Citrate-Induced p85α⁻PTEN Complex Formation Causes G2/M Phase Arrest in Human Pharyngeal Squamous Carcinoma Cell Lines.

Kuang-Chen Hung,Shih-Shun Chen,Shyang-Guang Wang,Meng-Liang Lin

doi:10.3390/ijms20092105

Abstract

Citrate is a key intermediate of the tricarboxylic acid cycle and acts as an allosteric signal to regulate the production of cellular ATP. An elevated cytosolic citrate concentration inhibits growth in several types of human cancer cells; however, the underlying mechanism by which citrate induces the growth arrest of cancer cells remains unclear. The results of this study showed that treatment of human pharyngeal squamous carcinoma (PSC) cells with a growth-suppressive concentration of citrate caused cell cycle arrest at the G2/M phase. A coimmunoprecipitation study demonstrated that citrate-induced cell cycle arrest in the G2/M phase was associated with stabilizing the formation of cyclin B1–phospho (p)-cyclin-dependent kinase 1 (CDK1) (Thr 161) complexes. The citrate-induced increased levels of cyclin B1 and G2/M phase arrest were suppressed by the caspase-3 inhibitor Ac-DEVD-CMK and caspase-3 cleavage of mutant p21 (D112N). Ectopic expression of the constitutively active form of protein kinase B (Akt1) could overcome the induction of p21 cleavage, cyclin B1–p-CDK1 (Thr 161) complexes, and G2/M phase arrest by citrate. p85α–phosphatase and tensin homolog deleted from chromosome 10 (PTEN) complex-mediated inactivation of Akt was required for citrate-induced G2/M phase cell cycle arrest because PTEN short hairpin RNA or a PTEN inhibitor (SF1670) blocked the suppression of Akt Ser 473 phosphorylation and the induction of cyclin B1–p-CDK1 (Thr 161) complexes and G2/M phase arrest by citrate. In conclusion, citrate induces G2/M phase arrest in PSC cells by inducing the formation of p85α–PTEN complexes to attenuate Akt-mediated signaling, thereby causing the formation of cyclin B1–p-CDK1 (Thr 161) complexes.

Highlights

Reprogramming of cellular metabolism from respiration toward aerobic glycolysis is recognized to supply the nucleotides, proteins, and lipids needed for rapid cell division, continuous growth, invasion, metastasis, and resistance to chemotherapeutic agents [1,2,3]
We first examined the effect of citrate on the growth of human pharyngeal squamous carcinoma (PSC) cells using the MTT assay and flow cytometric analysis of propidium iodide (PI) uptake
The coimmunoprecipitation assay of membrane fractions of vehicle-treated cells using an antibody specific for p85α or PTEN revealed that p-p85α (Tyr 508) formed a complex with p110α in the plasma membrane but did not interact with PTEN

Summary

Introduction

Reprogramming of cellular metabolism from respiration toward aerobic glycolysis is recognized to supply the nucleotides, proteins, and lipids needed for rapid cell division, continuous growth, invasion, metastasis, and resistance to chemotherapeutic agents [1,2,3]. An elevated level of the aerobic glycolysis-derived pyruvate is preferentially converted into lactic acid, some of the pyruvate is transported to the mitochondrial matrix, where it is diverted into acetyl-coenzyme A (acetyl-CoA) [4]. Mitochondrial acetyl-CoA and oxaloacetate (OAA) undergo a condensation reaction by citrate synthase to form citrate, which is transported to the cytosol to convert acetyl-CoA by ATP-citrate lyase (ACL). High levels of citrate are present in the cytosol that suppress phosphofructokinase, a key enzyme of glycolysis, inhibiting ATP production and the de novo synthesis of phospholipids [8]. Deprivation of ATP by glycolytic inhibition can induce apoptosis and inhibit cell growth in drug-resistant cancer cells [11,12], indicating that depletion of the cellular ATP supply may be an effective way to overcome chemoresistance in cancer therapy

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Conclusion