title: L- and D-lactate enhance DNA repair and modulate the resistance of cervical carcinoma cells to anticancer drugs via histone deacetylase inhibition and hydroxycarboxylic acid receptor 1 activation.
abstract: BACKGROUND
  The 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.
  
  
  METHODS
  Three 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.
  
  
  RESULTS
  L- 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).
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Laboratory of Cellular Immunology, Institute of Medical Biology, Polish Academy of Science, Lodz, Poland. wwagner@cbm.pan.pl.
authors.forename: Waldemar
authors.initials: W
authors.lastname: Wagner
authors.affiliation: Laboratory of Cellular Immunology, Institute of Medical Biology, Polish Academy of Science, Lodz, Poland. wciszewski@gmail.com.
authors.forename: Wojciech M
authors.initials: WM
authors.lastname: Ciszewski
authors.affiliation: Laboratory of Transcriptional Regulation, Institute of Medical Biology, Polish Academy of Science, Lodz, Poland. kkania@cbm.pan.pl.
authors.forename: Katarzyna D
authors.initials: KD
authors.lastname: Kania
mesh_terms: D000107:Acetylation; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002584:Cervix Uteri / Q000187:drug effects / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004260:DNA Repair; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006655:Histone Deacetylases / Q000378:metabolism*; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019344:Lactic Acid / Q000378:metabolism*; D027501:Monocarboxylic Acid Transporters / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism*; D009353:Zinostatin / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; C533610:HCAR1 protein, human; D006657:Histones; D027501:Monocarboxylic Acid Transporters; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; D019344:Lactic Acid; D004317:Doxorubicin; D009353:Zinostatin; D006655:Histone Deacetylases; D002945:Cisplatin