GP-2250, a novel anticancer agent, inhibits the energy metabolism, activates AMP-Kinase and impairs the NF-kB pathway in pancreatic cancer cells

Abstract

GP-2250, a novel anticancer agent, severely limits the energy metabolism, as demonstrated by the inhibition of hexokinase 2 and glyceraldehyde-3-phosphate dehydrogenase and a decrease of ATP. Rescue experiments with supplementary pyruvate or oxaloacetate demonstrated that a TCA cycle deficit largely contributed to cytotoxicity. Activation of the energy-deficit sensor, AMP-dependent protein kinase, was associated with increased phosphorylation of acetyl-CoA carboxylase and Raptor, pointing to a possible deficit in the synthesis of fatty acids and proteins as essential cell components. Binding of p65 to DNA was dose-dependently reduced in nuclear lysates. A transcriptional deficit of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) was substantiated by the downregulation of cyclin D1 and of the anti-apoptotic Bcl2, in line with reduction in tumour cell proliferation and induction of apoptosis, respectively. The upregulation of p53 concomitant with an excess of ROS supported apoptosis. Thus, the anticancer activity of GP-2250 is a result of disruption of energy metabolism and inhibition of tumour promotion by NF-κB.

Keywords: GP-2250; NFκB; ROS; aerobic glycolysis; apoptosis; pancreatic cancer; proliferation.

Conflict of interest statement

BM‐S, MB, IP, DW, JS, PH, WU are employees of Department of General and Visceral Surgery, St. Josef‐Hospital, Germany, which received research funding from Geistlich Pharma AG to conduct the study.HM received consultancy fees from Geistlich Pharma AG. TM is employed by Geistlich Pharma AG, Wolhusen, Switzerland (Geistlich). Beyond the contribution of one author (who provided chemical expertise of the analysed agent) Geistlich had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. All other authors declare no competing interests.

© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.

Figures

FIGURE 1

Impact of GP‐2250 on ATP level. Structure of GP‐2250 (A). Decrease of ATP in BxPC3 cells and (B) Panc Tul cells (C) compared to cell viability (MTT test) following incubation with GP‐2250 (250 and 500 μM) for 6 h. The level of ATP and cell viability were assessed in the same test samples. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5 diphenyltetrazoliumbromid; NC, negative control.**p ≤ 0.01; ***p ≤ 0.001.

FIGURE 2

Inhibition of glycolytic and TCA cycle‐related enzymes. (A) Recombinant human GAPDH treated with 100 and 250 μM GP‐2250 for 30 and 60 min, followed by GAPDH enzyme activity assay. (B) Inhibition of GAPDH activity in Panc Tul and BxPC3 cells following incubation with GP‐2250 (500 μM) for 24 h. The GAPDH inhibitor DMF, 100 μM, was included as control. (C) Human recombinant hexokinase 2 (HK2) incubated with 250 and 500 μM GP‐2250 for 60 min. Formation of NADH was measured every 5 min. (D) Inhibition of hexokinase 2 (HK2)activity in Panc Tul and BxPC3 cells following incubation with GP‐2250 (250, 500 and 1000 μM) for 24 h. (E) Inhibition of alpha‐ketoglutarate dehydrogenase (αKGDH) following treatment of Panc Tul and BxPC3 cells for 24 h with GP‐2250 (250, 500 and 1000 μM). (F) Inhibition of pyruvate dehydrogenase (PDH) following treatment of Panc Tul and BxPC3 cells for 24 h with GP‐2250 (250 and 500 μM). Concentrations of GP‐2250 ranging from 100 to 1000 μM were used in the cellular assays of GAPDH, HK2, αKGDH and PDH, Shown are the results of the lowest effective concentrations, respectively. *p ≤ 0.05, significant; **p ≤ 0.01, highly significant; ***p ≤ 0.001, extremely significant. DMF, dimethylfumarate; GAPDH, glyceraldehyde‐3‐phosphate‐dehydrogenase; HK2, hexokinase 2; NADH, nicotinamide adenine dinucleotide + hydrogen; NC, negative control; U, untreated.

FIGURE 3

Rescue from GP‐2250‐induced cytotoxicity by oxaloacetate or pyruvate. (A) Panc Tul and (B) BxPC3 cells treated with different concentrations of GP‐2250 (50–1000 μM) for 24 h in the absence (black columns) or presence (white columns) of supplementary oxaloacetate (OAA, 5 mM). (C) Panc Tul and (D) BxPC3 cells were treated with different concentrations of GP‐2250 (100–1000 μM) for 24 h in the absence (black columns) or presence (white columns) of pyruvate (PYR 5 mM). Cell viability was tested with MTT. There was major significant protection from GP‐2250‐induced cytotoxicity by OAA and PYR nearly over the entire range of active drug concentrations. NC, negative control. *p ≤ 0.05, significant; **p ≤ 0.01, highly significant; ***p ≤ 0.001, extremely significant.

FIGURE 4

Generation of ROS and inhibition of NF‐κB. Generation of ROS in Panc TuI (A) and BxPC3 (B) cells following incubation with various concentrations of GP‐2250 for 90 min compared to an untreated control (UC). Negative control (NC) contained NAC (5 mM) in the presence of 1000 μM GP‐2250. Inhibition of NF‐kB (C, D) p65/DNA binding was dose‐dependently inhibited following incubation of Panc Tul and BxPC3 cells with GP‐2250 (250–1000 μM) for 24 h, as tested in lysates of the nuclear fraction. Bay 117082 (10 μM) served as control. (E, F) p65/DNA binding in lysates of the nuclear fraction from untreated Panc TuI and BxPC3 cells incubated directly with GP‐2250 (250, 500 and 1000 μM). C, non‐treated control; NAC, n‐acetylcysteine; NC, negative control; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; ROS, reactive oxygen species; SD, standard deviation; UC, untreated control. *p ≤ 0.05, significant; **p ≤ 0.01, highly significant; ***p ≤ 0.001, extremely significant.

FIGURE 5

Representative western blot (A) and quantitative analysis (B) of pAMPK and cyclin D1. pAMPK: Time dependency of AMPK phosphorylation at T172 following incubation of Panc Tul with GP‐2250 (250 μM).) Dose‐dependency of AMPK phosphorylation after incubation of BxPC3 cells+ for 24 h. Cyclin D1: Time‐dependent decrease of cyclin D1 following incubation of Panc Tul (500 μM GP‐2250) and BxPC3 cells (250 μM GP‐2250). ß‐Actin/HSP‐90 used as internal controls. NC, negative control.

FIGURE 6

Representative western blot (A) and quantitative analysis (B) of regulatory proteins in Panc TuI (A) and BxPC3 (B) cells. pACC: Time‐dependent increase of ACC‐1 phosphorylation at Ser79 and of Raptor at Ser792 following incubation of Panc Tul (250 μM GP‐2250) and BxPC3 (500 μM GP‐2250). pRaptor: Time‐dependent Raptor phosphorylation at Ser 792 in PancTul and BxPc3 cells incubated with 500 and 1000 μM GP‐2250. p53: Time‐dependent increase of protein level of p53 in Panc Tul and BxPC3 cells (500 μM GP‐2250). Akt and mTor: Time‐dependent decrease of the protein level of Akt and mTor at 1000 μM GP‐2250 in Panc TuI and BxPC3 cells. Bcl2: Time‐dependent downregulation of protein level of Bcl2 following incubation of Panc Tul and BxPC3 cells (500 μM GP‐2250). ß‐Actin/HSP‐90 used as internal controls.

FIGURE 7

Proposed scheme of the alteration of cancer cell metabolism and NF‐κB inhibition by GP‐2250. Bold black arrows indicate metabolic pathways; red and green arrows indicate drug‐induced changes. By limiting the energy metabolism through the inhibition of hexokinase 2 and GAPDH, GP‐2250 induces an energy deficit in line with an impairment of the TCA cycle. The reduction of ATP triggers the activation of the energy‐deficit sensor AMPK. Its downstream events include the inhibition of mTOR, a major driver of tumour cell growth and potential impairment of fatty acid synthesis (FAS) through ACC‐1 inhibition. The inhibition of NF‐κB by GP‐2250 limits the rate of tumour cell proliferation through cyclin D1 downregulation. It also promotes apoptosis through downregulation of the anti‐apoptotic Bcl2. ROS contributes to the upregulation of the transcription factor p53, which supports apoptosis. AMPK, adenosine monophosphate‐dependent protein kinase; FAS, fatty acid synthesis; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; PDH, pyruvate dehydrogenase.