Abstract
BackgroundThe consideration of lactate as an active metabolite is a newly emerging and attractive concept. Recently, lactate has been reported to regulate gene transcription via the inhibition of histone deacetylases (HDACs) and survival of cancer cells via hydroxycarboxylic acid receptor 1 (HCAR1). This study examined the role of L- and D-lactate in the DNA damage response in cervical cancer cells.MethodsThree cervical cancer cell lines were examined: HeLa, Ca Ski and C33A. The inhibitory activity of lactate on HDACs was analysed using Western blot and biochemical methods. The lactate-mediated stimulation of DNA repair and cellular resistance to neocarzinostatin, doxorubicin and cisplatin were studied using γ-H2AX, comet and clonogenic assays. HCAR1 and DNA repair gene expression was quantified by real-time PCR. DNA-PKcs activity and HCAR1 protein expression were evaluated via immunocytochemistry and Western blot, respectively. HCAR1 activation was investigated by measuring intracellular cAMP accumulation and Erk phosphorylation. HCAR1 expression was silenced using shRNA.ResultsL- and D-lactate inhibited HDACs, induced histone H3 and H4 hyperacetylation, and decreased chromatin compactness in HeLa cells. Treating cells with lactate increased LIG4, NBS1, and APTX expression by nearly 2-fold and enhanced DNA-PKcs activity. Based on γ-H2AX and comet assays, incubation of cells in lactate-containing medium increased the DNA repair rate. Furthermore, clonogenic assays demonstrated that lactate mediates cellular resistance to clinically used chemotherapeutics. Western blot and immunocytochemistry showed that all studied cell lines express HCAR1 on the cellular surface. Inhibiting HCAR1 function via pertussis toxin pretreatment partially abolished the effects of lactate on DNA repair. Down-regulating HCAR1 decreased the efficiency of DNA repair, abolished the cellular response to L-lactate and decreased the effect of D-lactate. Moreover, HCAR1 shRNA-expressing cells produced significantly lower mRNA levels of monocarboxylate transporter 4. Finally, the enhancement of DNA repair and cell survival by lactate was suppressed by pharmacologically inhibiting monocarboxylate transporters using the inhibitor α-cyano-4-hydroxycinnamic acid (α-CHCA).ConclusionsOur data indicate that L- and D-lactate present in the uterine cervix may participate in the modulation of cellular DNA damage repair processes and in the resistance of cervical carcinoma cells to anticancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0114-x) contains supplementary material, which is available to authorized users.
Highlights
The model of lactate as an active metabolite has emerged as an attractive concept
We demonstrated that concentrations of L- and D-lactate consistent with those observed in the uterine cervix inhibit class I and II histone deacetylases (HDACs), induce the hyperacetylation of H3 and H4 histones, increase chromatin accessibility and significantly enhance the DNA repair rate in cervical cancer cells, as evaluated by γ-H2AX and comet assays
Vaginal and ectocervical microbiota protect against pathogen colonisation by acidifying the mucosa using lactic acid and, according to our results, appear to modulate the activity of the cervical cancer cells in a manner that alters its resistance to chemotherapeutics
Summary
The model of lactate as an active metabolite has emerged as an attractive concept. The role of lactate as a signalling factor is supported by observations that lactate mimics hypoxic conditions, stimulates connective tissue synthesis and enhances endothelial cell mobility and tumour angiogenesis [1,2,3]. HCAR1 has been implicated in the regulation of lactate transport mechanisms. HDACs are involved in acetylation, an important posttranslational protein modification, and their activity opposes that of histone acetyltransferases (HATs). One of the primary implications of chromatin-HAT/ HDAC remodelling complex interactions is its important but poorly characterised role in regulating the DNA damage response (DDR) [10, 11]. Recent evidence suggests that the coordinated action of HATs/HDACs may directly affect the DDR by modulating the activity of key proteins involved in DNA damage detection and repair, such as DNA-PK [15] and ATM [16]. Lactate has been reported to regulate gene transcription via the inhibition of histone deacetylases (HDACs) and survival of cancer cells via hydroxycarboxylic acid receptor 1 (HCAR1). This study examined the role of L- and D-lactate in the DNA damage response in cervical cancer cells
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