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.

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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.

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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.

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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.

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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].

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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.

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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.

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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.

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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).

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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).

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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

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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.

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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.

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핵심어: 간암세포주, 미토콘드리아 기능손상, 해당과정, LDHB (lactate dehydrogenase B), PDH (pyruvate dehydrogenase), Lactic acidosis, Hepatoma cell