INTRODUCTION - Regulation of mitochondrial respiration by LDHB suppression in hepatoma cell lin

Aerobically growing normal cells oxidize glucose into pyruvate through glycolysis with release of 2 ATP in cytosol, and then pyruvate is further oxidized into CO2 through tricarboxylic acid (TCA) cycle and oxidative phosphorylation in mitochondria, thereby generating total 36 or 38 ATP depending on tissue type. However, when oxygen is limiting, cells cannot operate mitochondrial TCA cycle and oxidative phosphorylation, so produce ATP by activating glycolysis alone, termed anaerobic glycolysis. (Kim and Dang, 2006; Vander Heiden et al., 2009). Interestingly, many cancer cells depend on glycolytic ATP generation even in the presence of sufficient oxygen in culture or in tissue. This unexpected phenomenon was termed aerobic glycolysis. In fact, this phenomenon was first reported by Warburg in the 1920s, leading him to the hypothesis that cancer results from irreversibly impaired mitochondrial metabolism (Warburg, 1956).

In the tumorigenesis, cancer cells rapidly increase their cell growth and proliferation, forming tumor nodular mass. Therefore, these cancer cells within certain size of the nodule must experience insufficient nutrient and oxygen due to limited blood vessel formation. This idea well supports the aerobic glycolysis of cancer cell. In addition, many studies also reported that mitochondrial respiration of cancer cell was damaged due to mitochondrial DNA mutation or altered mitochondrial biogenesis, even in aerobic condition (Taylor and Turnbull, 2005).As a result, cancer cells try to survive by maintaining cellular energy through activating glycolysis in the present of sufficient oxygen.

Although ATP generation rate per glucose of glycolysis is relatively low, its timely fast process may compensate total cellular energy need. However, to satisfy the cellular energy level through aerobic glycolysis, two additional conditions are required. First, is efficient and effective uptake of glucose in the condition of limited blood supply. For example, normal liver cell possesses Glut2, of which Km is 15 to 20 mM, so the cell uptakes glucose only high blood glucose condition. However, it is reported that hepatoma cell overexpressed Glut1, of which Km is 1 mM, thereby hepatoma cells can efficiently uptake glucose even in low glucose environment that is induced by nodular formation-induced hypoxia (Amann et al., 2009).

Second condition is continuous re-supply of NAD+, oxidized form of NADH, to maintain the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase)-mediated reaction step. This critical condition is fulfilled by the LDH (lactate dehydrogenase), the final fermenting step of glycolysis.

LDH is an enzyme that catalyzes the reversible conversion between pyruvate to lactate, using NADH or NAD⁺ as a co-substrate. LDH is composed of tetrameric subunits of two different gene products, LDH-H (heart type) and LDH-M (muscle type). LDH-H is encoded by the LDHB gene and subunit LDH-M is by LDHA gene. In general, cells contain five different LDH isoenzymes as a result of the five different combinations of H and LDH-M: LDH1 (H4); LDH2 (MH3); LDH3 (M2H2); LDH4 (M3H); LDH5 (M4) (Drent et al., 1996).

These isoenzymes show different substrate reactivity. LDH5 preferentially converts pyruvate into lactate and LDH1 has reverse reactivity, converting lactate into pyruvate (Kim et al., 2011).

In the early 2000, oncogenes, such as c-Myc and HIF-1 (hypoxia-inducible factor-1α), are

known to be the key upstream regulators for both mitochondrial suppression and glycolytic activation through LDHA gene induction (Lewis et al., 2000). Therefore, LDHA gene induction and resultant LDH5 activity are known to play a key role to maintain glycolytic ATP generation in cancer cell. Recently, it has been reported that suppression of LDHB gene transcription effectively activates LHD5 formation and glycolytic activation in hepatoma cell.

In addition, this phenomenon was associated with mitochondrial dysfunction (Kim et al., 2011).

This observation remains a question of how LDHB suppression is linked with the mitochondrial respiratory defect.

PDC (pyruvate dehydrogenase complex) is the key enzyme of glucose metabolism which starts mitochondrial oxidation process, generating acetyl-CoA from pyruvate, the final of glycolysis.

Pyruvate + CoA + NAD⁺ → CO2 + acetyl-CoA + NADH + H⁺.

This reaction links glycolysis and the citric acid cycle in cell containing mitochondria (Harris et al., 2002). PDC is a large complex that is organized around a 60-meric dodecahedral core formed by PDH (pyruvate dehydrogenase, E1), acetyltransferase (E2) and E3-binding protein (E3BP), dihydrolipoamide dehydrogenase (E3), pyruvate dehydrogenase kinase (PDK), and pyruvate dehydrogenase phosphatase (PDP) (Read, 2001; Hiromasa et al., 2004; Hitosugi et al., 2011). PDH (pyruvate dehydrogenase) is the most important enzyme component of PDC

(Fan et al., 2014a). The enzymic activity of the PDC is regulated by the phosphorylation of three serine residues (sites 1, 2, and 3 are Ser-293, Ser-300, and Ser-232, respectively) located on the E1 component (Kolobova et al., 2001). Among these three sites, phosphorylation of Ser-293 was suggested to prevent active site accessibility of PDH to its substrate pyruvate (Seifert et al., 2007). The phosphorylation of these sites is catalyzed by PDK, a Ser/Thr kinase, whereas dephosphorylation of PDH is by PDP (Yeaman et al., 1978; Roche et al., 2001).

Several studies reported that PDKs play key roles in the metabolic alteration of cancer cell.

Expression of the PDK1 gene, as well as several glycolytic enzymes, is upregulated by c-Myc and HIF-1α (Kim et al., 2006; Pardo et al., 2006; Dang et al., 2008).

Collectively, these observations suggest that PDH may play a role to connect between mitochondrial respiratory regulation and LDHB suppression.

II. MATERIALS AND METHODS

A. Cell cultures and cell growth rate

Human hepatoma cells (SNU-387, SNU-354, and SNU-423) were purchased from Korean Cell Line Bank (Seoul, Korea) and were cultured in GIBCO^® RPMI1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% GIBCO^® fetal bovine serum (FBS) (Invitrogen) and GIBCO^® antibiotics (Invitrogen) at 37°C in a humidified incubator with 5% CO2. Chang cell was obtained from American Tissue Culture Collections (ATCC, Rockville, MD) and Chang cell clone, denoted as Chang-L, which has higher hepatic characteristics (albumin production and liver-specific carbamoyl-phosphate synthase-1 expression) were isolated by single cell dilution and expansion, and used for this study (Kim et al., 2011). Chang-L clones were cultured in GIBCO^® Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% GIBCO^® FBS. Cell growth rates were monitored by counting the trypan blue-negative viable cells.

B. Western blot analysis

Cultured cells were washed twice with PBS, and lysed with lysis buffer (1 M Tris-HCl [pH7.4], 150 mM NaCl, 0.1% SDS, 1% NP40) containing phosphatase inhibitor (200 mM NaF, 200 mM NaVO4) and protease inhibitor (200 mM PMSF, 500 mM leupeptin, 500 mM pepstatin A) at 4℃ for 30min. The isolated proteins were separated on 8-12% SDS polyacrylamide gels and then transferred onto nitrocellulose membrane (Protran; Schleicher

and Schuell) using. These membranes were then incubated for 12 hours with the following primary antibodies. Next, the membranes were washed with 0.1% PBS-T buffer 3 times for 15 minutes each, after which, they were incubated with HRP-conjugated mouse, anti-rabbit, anti-sheep, and anti-goat antibody (1:3000-5000, Santa Cruz) for 1-2hours. The membranes were then washed 3 times with 0.1% PBS-T buffer for 15 minutes.

Immunoreactive bands were visualized with the ECL system (West Save, Lab Frontier).

Antibodies against phospho-PDH pSer²⁹³ (1:1000, AP1062) were purchased from Calbiochem (San Diego, CA). Antibodies against PDH-E1α (1:2000, ab110330), PDK1 (1:2000, ab90444), LDHA (1:3000, ab9002) were purchased from Abcam Ltd (Cambridge, UK). Antibodies against phospho-p44/42 MAPK (Erk1/2) (1:1000, #9106), p44/42 MAPK (Erk1/2) (1:2000,

#9102) were purchased from Cell signaling Technology, Inc (Danvers, MA). Antibodies against for NDUFA9 of complex I (A21344), flavoprotein (A11142) of complex II, UQCRC2 of complex III (A11143), and ATP5A1 of complex V (A21350) were from Molecular Probes Corp. (Eugene, OR) and labeled as Comp I, II, III, and V, respectively, in the figures.

C. Transfection of siRNAs

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.

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).

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.

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)].

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)].

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.

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.

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.

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].

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

reversed by DCA. These results imply that LDHB suppression-mediated PDH inactivation is mediated by activated PDK activity without induction of PDK expression.

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.

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.

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.

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.

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.

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.

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.

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].

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.

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.

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.

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).

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).

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.

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

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