Toyonobu Maeda

Acidic extracellular microenvironment and cancer

Acidic extracellular pH is a major feature of tumor tissue, extracellular acidification being primarily considered to be due to lactate secretion from anaerobic glycolysis. Clinicopathological evidence shows that transporters and pumps contribute to H+ secretion, such as the Na+/H+ exchanger, the H+-lactate co-transporter, monocarboxylate transporters, and the proton pump (H+-ATPase); these may also be associated with tumor metastasis. An acidic extracellular pH not only activates secreted lysosomal enzymes that have an optimal pH in the acidic range, but induces the expression of certain genes of pro-metastatic factors through an intracellular signaling cascade that is different from hypoxia. In addition to lactate, CO2 from the pentose phosphate pathway is an alternative source of acidity, showing that hypoxia and extracellular acidity are, while being independent from each other, deeply associated with the cellular microenvironment. In this article, the importance of an acidic extracellular pH as a microenvironmental factor participating in tumor progression is reviewed.

Induction of osteoblast differentiation indices by statins in MC3T3‐E1 cells

Statins inhibit 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase, which catalyzes conversion of HMG‐CoA to mevalonate, a rate‐limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10−7 M markedly increased mRNA expression for bone morphogenetic protein‐2 (BMP‐2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3‐E1), while suppressing gene expression for collagenase‐1, and collagenase‐3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase‐digestive proteins, and noncollagenous proteins was increased in the cells treated with 10−7 M simvastatin, or 10−8 M cerivastatin. In the culture of MC3T3‐E1 cells, statins stimulated mineralization; pretreating MC3T3‐E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin‐induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.

Leptin stimulates fibroblast growth factor 23 expression in bone and suppresses renal 1α,25-dihydroxyvitamin D3 synthesis in leptin-deficient ob/ob Mice

Leptin is the LEP (ob) gene product secreted by adipocytes. We previously reported that leptin decreases renal expression of the 25‐hydroxyvitamin D3 1α‐hydroxylase (CYP27B1) gene through the leptin receptor (ObRb) by indirectly acting on the proximal tubules. This study focused on bone‐derived fibroblast growth factor 23 (FGF‐23) as a mediator of the influence of leptin on renal 1α‐hydroxylase mRNA expression in leptin‐deficient ob/ob mice. Exposure to leptin (200 ng/mL) for 24 hours stimulated FGF‐23 expression by primary cultured rat osteoblasts. Administration of leptin (4 mg/kg i.p. at 12‐hour intervals for 2 days) to ob/ob mice markedly increased the serum FGF‐23 concentration while significantly reducing the serum levels of calcium, phosphate, and 1α,25‐dihydroxyvitamin D3 [1,25(OH)2D3]. Administration of FGF‐23 (5 µg i.p. at 12‐hour intervals for 2 days) to ob/ob mice suppressed renal 1α‐hydroxylase mRNA expression. The main site of FGF‐23 mRNA expression was the bone, and leptin markedly increased the FGF‐23 mRNA level in ob/ob mice. In addition, leptin significantly reduced 1α‐hydroxylase and sodium‐phosphate cotransporters (NaPi‐IIa and NaPi‐IIc) mRNA levels but did not affect Klotho mRNA expression in the kidneys of ob/ob mice. Furthermore, the serum FGF‐23 level and renal expression of 1α‐hydroxylase mRNA were not influenced by administration of leptin to leptin receptor–deficient (db/db) mice. These results indicate that leptin directly stimulates FGF‐23 synthesis by bone cells in ob/ob mice, suggesting that inhibition of renal 1,25(OH)2D3 synthesis in these mice is at least partly due to elevated bone production of FGF‐23.

Simvastatin Suppresses Leptin Expression in 3T3-L1 Adipocytes via Activation of the Cyclic AMP-PKA Pathway Induced by Inhibition of Protein Prenylation

Simvastatin inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyses conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. We demonstrated that simvastatin at 1 microM markedly inhibited adipocyte differentiation measured by Oil Red O staining in preadipocyte cells (3T3-L1), while expression of leptin, a marker of adipocyte differentiation, was suppressed by 1 muM simvastatin for up to 12 days of culture. Next, to elucidate mechanisms underlying the reduction of leptin expression induced by simvastatin, differentiated 3T3-L1 adipocytes were treated with various inhibitors with mevalonate or its metabolite in the presence or absence of simvastatin. Simvastatin time- and dose-dependently suppressed leptin mRNA expression. Heterogeneous nuclear RNA related to leptin mRNA was inhibited by 10 muM simvastatin, while stability of the mRNA was not changed by treatment with simvastatin in transcription-arrested 3T3-L1 cells. Simvastatin inhibition of leptin gene transcription was not abrogated by pre-treatment with cycloheximide, an inhibitor of protein synthesis. Addition of mevalonate or geranylgeranyl pyrophosphate (GGPP), a mevalonate metabolite, abolished simvastatin-induced inhibition of leptin expression in 3T3-L1 cells. Suppression of expression was observed upon addition of GGTI-298, a geranylgeranyl transferase I inhibitor, but not FTI-277, a farnesyl transferase inhibitor. Expression was suppressed by treatment with hydroxyfasudil, a protein prenylation inhibitor. Treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, reduced leptin expression in 3T3-L1 cells. Simvastatin dose-dependently increased intra-cellular cyclic AMP (cAMP) concentrations in 3T3-L1 cells, with maximal stimulation obtained at 10 muM. Addition of GGPP abolished simvastatin-induced stimulation of cAMP accumulation and protein kinase A (PKA) activity. H89, an inhibitor of PKA, completely abolished simvastatin-induced suppression of leptin expression. These results suggested that simvastatin reduced geranylgeranylprotein prenylation followed by deactivation of PI3K, leading to cAMP accumulation and subsequent activation of PKA in differentiated 3T3-L1 adipocytes. Finally, PKA inhibited leptin gene transcription without new protein synthesis.

Deguelin Induces Apoptosis by Targeting Both EGFR-Akt and IGF1R-Akt Pathways in Head and Neck Squamous Cell Cancer Cell Lines

Deguelin, a rotenoid compound from the African plant Mundulea sericea (Leguminosae), has been shown to possess antitumor activities but the exact role for the growth factor receptor mediated signaling pathway in head and neck squamous cell carcinoma (HNSCC) is currently still unclear. In the present study, we investigated the effect of deguelin on epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor (IGF1R) pathways in HNSCC cell lines. Flowcytometric analysis revealed accumulation of annexin V positivity in deguelin-treated cells, showing that deguelin induced apoptosis. The deguelin-induced apoptosis was accompanied by the reduction of constitutive phosphorylated levels of IGF1R, Akt, and extracellular signal-regulated kinase1/2 (ERK1/2). LY294002-mediated inhibition of phosphatidylinositol-3 kinase, which is an upstream effector for Akt activation, increased cleavage of poly(ADP-ribosyl) polymerase (PARP) but ERK inhibition by U0126 did not. Deguelin inhibited both IGF-1- and EGF-induced Akt activation. These results showed that deguelin possessed antitumor effect by targeting Akt in dual axis such as EGFR and IGF1R signaling pathways and suggested that it provides an applicable therapeutic strategy for HNSCC patients.

Cancer metabolism: New insights into classic characteristics

Summary Initial studies of cancer metabolism in the early 1920s found that cancer cells were phenotypically characterized by aerobic glycolysis, in that these cells favor glucose uptake and lactate production, even in the presence of oxygen. This property, called the Warburg effect, is considered a hallmark of cancer. The mechanism by which these cells acquire aerobic glycolysis has been uncovered. Acidic extracellular fluid, secreted by cancer cells, induces a malignant phenotype, including invasion and metastasis. Cancer cells survival depends on a critical balance of redox status, which is regulated by amino acid metabolism. Glutamine is extremely important for oxidative phosphorylation and redox regulation. Cells highly dependent on glutamine and that cannot survive with glutamine are called glutamine-addicted cells. Metabolic reprogramming has been observed in cancer stem cells, which have the property of self-renewal and are resistant to chemotherapy and radiotherapy. These findings suggest that studies of cancer metabolism can reveal methods of preventing cancer recurrence and metastasis.

RhoA mediates the expression of acidic extracellular pH-induced matrix metalloproteinase-9 mRNA through phospholipase D1 in mouse metastatic B16-BL6 melanoma cells

Solid tumors are characterized by acidic extracellular pH (pHe). The present study examined the contribution of small GTP-binding proteins to phospholipase D (PLD) activation of acidic pHe-induced matrix metalloproteinase-9 (MMP-9) production. Acidic pHe-induced MMP-9 production was reduced by C3 exoenzyme, which inhibits the Rho family of GTPases; cytochalasin D, which inhibits actin reorganization; and simvastatin, which inhibits geranylgeranylation of Rho. Small interfering RNA (siRNA) against RhoA, but not against Rac1 or Cdc42, significantly inhibited acidic pHe induction of MMP-9. Pull-down assays showed that acidic pHe increased the activated form of RhoA. Forced expression of constitutively active RhoA induced MMP-9 production, even at neutral pHe. RhoA siRNA also reduced acidic pHe induced PLD activity. Specific inhibition of PLD1 and Pld1 gene knockout significantly reduced acidic pHe-induced MMP-9 expression. In contrast, PLD2 inhibition or knockout had no effect on MMP-9 expression. These findings suggested that RhoA-PLD1 signaling is involved in acidic pHe induction of MMP-9.

Mineral trioxide aggregate induces osteoblastogenesis via Atf6

Mineral trioxide aggregate (MTA) has been recommended for various uses in endodontics. To understand the effects of MTA on alveolar bone, we examined whether MTA induces osteoblastic differentiation using MC3T3-E1 cells. MTA enhanced mineralization concomitant with alkaline phosphatase activity in a dose- and time-dependent manner. MTA increased production of collagens (Type I and Type III) and matrix metalloproteinases (MMP-9 and MMP-13), suggesting that MTA affects bone matrix remodeling. MTA also induced Bglap (osteocalcin) but not Bmp2 (bone morphogenetic protein-2) mRNA expression. We observed induction of Atf6 (activating transcription factor 6, an endoplasmic reticulum (ER) stress response transcription factor) mRNA expression and activation of Atf6 by MTA treatment. Forced expression of p50Atf6 (active form of Atf6) markedly enhanced Bglap mRNA expression. Chromatin immunoprecipitation assay was performed to investigate the increase in p50Atf6 binding to the Bglap promoter region by MTA treatment. Furthermore, knockdown of Atf6 gene expression by introduction of Tet-on Atf6 shRNA expression vector abrogated MTA-induced mineralization. These results suggest that MTA induces in vitro osteoblastogenesis through the Atf6–osteocalcin axis as ER stress signaling. Therefore, MTA in endodontic treatment may affect alveolar bone healing in the resorbed region caused by pulpal infection.

TRPM5 mediates acidic extracellular pH signaling and TRPM5 inhibition reduces spontaneous metastasis in mouse B16-BL6 melanoma cells

Extracellular acidity is a hallmark of solid tumors and is associated with metastasis in the tumor microenvironment. Acidic extracellular pH (pHe) has been found to increase intracellular Ca2+ and matrix metalloproteinase-9 (MMP-9) expression by activating NF-κB in the mouse B16 melanoma model. The present study assessed whether TRPM5, an intracellular Ca2+-dependent monovalent cation channel, is associated with acidic pHe signaling and induction of MMP-9 expression in this mouse melanoma model. Treatment of B16 cells with Trpm5 siRNA reduced acidic pHe-induced MMP-9 expression. Enforced expression of Trpm5 increased the rate of acidic pHe-induced MMP-9 expression, as well as increasing experimental lung metastasis. This genetic manipulation did not alter the pHe critical for MMP-9 induction but simply amplified the percentage of inducible MMP-9 at each pHe. Treatment of tumor bearing mice with triphenylphosphine oxide (TPPO), an inhibitor of TRPM5, significantly reduced spontaneous lung metastasis. In silico analysis of clinical samples showed that high TRPM5 mRNA expression correlated with poor overall survival rate in patients with melanoma and gastric cancer but not in patients with cancers of the ovary, lung, breast, and rectum. These results showed that TRPM5 amplifies acidic pHe signaling and may be a promising target for preventing metastasis of some types of tumor.

Deguelin Potentiates Apoptotic Activity of an EGFR Tyrosine Kinase Inhibitor (AG1478) in PIK3CA-Mutated Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is known to be intrinsically resistant to inhibitors for epidermal growth factor receptor (EGFR). Until now, clinical outcomes for HNSCC using EGFR inhibitors as single agents have yielded disappointing results. Here, we aimed to study whether combinatorial treatment using AG1478 (EGFR tyrosine kinase inhibitor) and deguelin, which is a rotenoid isolated from the African plant Mundulea sericea, could enhance the anti-tumor effects of AG1478 in HNSCC. For Ca9-22 cells with EGFR, KRAS, and PIK3CA wild types, AG1478 alone suppressed both phosphorylated levels of ERK and AKT and induced apoptosis. On the contrary, for HSC-4 cells with EGFR and KRAS wild types, and a PIK3CA mutant, AG1478 alone did not suppress the phosphorylated level of AKT nor induce apoptosis, while it suppressed ERK phosphorylation. Forced expression of constitutively active PIK3CA (G1633A mutation) significantly reduced the apoptotic effect of AG1478 on the PIK3CA wild-type Ca9-22 cells. When HSC-4 cells with the PIK3CA G1633A mutation were treated with a combination of AG1478 and deguelin, combination effects on apoptosis induction were observed through the inhibition of the AKT pathway. These results suggest that the combination of EGFR tyrosine kinase inhibitor with deguelin is a potential therapeutic approach to treat PIK3CA-mutated HNSCC.