Wagner Waldemar, Ciszewski Wojciech M, Kania Katarzyna D
Laboratory of Cellular Immunology, Institute of Medical Biology, Polish Academy of Science, Lodz, Poland.
Laboratory of Transcriptional Regulation, Institute of Medical Biology, Polish Academy of Science, Lodz, Poland.
Cell Commun Signal. 2015 Jul 25;13:36. doi: 10.1186/s12964-015-0114-x.
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.
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.
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).
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.
将乳酸视为一种活性代谢物是一个新出现且颇具吸引力的概念。最近，有报道称乳酸可通过抑制组蛋白脱乙酰酶（HDACs）来调节基因转录，并通过羟基羧酸受体1（HCAR1）促进癌细胞存活。本研究探讨了L - 乳酸和D - 乳酸在宫颈癌细胞DNA损伤反应中的作用。
检测了三种宫颈癌细胞系：HeLa、Ca Ski和C33A。采用蛋白质免疫印迹法和生化方法分析乳酸对HDACs的抑制活性。使用γ-H2AX、彗星实验和克隆形成实验研究乳酸介导的DNA修复刺激作用以及细胞对新制癌菌素、阿霉素和顺铂的耐药性。通过实时定量PCR对HCAR1和DNA修复基因表达进行定量分析。分别通过免疫细胞化学和蛋白质免疫印迹法评估DNA依赖性蛋白激酶催化亚基（DNA-PKcs）活性和HCAR1蛋白表达。通过测量细胞内cAMP积累和细胞外调节蛋白激酶（Erk）磷酸化来研究HCAR1的激活情况。使用短发夹RNA（shRNA）使HCAR1表达沉默。
L - 乳酸和D - 乳酸抑制HDACs，诱导组蛋白H3和H4高度乙酰化，并降低HeLa细胞中的染色质紧密程度。用乳酸处理细胞使连接酶4（LIG4）、NBS1和脱嘌呤嘧啶核酸内切酶（APTX）表达增加近2倍，并增强DNA-PKcs活性。基于γ-H2AX和彗星实验，在含乳酸的培养基中培养细胞可提高DNA修复率。此外，克隆形成实验表明乳酸介导细胞对临床使用的化疗药物产生耐药性。蛋白质免疫印迹法和免疫细胞化学显示，所有研究的细胞系在细胞表面均表达HCAR1。通过百日咳毒素预处理抑制HCAR1功能可部分消除乳酸对DNA修复的影响。下调HCAR1会降低DNA修复效率，消除细胞对L - 乳酸的反应，并减弱D - 乳酸的作用。此外，表达HCAR1 shRNA的细胞中单羧酸转运蛋白4的mRNA水平显著降低。最后，使用抑制剂α-氰基-4-羟基肉桂酸（α-CHCA）通过药理学方法抑制单羧酸转运蛋白可抑制乳酸对DNA修复和细胞存活的增强作用。
我们的数据表明，子宫颈中存在的L - 乳酸和D - 乳酸可能参与细胞DNA损伤修复过程的调节以及宫颈癌细胞对抗癌治疗的耐药性。
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