Journal: Molecular cancer research : MCR

Article Title: Targeting the kynurenine pathway for the treatment of cisplatin resistant lung cancer

doi: 10.1158/1541-7786.MCR-19-0239

Figure Lengend Snippet: Modulator role of KYN/AHR/ARNT axis in CR cells metabolism. (A) Immunofluorescence (IF) staining of cells with 1:1000 AHR antibody (red) and DAPI (blue nuclei). CR cells (ALC) possessed significantly higher intensity of AHR expression in the nucleus when compared to parental cells (*p=0.008). DMF or CH223191 significantly reduced AHR accumulation in CR cells (**p=0.007, ***p=0.001; respectively) while exposure to KYN markedly increased AHR accumulation in the nucleus of both parental and CR cells. Bar graph indicated quantification of IF intensity (RFU/cell) using hybrid cell count. (B) Addition of 100μM of KYN increased IDO1 activity in CR cells substantially (*p=0.03), but not significantly (NS) in parental cells. Treatment of 10μM of DMF or 1μM of CH223191 resulted in significant suppression of IDO1 activities (**p=0.006, ***p=0.002, respectively). (C) Immunoblot of lung cancer cell lines showed that resistant variants did not possess HIF1α, but expressed higher levels of AHR and LAT1. Actin was used as a loading control. (D) Flow cytometry analysis of surface LAT1 in lung cancer cell lines. D1:CR cells possessed higher surface LAT1 when compared to parental counterparts. D2:Treatment of KYN at 100μM for 48h further enhanced LAT1 expression in CR cells. D3:KYN treatment did not increase LAT1 expression in parental cells. D4:Knocking down HIF1α in parental cells resulted in increased LAT1 expression after KYN treatment. (E) Knocking down HIF1α in parental cells increased TRP uptake and further increased TRP uptake upon exposure to KYN (100μM). Treatment of KYN further heightened TRP uptake in CR cells (48h; *p=0.02, **p=0.006). (F) Knocking down IDO1 (shIDO1) in ALC suppressed LAT1 expression. Sh-A to D represent 4 unique shRNA sequences. (G) Diagram illustrating the binding partners of ARNT (HIF1β). When HIF1α is down-regulated, ARNT formed a new binding partner with AHR/KYN and initiated the transcription of genes that favored proliferation and increased TRP uptake which can lead to further KYN secretion in CR cells.

Article Snippet: IDO1 inhibitors (Epacadostat (cat#T3545), PF-06840003 (cat#T4307), NLG-919 (cat#T1806), and Indoximod (cat# T6543)) were purchased from TargetMol.

Techniques: Immunofluorescence, Staining, Expressing, Cell Counting, Activity Assay, Western Blot, Flow Cytometry, shRNA, Binding Assay

Journal: Molecular cancer research : MCR

Article Title: Targeting the kynurenine pathway for the treatment of cisplatin resistant lung cancer

doi: 10.1158/1541-7786.MCR-19-0239

Figure Lengend Snippet: Increase IDO1 activity and immune suppressive phenotype were found in CR tumor. (A) ROS analysis of mouse (LLC vs LLC-CR) cells. LLC-CR expressed 2 fold higher basal level of ROS. (B) Immunoblot showed that LLC-CR also expressed higher levels of LAT1 protein. (C) LLC-CR possessed higher IDO1 activity (*p=0.04). (D) Immunohistochemistry (IHC) staining of intratumoral CD4+, CD25+, FoxP3+ (arrow), TGFβ, and IDO1. Higher T-reg densities were found in mice bearing CR tumor than control. Box graph indicated quantification of IHC (intensity/μM2) using hybrid cell count.

Article Snippet: IDO1 inhibitors (Epacadostat (cat#T3545), PF-06840003 (cat#T4307), NLG-919 (cat#T1806), and Indoximod (cat# T6543)) were purchased from TargetMol.

Techniques: Activity Assay, Western Blot, Immunohistochemistry, Cell Counting

Journal: Molecular cancer research : MCR

Article Title: Targeting the kynurenine pathway for the treatment of cisplatin resistant lung cancer

doi: 10.1158/1541-7786.MCR-19-0239

Figure Lengend Snippet: IDO1 expression alone is not the only factor in determining its activity. (A) Diagram illustrated that tryptophan can be used to generate serotonin and KYN. (B) Relative mRNA levels of IDO1. Total RNAs extracted from these cells were reverse-transcribed and subsequently used as template for real-time quantitative PCR. GAPDH was used as internal control. The results shown in the graph were calculated with the ΔΔCt method by setting the IDO1 mRNA level of LL24 cells as 1 (*p=0.005, **p=0.01). (C) Immunoblot of lung cancer cell lines treated with and without IFNγ (20ng/ml) for 24h. No significant differences in IDO1 protein expression levels were observed between parental and CR cells. (D) Significantly higher IDO1 activity was found in CR cells when compared to parental cell counterparts (*p=0.02, **p=0.03) and was further enhanced with IFNγ (20ng/ml) treatment for 24h (***p=0.03, ****p=0.008).

Article Snippet: IDO1 inhibitors (Epacadostat (cat#T3545), PF-06840003 (cat#T4307), NLG-919 (cat#T1806), and Indoximod (cat# T6543)) were purchased from TargetMol.

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Targeting the kynurenine pathway for the treatment of cisplatin resistant lung cancer

doi: 10.1158/1541-7786.MCR-19-0239

Figure Lengend Snippet: Antitumor activity of IDO inhibitors in CR cells. (A) Growth inhibitory effect of various IDO inhibitors (Epacadostat: EPA, NLG-919: NLG, PF-06840003: PF, or Indoximod: INDO) for 48hrs showed that CR cells were sensitive to IDO1 inhibitors with EPA yielding the best efficacy (*p=0.03, **p=0.02). (B) ID50 dosage of EPA alone and in combination with 20ng/ml of IFNγ. Combination treatment enhance EPA toxicity only in CR cells. (Mean SD of three experiments, 48h). (C) ROS analysis detected by CM-H2DCFDA probe indicated that CR cells expressed higher basal levels of ROS. (D) ROS levels were heightened when treated with 15μM of EPA for 48hrs (*p=0.009, **p=0.005). Bar graph represents the relative fluorescent units/cell via fluorometer plate reader. (E) Immunoblot of lung cancer cell lines treated with and without EPA (15μM/ml) for 48h.

Article Snippet: IDO1 inhibitors (Epacadostat (cat#T3545), PF-06840003 (cat#T4307), NLG-919 (cat#T1806), and Indoximod (cat# T6543)) were purchased from TargetMol.

Techniques: Activity Assay, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Targeting the kynurenine pathway for the treatment of cisplatin resistant lung cancer

doi: 10.1158/1541-7786.MCR-19-0239

Figure Lengend Snippet: Sensitivity to IDO1 inhibitor is dependent with higher ROS levels. (A) ID50 dosage of cisplatin in parental cell line “A” and its cisplatin resistance variants. (B) Intracellular ROS production measured by fluorescence intensity using CM-H2DCFDA probe. (C) ID50 dosage of EPA and its cisplatin resistance variants. CR2, CR4, and CR6 possessed 2, 4, and 6 fold resistance to cisplatin, respectively (Mean SD of three experiments, 48h). (D) Knocking down of IDO1 (shIDO-A and shIDO-B) further enhanced ROS level in CR cells (*p=0.02,**p=0.03); however, treatment with 15μM of EPA for 48hrs did not produce additional ROS accumulation in ALCshIDO. (E) Growth inhibitory effect of EPA (48hrs) on ALC and ALCshIDO. EPA significantly suppressed ALC cells’ growth (*P=0.002), and no significant growth inhibition were observed in the knocked down clones. (F) Intracellular ROS production. Antioxidant (TIRON) suppressed ROS production in CR cells (*p=0.003, **p=0.006). (G) Treatment of TIRON (48hrs) attenuated IDO1 activity (*p=0.03) in CR cells. (H) TIRON treatment conferred resistance to EPA in CR cells (48h; **p=0.02). (I) CR cells do not primarily utilize glucose, but rather consume amino acids such as glutamine and tryptophan for survival. This metabolic switch is due to increased ROS production hyper-activating kynurenine pathway (KP) to balance oxidative stress and maintain cellular growth and proliferation. Kynurenine (KYN) is oxidized through indoleamine 2,3-dioxygenase (IDO), and plays a key role in reprogramming naïve T-cells to the immune suppressive regulatory T-cell (T-reg) phenotype. Further increase in ROS by interfering with tumor metabolism via IDO1 inhibiting will selectively target these cisplatin resistant lung cancer cells.

Article Snippet: IDO1 inhibitors (Epacadostat (cat#T3545), PF-06840003 (cat#T4307), NLG-919 (cat#T1806), and Indoximod (cat# T6543)) were purchased from TargetMol.

Techniques: Fluorescence, Inhibition, Clone Assay, Activity Assay

Journal: Cancers

Article Title: OATD-02 Validates the Benefits of Pharmacological Inhibition of Arginase 1 and 2 in Cancer

doi: 10.3390/cancers14163967

Figure Lengend Snippet: OATD-02 shows superior efficacy in the CT26 tumour model when combined with immune checkpoint inhibitor (anti-PD-L1 antibody) and IDO inhibitor (epacadostat). ( A )—BALB/c mice were inoculated with CT26 cells and upon 24 h dosed with OATD-02 (50 mg/kg, PO, BID) or epacadostat (30 mg/kg, PO, BID) till the end of the experiment; anti-PD-L1 antibody was administered at 2.5 mg/kg (IP, QD at days: 8, 10, 12, 14 and 16); ( B )—final measurements of tumour volumes were taken at day 24 post-inoculation and TGI was calculated (** 0.0042 < p < 0.0060, U Mann–Whitney test).

Article Snippet: The IDO1 inhibitor (epacadostat, INCB024360) was purchased from Shanghai Sunshine Chemical Technology (lot SSC151112) and used for oral gavage as a formulation containing 2% DMSO and 18% PEG400 in sterile saline.

Techniques: MANN-WHITNEY