The inhibition of lactate dehydrogenase A hinders the transcription of histone 2B gene independently from the block of aerobic glycolysis
Elisa Brighenti, Domenica Carnicelli, Maurizio Brigotti*, Luigi Fiume
Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy
a r t i c l e i n f o
Received 17 February 2017
Accepted 23 February 2017
Available online 28 February 2017
Anti-proliferative agents Nuclear LDHA inhibition
Histone 2B gene transcription inhibitors Oxamic acid
a b s t r a c t
Most cancer cells use aerobic glycolysis to fuel their growth and many efforts are made to selectively block this metabolic pathway in cancer cells by inhibiting lactate dehydrogenase A (LDHA). However, LDHA is a moonlighting protein which exerts functions also in the nucleus as a factor associated to transcriptional complexes. Here we found that two small molecules which inhibit the enzymatic activity of LDHA hinder the transcription of histone 2B gene independently from the block of aerobic glycolysis. Moreover, we observed that silencing this gene reduces cell replication, hence suggesting that the in- hibition of LDHA can also affect the proliferation of normal non-glycolysing dividing cells.
© 2017 Elsevier Inc. All rights reserved.
Inhibition of lactate dehydrogenase A (LDHA) aiming at blocking aerobic glycolysis and selectively damaging tumour cells is an approach to antineoplastic chemotherapy proposed several years ago [1,2] and presently actively pursued by Academic Institutions (for reviews see Refs. [3,4]) and Pharmaceutical Companies [5e9]. However, it is worth noting that LDHA is directly involved in transcriptional (reviewed in Ref. ) and post-transcriptional  regulation of expression of genes. In particular, Zheng, Luo and collaborators demonstrated that LDHA is an essential component of the complex activating the octamer-binding factor 1 which tran- scribes the histone 2B (H2B) gene [11e13].
Here we investigated whether inhibitors of LDHA impair the transcription of this gene.
⦁ Materials and methods
⦁ Cell lines and materials
HCT116 (human colon cancer cell line) was obtained from ATCC. NCM460 (human colon non-cancer cell line) was purchased from
Abbreviations: FBS, foetal calf serum; LDHA, lactate dehydrogenase A; H2B, histone 2B; OXA, oxamic acid; GSKA, GSK 2837808A.
* Corresponding author. Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Universita` di Bologna, Via San Giacomo 14, 40126, Bologna, Italy.
E-mail address: [email protected] (M. Brigotti).
INCELL Corporation (San Antonio, TX, USA). Cells were cultured in DMEM containing 25 mM glucose and supplemented with 10% foetal calf serum (FCS). GSK 2837808A (GSKA) (CAS No 1445879- 21-9) was purchased from Tocris Bioscence Bristol, United Kingdom). Oxamic acid (OXA) was purchased from Sigma-Aldrich.
⦁ Aerobic glycolysis
Cells (5 105 in 2 ml of culture medium) were seeded in 35 mm diameter-well (6-well Nunclon Plate). For each determination 3 wells were used. Cells were cultured in the presence of glucose or substituting galactose for glucose. In the former case, cells were maintained in DMEM containing glucose (25 mM) and supple- mented with 10% FCS dialysed (Sigma-Aldrich) to remove lactic acid. In the latter case, cells were maintained in Leibovitz-15 me- dium, which contained galactose (5 mM) instead of glucose, sup- plemented with dialysed FCS. After 15e16 h the medium was removed and replaced with the same fresh medium without FCS in order to allow the measure of cell proteins at the end of the
experiment. Lactic acid (intracellular and released in the medium) was measured after 3 h incubation at 37 ◦C. To each well, 100 ml of 100% trichloroacetic acid cold solution was added, cell lysate was collected and the well washed with 1 ml of cold 10% trichloroacetic acid. After centrifugation, the concentration of lactic acid in the supernatant was measured on 0.2e0.4 aliquots according to the method of Barker and Summerson . The precipitated proteins were dissolved in KOH and measured by using Bradford Reagent (Sigma-Aldrich).
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E. Brighenti et al. / Biochemical and Biophysical Research Communications 485 (2017) 742e745 743
Cells Glucose Galactose
Lactic acid (mg/well)a Cell proteins (mg/well)b Lactic acid (mg/well) Cell proteins (mg/well)
HCT116 NCM460 69.3 ± 12.1
115.4 ± 12.9 107.0 ± 16.5
174.7 ± 20.1 n.d.
n.d. 81.1 ± 10.8
109.9 ± 7.9
Lactic acid formed by cells after 3 h incubation in the presence of glucose or galactose.
n.d. not detected (<2 mg/well).
a Lactic acid was determined as described in Ref.  (mean ± SD, n ¼ 3).
b Protein content in the wells at the end of the experiment (mean ± SD, n ¼ 3).
⦁ Determination of H2B mRNA transcription
The cells were cultured for 15e16 h in the presence of glucose or galactose as described above for the measure of aerobic glycolysis. For each determination 3 wells were used. Thereafter, the medium was removed and replaced with the same fresh medium containing OXA or GSKA, two small molecules which inhibit LDHA. OXA and GSKA were used at concentrations (60 mM and 10 mM, respectively) found to impair aerobic glycolysis [2,7]. At 0 time and after 0.5 and 3 h of incubation at 37 ◦C, the cells were collected and total RNA extracted with TRIreagent (Ambion, Austin, TX,USA), according to manufacturer’s instructions. Total cell RNA was quantiﬁed spec- trophotometrically and 2 mg of RNA for each sample were reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), following the manu- facturer’s protocol. The relative amounts of human H2B and beta-
glucuronidase RNAs were evaluated by Real-time RT-PCR per- formed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with the 20 deltadeltaCT method . The mean del- taCT value of the 0 time samples was used in each experiment to calculate the deltadeltaCT value of sample replicates. Primers for SYBR Green Real-Time RT-PCR analysis of human H2B mRNA were
designed using the Roche online primers design tool. Beta- glucuronidase mRNA was quantiﬁed with TaQMan Gene Expres- sion Assays kit (Applied Biosystems). Sequences are available on request.
⦁ Gene silencing by RNAi transfection
NCM460 and HCT116 cells were seeded in 6-well plates. Vali- dated stealth RNAi siRNA (Invitrogen) targeting H2B was used while silenced control cells (scrambled) were transfected with
Fig. 1. Effect of the LDHA inhibitors GSKA and OXA on H2B mRNA expression. HCT116 and NCM460 cells were exposed to the LDHA inhibitors, GSKA or OXA, in the presence of glucose or galactose. Cells were collected at 0 time and after 0.5 and 3 h of treatment. (A) Real-time RT-PCR evaluation of H2B mRNA in HCT116 and NCM460 cells cultured in the medium containing glucose. (B) Real-time RT-PCR evaluation of H2B mRNA expression in HCT116 and NCM460 cells cultured in the medium containing galactose in the absence of glucose. Histograms show the value (mean ± SD) of three experiments. *p < 0.05; **p < 0.01; ***p < 0.001.
744 E. Brighenti et al. / Biochemical and Biophysical Research Communications 485 (2017) 742e745
equivalent amounts of Stealth RNAi negative control (Invitrogen). Cells were transfected with lipofectamine RNAiMAX (Invitrogen) in opti-MEM medium (Invitrogen) following the manufacturer’s procedures. The silencing procedures were carried out every day for three days.
⦁ Evaluation of cell proliferation rate
NCM460 and HCT116 were seeded in 24-well plates 24 h after the end of the silencing procedure. The cells were cultured for 96 h, then cell population was evaluated according to the Crystal Violet method as described in Ref. .
⦁ Statistical analysis
Statistical analysis of the data was performed by using the paired t-Test on three experimental replicates. P values < 0.05 were regarded as statistically signiﬁcant.
In agreement with previous data obtained using other cell lines [17,18] we found that HCT116 and NCM460 cells when cultured in the presence of galactose without glucose did not produce any detectable amounts of lactic acid (Table 1). To inhibit LDHA we used GSKA which is a selective NADH competitive inhibitor of LDHA  and oxamic acid which is the molecule mostly used to inhibit LDHA. The latter, unlike GSKA, blocks the enzyme by occupying the py- ruvic acid binding site . Both GSKA and oxamic acid caused an early (0.5e3 h) impairment of H2B gene transcription (Fig. 1), a result in keeping with the observation that the expression of this gene is inhibited by silencing LDHA gene . The inhibition of H2B gene transcription was independent from a block of aerobic glycolysis since it was also observed in the cells cultured without glucose which do not produce appreciable amounts of lactic acid
(Table 1). The inhibitions by GSKA or oxamic acid could be ascribed to the block of the catalytic activity of LDHA, which is maintained in the transcriptional complex , or to conformational changes of LDHA not allowing its assembly with the other components of the complex. We also observed that H2B gene silencing, obtained by using RNA interference procedure caused a reduction of the pro- liferative rate of both HCT116 and NCM460 cells (Fig. 2). This ﬁnding is in agreement with the observation that histone expres- sion is tightly coupled to S-phase progression .
The present results suggest that the impairment of cell prolif- eration observed in cells treated with LDHA inhibitors are not only due to the block of aerobic glycolysis, as assumed up to now, but are in part a consequence of the hindered expression of H2B gene which can also impair the proliferation of normal dividing cells. One argument supporting the concept that LDHA inhibitors could be safely given in humans is based on the knowledge that subjects with a complete genetic deﬁciency of the enzymatic activity of LDHA in blood and in cell cytoplasm do not show any symptoms under ordinary circumstances . However, this does not neces- sarily imply that also the transcriptional activity of LDHA is missing in these subjects and that administration of the enzyme inhibitors to humans will not impair H2B gene expression in all dividing cells. In fact, these subjects produce an incomplete LDHA protein lacking the terminal eCOOH subdomain [21,22] which is mandatory for the formation of the tetrameric structure . While the tetramer formation is critical to LDHA enzymatic function in solution and in the cytoplasm , in the transcriptional complex LDHA is active in the monomeric form  which may be operative also in humans lacking its enzymatic activity in cytoplasm.
In conclusion, the herein described results provide a novel
perspective to the action of known inhibitors of LDHA fostering our understanding of their action as anti-proliferative agents.
Fig. 2. Effect of H2B mRNA silencing on cell proliferation rate. The expression of H2B mRNA was down-regulated in HCT116 and NCM460 cells using the mRNA Interference procedure. The cells were interfered with scrambled (SCR) or H2B siRNA (H2Bi) for 72 h. 24 h after the end of the silencing procedure, cells were seeded in a 24-well plates for 96 h.
⦁ Real-time RT-PCR evaluation of H2B mRNA expression, and long-term effect (96 h) of H2B interference on the proliferation rate of HCT116 cells. (B) Real time RT-PCR evaluation of H2B mRNA expression, and long-term effect (96 h) of H2B interference on the proliferation rate of NCM460 cells. O.D.: optical density. **p < 0.01; ***p < 0.001.
E. Brighenti et al. / Biochemical and Biophysical Research Communications 485 (2017) 742e745 745
The authors thank Prof. Massimo Derenzini for his critical dis- cussion and revision of the ﬁnal manuscript. This work was sup- ported by the Associazione Italiana per la Ricerca sul Cancro (AIRC, grant n IG13480).
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.02.119.
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