0.5% CMC, i.p.), rolipram (PDE4 inhibitor, 10 mg/kg in 0.5% CMC, i.p.), or SR1078 (RORα/γ agonist, 10 mg/kg in 0.5% CMC, i.p.) at ZT3. Serum corticosterone and adrenaline levels 1 h after treatment ( n = 4 per group). * p < 0.05, ** p < 0.01 vs. control; # p < 0.05 vs. SR1078 by Dunn’s multiple comparison test. ( C ) Relative clock gene mRNA levels in the stomach and lungs 2 h after treatment ( n = 5 per group). ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. SR1078 by Tukey’s multiple comparisons test.

Article Snippet: Rolipram (PDE4 inhibitor, FUJIFILM Wako Pure Chemical Corporation) and SR1078 (RORα/γ agonist, FUJIFILM Wako Pure Chemical Corporation) were also used for the experiment at 10 mg/kg in 0.5% CMC [ ].

Techniques: Gene Expression, Control, Comparison