Small interfering RNAs (siRNA) into cells, cells were transfected with the siRNA duplexes using Oligofectamine™ Reagent (Invitrogen), respectively, according to the manufacturer’s instructions. Non-specific siRNA (siNC) were used as mock control. Target siRNAs were generated from Bioneer and single strand sequences of the siRNAs were as follows: 5’-GGAUAUACCAACUGGGCUA-3’ for LDHB #1,
5’-7
GCAGAUACCCUGUGGGACA-3’ for LDHB #2, 5’-CGUGCAUUCCCGAUUCCUU-3’
for LDHA #1, 5’-CUGGUUAGUGUGAAAU-3’ for LDHA #2,
GAGGAUGGGCUCAAAUACU-3’ for PDHA1 #1, CGUUACCACGGACACAGUA for PDHA1 #2.
D. LDH isozyme in-gel activity
Activity profiles of the five LDH isozymes are estimated by in gel activity analysis after separating the lysates (5 μg) on agarose native gel electrophoresis according to the protocol provided with an REP LDH Isoenzyme Assay Kit (Helena Laboratories, Beanmont, TX, USA) with Rapid Electrophoresis (Helena Lab.).
E. Lactate level in cultured medium
Lactate levels in cultured media were assessed using standard spectrophotometric assays (Robinson et al., 1985) with slight modification. Briefly, a portion (100 μl) of cultured medium was mixed with 200 μl of ice cold 1 M perchloric acid and incubated on ice. After centrifugation at 4000×g for 15 min, the supernatant was neutralized to pH 7.0 by adding the same volume of 0.7 M tripotassium phosphate solution. The neutralized supernatant (10 μl) was applied to spectophotometric lactate assay using ThermoMax microplate reader (Molecular Devices Co., Sunnyvale, CA, USA). Lactate level was estimated from standard lactate calibration curve prepared at the same condition and expressed as lactate (nmole) released from 1 μg of cell lysate.
8
F. Real-time reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated using Trizol (Invitrogen) and cDNA was prepared using AMV reverse transcriptase (Promega). For real-time reverse transcription (RT)-PCR , it was performed with 40 cycles of the reaction involving 95°C for 15 seconds and 60°C for 60 seconds using THUNDERBIRDTM SYBRTM qPCR Mix (Toyobo Co., Ltd, Osaka, Japan) according to the manufacturer’s protocol. The PCR primer sets were produced by Bioneer as follows: PDK1,5’- CTATGAAAATGCTAGGCGTCTGT-3’ and 5’- AACCACTTGTATTGGCTGTCC-3’;
PDK2,5’- AGGACACCTACGGCGATGA-3’ and 5’- TGCCGATGTGTTTGGGATGG-3’;
PDK3,5’- GCCAAAGCGCCAGACAAAC-3’ and 5’- CAACTGTCGCTCTCATTGAGT-3’;
PDK4, 5’- TTATACATACTCCACTGCACCA-3’ and 5’-
ATAGACTCAGAAGACAAAGCCT-3’; ß-actin, 5'-CCTTCCTGGGCATGGAGTCCTGT-3’
and 5'-GGAGCAATGATCTTGATCTTC-3’. Expression levels of target mRNAs were normalized by ß-actin mRNA level.
G. Mitochondrial membrane potential
To estimate mitochondrial membrane potential, cells were treated with TTFA for the indicated periods, further incubated in media containing 10 mg/ml JC-1 (Molecular probe), and washed with PBS for 20 min at 37.8℃. The average cellular red and green fluorescence intensities were obtained by Axiovision 4.2 software with ratio module after visualization with 100 ms on Axiovert 200 M equipped with HBO103 using a LD Plan-Neofluar 40_ objective (Carl Zeiss AG, Gottingen, Germany). The ratio of red fluorescence to green one was
9 estimated and expressed as percentage of control.
H. Endogenous cellular oxygen consumption rate
Endogenous cellular oxygen consumption rate was measured in situ with cultured cells using XF-24 Extracellular Flux Analyzer (Seahorse Bioscience) according to the protocol provided. Briefly, cells were seeded on XF24c ell culture microplates (Seahorse Bioscience) at a density of 10,000 cells per well and preincubated with XF assay medium (Seahorse Bioscience) containing 1 mM pyruvate and 5 mM glucose. Its mitochondrial specificity was confirmed by adding 100 nM antimycin A.
I. Chemicals
Dichloroacetate (DCA) (347795), PD98059 (P-215), L-(+)-lactic acid solution (L1875), and Sodium lactate (L7022) were purchased from Sigma-Aldrich (St. Louis,MO).
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III. RESULTS
1. Decreased LDHB expression-mediated lactic acidosis is associated with mitochondrial respiratory activity.
First, the relationship between LDHB suppression and mitochondrial dysfunction of hepatoma cells was investigated by using three different SNU hepatoma cells, 387, SNU-354, and SNU-423, derived from human hepatocellular carcinomas. Chang-L (Ch-L) clone, which was derived from Chang cell and showed liver-characteristics and active mitochondrial respiration (Kim et al., 2011), was also used as a cell with active mitochondrial activity. SNU-354 and SNU-423 showed decreased LDHB expression, compared to Ch-L cell and SNU-387 cell (Fig. 1A). These two hepatoma cells with low LDHB expression showed a clear isoenzyme shift toward LDH5 (Fig. 1B), increased lactate level and decreased pH in medium (Fig. 1C). Interestingly, these two cells also showed decreased cellular oxygen consumption rates (Fig. 2). These data suggested that LDHB expression is associated with mitochondrial respiratory function.
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Fig. 1. Decreased LDHB expression is linked with lactic acidosis.
Chang cell clone (Ch-L) and three different SNU hepatoma cell lines (SNU-387, SNU-354, and SNU-423) were cultured for 2 days to maintain exponentially growing state. A) Western blot analysis for LDHA and LDHB expression. B) In-gel activity profile of LDH isozymes was performed as described in the ‘Materials and methods’. C) Lactate levels in media. Media lactate levels released from the cells for 2 days were estimated and expressed as lactate (nmole) released from 1 μg of cell lysate protein. pH of media were immediately measured by using spectrophotometric analysis [**, < 0.01 vs.Ch-L (control)].
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Fig. 2. Decreased LDHB expression is associated with mitochondrial respiratory activity.
Endogenous cellular oxygen consumption rate was measured and its specificity for mitochondrial respiration was confirmed by adding 100 nM antimycin A. [**, <0.01 vs. Ch-L (control)].
13
2. LDHB suppression is an upstream event of decreased mitochondrial respiration.
To further investigate the relationship between LDHB suppression and mitochondrial dysfunction, Ch-L (with active mitochondria) was treated with subcytotoxic doses of respiratory inhibitors, rotenone (complex I inhibitor), TTFA (complex II inhibitor), antimycin A (complex III inhibitor), KCN (complex IV inhibitor), and oligomycin (complex V inhibitor) for 12 hours as previously reported (Byun et al., 2008). LDHB expression was not altered by these respiratory inhibitions (Fig. 3). When LDHB expression was knocked down using siRNA in SNU-387 cells (high LHDB level), LDH isozyme was effectively shifted toward LDH5, but expressions of respiratory complex subunits were unchanged (Fig. 4). Nevertheless, cellular oxygen consumption rate and mitochondrial membrane potential were significantly decreased (Fig. 5). These results indicated that LDHB suppression-mediated LDH5 activation is the upstream event to decrease mitochondrial respiration without altering their subunit expressions.
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Fig. 3. Mitochondrial respiratory inhibition does not affect LDHB expression.
Ch-L was treated with 10 μM Rotenone (Ro), 400 μM TTFA (TT), 10 μM antimycin A (AA), 10 mM KCN (Kcn), 10 μM Oligomycin (Oli) for 12 hours. Western blot analysis for mitochondrial respiratory complex subunits, complex I (NDUFA9), complex II (Fp), complex III (core2), complex IV (subunit II), LDHB, and LDHA expression.
15
Fig. 4. Down-expressed LDHB induced to LDH iosenzyme shift.
SNU-387 cells were transfected with siRNA for LDHB (siLDHB) and LDHA (siLDHA) for 3 days. A) Western blot analysis for mitochondrial respiratory complex expression. complex I (NDUFA9), complex II (Fp), complex III (core2), complex V (ATP). B) In-gel LDH isoezyme analysis.
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Fig. 5. LDHB suppression was associated with mitochondrial dysfunction
SNU387 cells were transfected with siRNA for LDHB (siLDHB) and LDHA (siLDHA) for 3 days. A) Cellular oxygen consumption rate. B) Mitochondrial membrane potential. [**, p<0.01 vs. negative control].
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3. LDHB knockdown induces PDK-mediated PDH phosphorylation without HIF1/PDK1 induction.
To understand the molecular mechanism of how LDHB suppression induces mitochondrial dysfunction, two hypotheses were proposed. One possibility is that LDHB suppression may decrease expressions of mitochondrial respiratory complex subunits. However, as shown in fig.4A, LDHB knock-down did not altered expression of respiratory subunits. The other possibility was placed on post-translational modification of mitochondrial respiration-linked enzymes. PDH was first targeted due to its important role as tumor-associated metabolic shift.
PDH is the key enzyme to turn on mitochondrial TCA cycle and resultant respiration by producing acetyl-CoA.
Interestingly, mitochondrial defective hepatoma cells, SNU354 and SNU423, showed increased PDH phosphorylation, which indicates PDH inactivation. It is well known that PDH is negatively regulated by PDK. There are four PDK isoforms that are distributed differently in tissues. Several studies reported that PDK1 play a key role in the metabolic alteration of cancer cell. Especially, PDK1 was known to be induced by HIF-1α and Myc. However, when LDHB expression was suppressed both HIF-1α and PDK1 expression remained unchanged (Fig. 6B). In 2011, Hitosugi T. et al reported that PDK1 activity was also regulated by post-translational modification, especially phosphorylation (Hitosugi et al., 2011). To check the involvement of PDK1 activity, Dichloroacetate (DCA), an inhibitor, was treated in LDHB down-expressed SNU-387 cell. Phosphorylation level by LDHB repression was clearly
18
reversed by DCA. These results imply that LDHB suppression-mediated PDH inactivation is mediated by activated PDK activity without induction of PDK expression.
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Fig. 6. LDHB suppression induces PDH phosphorylation without induction of HIF-1α and PDK1 expressions.
Western blot analysis for phosphorylation of PDH, HIF-1α, and PDK1 expression. A) Three different SNU hepatoma cell lines (SNU-387, SNU-354, and SNU-423) were cultured for 2 days to maintain exponentially growing state. B) SNU-387 cells were transfected with siRNAs for LDHB (siLDHB) and LDHA (siLDHA) for 3 days. Western blot analysis for phosphorylation of PDH expression.
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Fig. 7. Expressions of PDK isotypes are not induced by LDHB knockdown.
SNU-387 cells were transfected with siRNAs for LDHB (siLDHB) for 3 days. Quantitative analyses of the expression levels are shown.
21
Fig. 8. LDHB suppression-mediated PDH phosphorylation is regulated by PDK activation without induction of PDK expression.
SNU-387 cells were transfected with siRNAs for LDHB (siLDHB) for 3 days. Before cell harvest, 5 mM DCA treated for 1 hour. Western blot analysis for phosphorylation of PDH and PDK1 expression.
22
4. Lactic acidosis increases PDH phosphorylation.
Next, I investigated the mechanism of how LDHB suppression induces PDK activation.
LDHB suppression-mediated LDH5 induction must be linked with the hepatoma-associated lactic acidosis (Fig. 1C). Therefore, we tested whether PDH inactivation is mediated by lactic acidosis. Previous studies reported that the physiological concentration of lactate in the hypoxic tumor microenvironment reached about 5 to 20 mM (Rudrabhatla et al., 2006;
Rattigan et al., 2012). When SNU-387 cells were treated with lactic acid for 12 hours, PDH phosphorylation increased in a dose-dependent manner (fig. 9) without induction of PDK transcription (fig. 11). This PDH phosphorylation started to increase 1hour after treatment (fig.
10A). Similar results were observed when SNU-387 cells were treated with the media containing 20 mM lactic acid and adjusted pH to 6.5, but not with the media containing 20 mM lactic acid with pH 7.8 (fig. 10B and 10C) , indicating that lactic acidosis, not the organic matter of lactate alone, is involved in PDH inactivation. Sodium lactate (20 mM) did not increase PDH phosphorylation, but decreasing pH alone slightly increased the phosphorylation (fig. 11A and 11B). Finally, it was clearly confirmed that PDH phosphorylation by 20 mM lactic acid was the most effective (fig. 11C). These results indicated that LDHB suppression-associated PDH phosphorylation is mediated by lactic acidosis.
23
Fig. 9. Lactic acid induces PDH phosphorylation in a dose-dependent manner.
SNU-387 cell treated with the indicated concentrations of lactic acid for 12 hours. Western blot analysis for phosphorylation of PDH expression.
24
Fig. 10. Lactic acidosis, not lactate alone, increases PDH phosphorylation.
SNU-387 cell treated with 20 mM lactic acid in time-dependent manner. A) SNU-387 cell was treated only 20 mM lactic acid. B) SNU-387 cell was treated with 20 mM lactic acid and pH adjusted to 6.5 by 100 mM NaOH. C) SNU-387 cell was treated with 20mM lactic acid and pH adjusted to 7.8 by 100 mM NaOH.
25
Fig. 11. PDH phosphorylation is also slightly induced by acidification itself and further amplified by lactate-associated acidification.
A) SNU-387 cell was treated with sodium lactate for 12 hours. B) SNU-387 cell was treated with 5 N HCl for 12 hours. C) SNU-387 cell was treated with 20 mM lactic acid, 5 N HCl and 20 mM sodium lactate for 12 hours.
26
Fig. 12. Lactic acidosis is associated with mitochondrial respiratory activity.
Endogenous cellular oxygen consumption rate was measured and its specificity for mitochondrial respiration was confirmed by adding 100 nM antimycin A. [**, <0.01 vs.
control].
27
5. Lactate induced PDH phosphorylation through ERK activation.
Next, I investigated the mechanism of how increased lactate by LDHB suppression-mediated PDH phosphorylation. In 2011, Grassian AR. et al. reported that ERK regulated PDH flux through PDK4 modulates cell proliferation (Grassian et al., 2011). To check the involvement of ERK activation, PD98059, a MAP kinase inhibitor, was treated in LDHB down-expressed SNU-387 cell. Phosphorylation level by LDHB repression was clearly reversed by PD98059 (fig. 13). These results imply that LDHB suppression-mediated PDH inactivation is mediated by ERK activation.
Next, I investigated the mechanism of how lactic acidosis induces PDH phosphorylation.
When SNU-387 cell was treated with lactic acid, increased PDH and ERK phosphorylation (fig.14). These results imply that lactic acidosis-induced PDH phosphorylation is mediated through ERK activation.
28
Fig. 13. LDHB-suppression-induced PDH phosphorylation is mediated through ERK activation.
A) Western blot analysis for phosphorylation of PDH, ERK expression. SNU-387 cells were transfected with siRNAs for LDHB (siLDHB) for 3 days. B) Before cell harvest, 20 μM PD98059 treated for 1 and 4 hours.
29
Fig. 14. Lactic acidosis-induced PDH phosphorylation is mediated through ERK activation.
SNU-387 cell was treated with lactic acid (LA). Western blot analysis for phosphorylation of PDH and ERK expression. A) 15 and 20 mM in dose-dependent manner for 12 hours. B) 3, 6, and 12 hours in time-dependent manner for 20 mM.
30
6. Lactic acidosis effect on cell growth by PDH phosphorylation.
Finally, I investigated the phenotype of cell by lactate. When SNU-387 cells were treated with lactic acid for 12 hours, decreased the cell growth and change epithelial to fibroblastic morphology. These results indicated that similar to a low oxygen consumption rate and growth rate of SNU-354, SNU-423 cells (fig. 15). When PDH (PDHE1α) expression was knocked down using siRNA in SNU-387 cells, decreased the cell growth and change morphology.
These results indicated that inactivation of PDH by lactic acid effect on cell growth (fig. 16).
31
Fig. 15. Lactic acidosis delays cell growth with morphological changes.
A) SNU-387 cell treated with 15 and 20 mM lactic acid (LA) for 12, 24, and 48 hours. Cell growth rates were monitored by counting the trypan blue-negative viable cells (a). Live and death cell number (b). B) SNU-387 cell treated with 20 mM lactic acid for 12 hours. Cellular morphology of SNU-387 cell. C) Cellular morphology of Ch-L and three different SNU hepatoma cell lines (SNU-387, SNU-354, SNU-423).
32
Fig. 16. PDH suppression delays cell growth with morphological changes.
SNU-387 cells were transfected with siRNAs for PDHA1 (siPDHA1) for 3 days. A) Western blot analysis for phosphorylation of PDH expression. B) Cell growth rates were monitored by counting the trypan blue-negative viable cells (a). Live and death cell number (b). C) SNU-387 cell treated with 20 mM lactic acid for 12 hours. Cellular morphology of SNU-SNU-387 cell.
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Fig. 17. Schematic model for regulation of mitochondrial respiration by LDHB suppression in hepatoma cell.
These results suggest that activity of lactate increased by LDHB suppression. Accordingly, increased lactate was induced PDH inactivation. Also, increased lactate mediated PDH inactivation is mediated by ERK activation
34
IV. DISCUSSION
In this study, it was proved that LDHB suppression-mediated lactic acidosis induces mitochondrial respiratory dysfunction by inactivation of PDH in hepatoma cell lines. Thus, it was identified that several key functions: First, suppressed LDHB expression in hepatoma cell increased LDH5 activity and production and release. Accordingly, the increased extracellular lactate may be re-introduced back into the tumor cell and affect diverse cellular function.
Second, LDHB suppression is an upstream event of decreased mitochondrial respiration. Third, I clearly demonstrated that lactic acidosis formed by released lactate induced the mitochondrial respiratory dysfunction by inactivation of PDH. Finally, LDHB-suppression-induced PDH phosphorylation is mainly mediated by ERK activation.
Extracellular release of lactate and resultant microenvironmental acidification, even under aerobic conditions (i.e., aerobic glycolysis), is a common feature of cancer development.
Increased lactate levels often allows to predict for metastases and overall survival of patients, as shown by several studies (Holroyde et al., 1979; Walenta et al., 2000; Saraswathy et al., 2009). Lactate promotes tumor metastasis by inducing hyaluronan secretion from tumor-associated fibroblasts which generate a milieu favorable for migration (Rudrabhatla et al., 2006). Lactate itself also induces the tumor cell migration (Beckert et al., 2006; Baumann et al., 2009; Goetze et al., 2011). Lactate functions to stimulate production of VEGF, as well as a growth-promoting factors that assist in angiogenesis (Trabold et al., 2003). However, it remains unclear how lactate regulates mitochondrial respiratory function.
35
PDC is the key enzyme of glucose metabolism which starts mitochondrial oxidation process, generating acetyl-CoA from pyruvate, the final of glycolysis. The regulation of PDH activity through phosphorylation is well known in particular in cancer cell. Several studied have shown that new mechanisms regulate in the activity of PDH. For example, it is known that activity of PDH regulated TPKL/GSK3β (Hoshi et al., 1996). Also, phosphorylation of tyrosine residue through PDK1 and PDP1 inhibits PDH in EGF-stimulated cells (Fan et al., 2014a; Fan et al., 2014b).
Several studies reported that PDKs play key roles in the metabolic alteration of cancer cell.
Expression of the PDK1 gene is upregulated by c-Myc and HIF-1α (Kim et al., 2006; Pardo et al., 2006; Dang et al., 2008). Interestingly, it was also proved that LDHB suppression-mediated lactic acidosis phosphorylated PDH through PDK activation by ERK without inducing PDK expression.
ERK pathway are critical in the regulation of several biological processes such as proliferation. In 2011, Grassian AR. et al. reported that ERK regulated PDH flux through PDK4 modulates cell proliferation (Grassian et al., 2011).
Our present study emphasize that aerobic glycolysis is not only achieved as an adaptive cellular strategy to meet energy deficit induced by tumoral hypoxia or mitochondrial respiratory defect, but also an active tumoral strategy to shut down mitochondrial respiratory function and induce aggressive tumoral properties.
36
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