Aging-Related Metabolic Dysfunction in the Salivary Gland: A Review of the Literature.

Aging-related salivary dysfunction commonly induces the poor oral health, including decreased saliva flow and dental caries. Although the clinical significance of the salivary glands is well-known, the complex metabolic pathways contributing to the aging-dysfunction process are only beginning to be uncovered. Here, we provide a comprehensive overview of the metabolic changes in aging-mediated salivary gland dysfunction as a key aspect of oral physiology. Several metabolic neuropeptides or hormones are involved in causing or contributing to salivary gland dysfunction, including hyposalivation and age-related diseases. Thus, aging-related metabolism holds promise for early diagnosis, increased choice of therapy and the identification of new metabolic pathways that could potentially be targeted in salivary gland dysfunction.

HOXC6-Mediated miR-188-5p Expression Induces Cell Migration through the Inhibition of the Tumor Suppressor FOXN2.

Homeobox C6 (HOXC6) is a transcription factor that plays a role in the malignant progression of various cancers. However, the roles of HOXC6 and its regulatory mechanism remain unclear. In this study, we used microRNA (miRNA) regulatory networks to identify key regulatory interactions responsible for HOXC6-mediated cancer progression. In microarray profiling of miRNAs, the levels of miRNAs such as hsa-miR-188-5p, hsa-miR-8063, and hsa-miR-8064 were significantly increased in HOXC6-overexpressing cells. Higher positive expression rates of HOXC6 and miR-188-5p were observed in malignant cancer. We also found that HOXC6 significantly upregulated miR-188-5p expression. The underlying function of HOXC6-mediated miR-188-5p expression was predicted through TargetScan and the MiRNA Database. Overexpression of mir-188-5p inhibited the expression of forkhead box N2 (FOXN2), a tumor suppressor gene. Furthermore, in the luciferase assay, miR-188-5p bound to the 3'-UTR of FOXN2 and was mainly responsible for the dysregulation of FOXN2 expression. Silencing FOXN2 induced cell migration, and the effect of FOXN2 silencing was enhanced when the HOXC6/miR-188-5p axis was induced. These results suggest that HOXC6/miR-188-5p may induce malignant progression in cancer by inhibiting the activation of the FOXN2 signaling pathway.

Choline Acetyltransferase Induces the Functional Regeneration of the Salivary Gland in Aging SAMP1/Kl -/- Mice.

Salivary gland dysfunction induces salivary flow reduction and a dry mouth, and commonly involves oral dysfunction, tooth structure deterioration, and infection through reduced salivation. This study aimed to investigate the impact of aging on the salivary gland by a metabolomics approach in an extensive aging mouse model, SAMP1/Klotho -/- mice. We found that the salivary secretion of SAMP1/Klotho -/- mice was dramatically decreased compared with that of SAMP1/Klotho WT (+/+) mice. Metabolomics profiling analysis showed that the level of acetylcholine was significantly decreased in SAMP1/Klotho -/- mice, although the corresponding levels of acetylcholine precursors, acetyl-CoA and choline, increased. Interestingly, the mRNA and protein expression of choline acetyltransferase (ChAT), which is responsible for catalyzing acetylcholine synthesis, was significantly decreased in SAMP1/Klotho -/- mice. The overexpression of ChAT induced the expression of salivary gland functional markers (α-amylase, ZO-1, and Aqua5) in primary cultured salivary gland cells from SAMP1/Klotho +/+ and -/- mice. In an in vivo study, adeno-associated virus (AAV)-ChAT transduction significantly increased saliva secretion compared with the control in SAMP1/Klotho -/- mice. These results suggest that the dysfunction in acetylcholine biosynthesis induced by ChAT reduction may cause impaired salivary gland function.

Correction to: Psoralidin Stimulates Expression of Immediate-Early Genes and Synapse Development in Primary Cortical Neurons.

The original version of this article unfortunately contained a mistake. The funding information was incorrect in the Acknowledgement section of this article. The corrected text is given below.

Global analysis of gene expression profiles in the submandibular salivary gland of klotho knockout mice.

Salivary dysfunction commonly occurs in many older adults and is considered a physiological phenomenon. However, the genetic changes in salivary glands during aging have not been characterized. The present study analyzed the gene expression profile in salivary glands from accelerated aging klotho deficient mice (klotho-/-, 4 weeks old). Microarray analysis showed that 195 genes were differentially expressed (z-score > 2 in two independent arrays) in klotho null mice compared to wild-type mice. Importantly, alpha2-Na+ /K+ -ATPase (Atp1a2), Ca2+ -ATPase (Atp2a1), epidermal growth factor (EGF), and nerve growth factor (NGF), which have been suggested to be regulators of submandibular salivary gland function, were significantly decreased. When a network was constructed from the differentially expressed genes, proliferator-activated receptor-γ (PPAR γ), which regulates energy homeostasis and insulin sensitivity, was located at the core of the network. In addition, the expression of genes proposed to regulate various PPAR γ-related cellular pathways, such as Klk1b26, Egfbp2, Cox8b, Gpx3, Fabp3, EGF, and NGFß, was altered in the submandibular salivary glands of klotho-/- mice. Our results may provide clues for the identification of novel genes involved in salivary gland dysfunction. Further characterization of these differentially expressed genes will be useful in elucidating the genetic basis of aging-related changes in the submandibular salivary gland.

New metformin derivative HL156A prevents oral cancer progression by inhibiting the insulin-like growth factor/AKT/mammalian target of rapamycin pathways.

Metformin is a biguanide widely prescribed as an antidiabetic drug for type 2 diabetes mellitus patients. The purpose of the present study was to observe the effects of the new metformin derivative, HL156A, on human oral cancer cell and to investigate its possible mechanisms. It was observed that HL156A significantly decreased FaDu and YD-10B cell viability and colony formation in a dose-dependent way. HL156A also markedly reduced wound closure and migration of FaDu and YD-10B cells. We observed that HL156A decreased mitochondrial membrane potential and induced reactive oxygen species (ROS) levels and apoptotic cells with caspase-3 and -9 activation. HL156A inhibited the expression and activation of insulin-like growth factor (IGF)-1 and its downstream proteins, AKT, mammalian target of rapamycin (mTOR), and ERK1/2. In addition, HL156A activated AMP-activated protein kinase/nuclear factor kappa B (AMPK-NF-κB) signaling of FaDu and YD-10B cells. A xenograft mouse model further showed that HL156A suppressed AT84 mouse oral tumor growth, accompanied by down-regulated p-IGF-1, p-mTOR, proliferating cell nuclear antigen (PCNA) and promoted p-AMPK and TUNEL expression. These results suggest the potential value of the new metformin derivative HL156A as a candidate for a therapeutic modality for the treatment of oral cancer.

Psoralidin Stimulates Expression of Immediate-Early Genes and Synapse Development in Primary Cortical Neurons.

Upon synaptic stimulation and glutamate release, glutamate receptors are activated to regulate several downstream effectors and signaling pathways resulting in synaptic modification. One downstream intracellular effect, in particular, is the expression of immediate-early genes (IEGs), which have been proposed to be important in synaptic plasticity because of their rapid expression following synaptic activation and key role in memory formation. In this study, we screened a natural compound library in order to find a compound that could induce the expression of IEGs in primary cortical neurons and discovered that psoralidin, a natural compound isolated from the seeds of Psoralea corylifolia, stimulated synaptic modulation. Psoralidin activated mitogen-activated protein kinase (MAPK) signaling, which in turn induced the expression of neuronal IEGs, particularly Arc, Egr-1, and c-fos. N-methyl-D-aspartate (NMDA) receptors activation and extracellular calcium influx were implicated in the psoralidin-induced intracellular changes. In glutamate dose-response curve, psoralidin shifted glutamate EC50 to lower values without enhancing maximum activity. Interestingly, psoralidin increased the density, area, and intensity of excitatory synapses in primary hippocampal neurons, which were mediated by NMDA receptor activation and MAPK signaling. These results suggest that psoralidin triggers synaptic remodeling through activating NMDA receptor and subsequent MAPK signaling cascades and therefore could possibly serve as an NMDA receptor modulator.

Heat shock-induced interactions among nuclear HSFs detected by fluorescence cross-correlation spectroscopy.

The cellular response to stress is primarily controlled in cells via transcriptional activation by heat shock factor 1 (HSF1). HSF1 is well-known to form homotrimers for activation upon heat shock and subsequently bind to target DNAs, such as heat-shock elements, by forming stress granules. A previous study demonstrated that nuclear HSF1 and HSF2 molecules in live cells interacted with target DNAs on the stress granules. However, the process underlying the binding interactions of HSF family in cells upon heat shock remains unclear. This study demonstrate for the first time that the interaction kinetics among nuclear HSF1, HSF2, and HSF4 upon heat shock can be detected directly in live cells using dual color fluorescence cross-correlation spectroscopy (FCCS). FCCS analyses indicated that the binding between HSFs was dramatically changed by heat shock. Interestingly, the recovery kinetics of interaction between HSF1 molecules after heat shock could be represented by changes in the relative interaction amplitude and mobility.

Autophagy regulated by prolyl isomerase Pin1 and phospho-Ser-GSK3αß involved in protection of oral squamous cell carcinoma against cadmium toxicity.

Prolyl isomerase Pin1 plays an important role in cell proliferation and is overexpressed in many human tumors. However, its role in autophagy induction remains undefined. Here we show that Pin1 regulates cell survival via autophagy in cadmium (Cd)-exposed oral squamous cell carcinoma (OSCC). OSCC exposure to Cd induced autophagy, as demonstrated by the formation of green fluorescent punctae in transfected cells expressing GFP-conjugated microtubule-associated protein light chain 3 (LC3) and by LC3 flux in the presence of autophagy inhibitors. Suppression of Atg5 enhanced Cd-induced apoptosis, indicating that autophagy is involved in cell protection. In dose-response experiments, cleavage of procaspase-3, PARP-1, and LC3-II was induced by Cd with an IC50 of 45 µM. Expression of Pin1 was decreased at or above the Cd IC50 value and was inversely correlated with the level of phospho(p)-Ser-GSK3αß. Genetic or pharmacologic inhibition of Pin1 suppressed Cd-induced autophagy, but increased p-Akt-mediated p-Ser-GSK3αß; this was reversed by overexpression of Pin1. However, suppression of GSK3αß inhibited Cd-induced autophagy and induced apoptosis, which could be reversed by overexpression of GSK3ß. The PI3K inhibitor Ly294002 blocked p-Akt-mediated increases in p-Ser-GSK3αß and autophagy and induced apoptosis. Therefore, p-Ser-GSK3αß can directly regulate Cd-induced autophagy, although its function is suppressed by Pin1. Collectively, the present results indicate that targeting Pin1 and GSK3αß at the same time could be an effective therapeutic tool for Cd-induced carcinogenesis.

BAG3 affects the nucleocytoplasmic shuttling of HSF1 upon heat stress.

Bcl2-associated athoanogene (BAG) 3 is a member of the co-chaperone BAG family. It is induced by stressful stimuli such as heat shock and heavy metals, and it regulates cellular adaptive responses against stressful conditions. In this study, we identified a novel role for BAG3 in regulating the nuclear shuttling of HSF1 during heat stress. The expression level of BAG3 was induced by heat stress in HeLa cells. Interestingly, BAG3 rapidly translocalized to the nucleus upon heat stress. Immunoprecipitation assay showed that BAG3 interacts with HSF1 under normal and stressed conditions and co-translocalizes to the nucleus upon heat stress. We also demonstrated that BAG3 interacts with HSF1 via its BAG domain. Over-expression of BAG3 down-regulates the level of nuclear HSF1 by exporting it to the cytoplasm during the recovery period. Depletion of BAG3 using siRNA results in reduced nuclear HSF1 and decreased Hsp70 promoter activity. BAG3 in MEF(hsf1(-/-)) cells actively translocalizes to the nucleus upon heat stress suggesting that BAG3 plays a key role in the processing of the nucleocytoplasmic shuttling of HSF1 upon heat stress.

Transcriptional regulation, stabilization, and subcellular redistribution of multidrug resistance-associated protein 1 (MRP1) by glycogen synthase kinase 3αß: novel insights on modes of cadmium-induced cell death stimulated by MRP1.

Cadmium (Cd) resistance is associated with the suppression of autophagy in H460 lung cancer cells, which is regulated by phospho(p)serine-glycogen synthase kinase (GSK) 3αß. However, the involvement of multidrug resistance (MDR) in this signaling pathway and its underlying mechanisms remain to be elucidated. In this study, we used Cd-resistant cells (RH460), developed from H460 lung cancer cells, to demonstrate that the induction of MDR-associated protein (MRP1) in response to Cd is enhanced in H460 cells compared to RH460. Treating RH460 cells with Cd induced large cytoplasmic vacuoles, which was inhibited by the autophagy inhibitor 3-methyladenine. MRP1 was detected in the nuclear-rich membrane fractions and redistributed from the perinuclear to the cytoplasmic compartment following exposure to Cd. Cd-induced MRP1, p-Ser/p-Tyr GSK3αß, and LC3-II were all suppressed by the GSK3 inhibitor SB216763, but increased by lithium. Furthermore, MRP1 was upregulated by the Ser/Thr phosphatase inhibitor okadaic acid and downregulated by the tyrosine phosphatase inhibitor vanadate, suggesting that MRP1 protein was stabilized by p-Ser GSK3αß. In addition, co-immunoprecipitation and co-localization analyzes revealed a physical interaction between MRP1 and p-Ser GSK3αß. Genetic knockdown of GSK3ß decreased Cd-induced MRP1 mRNA and protein levels, whereas its overexpression upregulated MRP1 protein expression. MRP1 also co-localized with lysosomal membrane protein-2, which may cause lysosomal membrane permeabilization and the subsequent release of cathepsins into the cytosol. In mice chronically injected with Cd, MRP1 localized to the perinuclear region of bronchial and alveolar epithelial cells. Collectively, these data suggest that Cd toxicity is regulated by the transcriptional regulation, stabilization, and subcellular redistribution of MRP1 via the posttranslational modification of GSK3αß. Therefore, the serine phosphorylation of GSK3αß plays a critical role in MRP1-induced cell death.

Diverse Toll-like receptors mediate cytokine production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in macrophages.

Toll-like receptors (TLRs) orchestrate a repertoire of immune responses in macrophages against various pathogens. Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans are two important periodontal pathogens. In the present study, we investigated TLR signaling regulating cytokine production of macrophages in response to F. nucleatum and A. actinomycetemcomitans. TLR2 and TLR4 are redundant in the production of cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) in F. nucleatum- and A. actinomycetemcomitans-infected macrophages. The production of cytokines by macrophages in response to F. nucleatum and A. actinomycetemcomitans infection was impaired in MyD88-deficient macrophages. Moreover, cytokine concentrations were lower in MyD88-deficient macrophages than in TLR2/TLR4 (TLR2/4) double-deficient cells. An endosomal TLR inhibitor, chloroquine, reduced cytokine production in TLR2/4-deficient macrophages in response to F. nucleatum and A. actinomycetemcomitans, and DNA from F. nucleatum or A. actinomycetemcomitans induced IL-6 production in bone marrow-derived macrophages (BMDMs), which was abolished by chloroquine. Western blot analysis revealed that TLR2/4 and MyD88 were required for optimal activation of NF-κB and mitogen-activated protein kinases (MAPKs) in macrophages in response to F. nucleatum and A. actinomycetemcomitans, with different kinetics. An inhibitor assay showed that NF-κB and all MAPKs (p38, extracellular signal-regulated kinase [ERK], and Jun N-terminal protein kinase [JNK]) mediate F. nucleatum-induced production of cytokines in macrophages, whereas NF-κB and p38, but not ERK and JNK, are involved in A. actinomycetemcomitans-mediated cytokine production. These findings suggest that multiple TLRs may participate in the cytokine production of macrophages against periodontal bacteria.

Soluble klotho induces the heat shock factor 1 through EGR1 expression.

Klotho is an antiaging protein that has multiple functions. The purpose of this study is to investigate whether soluble klotho plays a role in cellular stress response pathways. We found that klotho deficiency (kl-/-) largely decreased HSF1 levels and impaired heat shock protein expression. Interestingly, recombinant soluble klotho-induced HSF1 and HSPs such as HSP90, HSP70, and HSP27 in kl-/- mouse embryonic fibroblasts (MEFs). Soluble Klotho treatment also induced cell proliferation and HSF1 promoter activity in MEF kl-/- cells in a concentration-dependent manner. Furthermore, using point mutagenesis, we identified regulatory/binding sites of transcription factors EGR1 regulated by soluble klotho in the HSF1 promoter. Taken together, our findings unravel the molecular basis of klotho and provide molecular evidence supporting a direct interaction between soluble klotho and HSF1-mediated stress response pathway.

Interleukin-17 induces AP-1 activity and cellular transformation via upregulation of tumor progression locus 2 activity.

Inflammatory conditions elicited by extrinsic environmental factors promote malignant transformation, tumor growth and metastasis. Although the role of T cells in cancer promotion has been examined, little is known about the underlying molecular mechanisms of interleukin-17 A (IL-17A), a proinflammatory cytokine produced by activated CD4(+) memory T cells, in carcinogenesis. Here, we report that IL-17A induces neoplastic transformation of JB6 Cl41 cells through activation of tumor progression locus 2 (TPL2). IL-17A dose- and time-dependently increases TPL2 phosphorylation in JB6 Cl41 cells through IL-17A receptor. IL-17A activates mitogen-activated protein kinase/extracellular signal-regulated kinase kinases, c-jun N-terminal kinases and STAT3 signaling pathways, which are inhibited by a TPL2 kinase inhibitor (TKI). Furthermore, IL-17A activates c-fos and c-jun promoter activity, resulting in increased activator protein-1 (AP-1) activity. When small interfering RNA of IL-17A receptor (IL-17R), IL-17A and TPL2 were introduced into JB6 Cl41 cells, respectively, IL-17A-induced AP-1 activity was significantly decreased compared with control cells. Similarly, TPL2 inhibition suppressed AP-1 activity induced by IL-17A. The knockdown of IL-17R and TKI treatment in JB6 Cl41 cells resulted in decreased IL-17A-induced cell transformation. The in vivo chorioallantoic membrane assay also showed that IL-17A increased tumor formation of JB6 Cl41 cells, whereas TKI inhibited the tumorigenesis promoted by IL-17A. Consistent with these observations, knockdown of IL-17A and/or inhibition of TPL2 attenuated tumorigenicity of human breast cancer MCF7 cells. Together, our findings point to a critical role for the IL-17A-induced TPL2 signaling pathway in supporting cancer-associated inflammation in the tumor microenvironment. Therapeutic approaches that target this pathway may, therefore, effectively inhibit carcinogenesis.

HOXC6 is deregulated in human head and neck squamous cell carcinoma and modulates Bcl-2 expression.

Homeobox C6 (HOXC6) genes belong to the homeoprotein family of transcription factors, which play an important role in morphogenesis and cellular differentiation during embryonic development. The aim of this study was to explore the role of HOXC6 in the regulation of Bcl-2 in human head and neck squamous cell carcinoma (HNSCC). The HOXC6 and Bcl-2 gene were identified as being overexpressed in HNSCC tissue and cell lines. Transfection assays demonstrated that HOXC6 increased the levels of Bcl-2 mRNA and protein. A luciferase reporter assay suggested that HOXC6 induced activity of the Bcl-2 promoter. A series of Bcl-2 promoter deletion mutants were examined and the minimal HOXC6-responsive region was identified to be in the TAAT motif (-420 bp) of the Bcl-2 promoter. Interestingly, the inhibition of HOXC6 using siRNA led to the repression of Bcl-2 expression and induced caspase-3-dependent apoptosis; overexpression of HOXC6 in HNSCC cells increased the resistance to paclitaxel-induced apoptosis. Together, our findings suggest that HOXC6 is an important mechanism of the anti-apoptotic pathway via regulation of Bcl-2 expression.

Synthesized Pheophorbide a-mediated photodynamic therapy induced apoptosis and autophagy in human oral squamous carcinoma cells.

BACKGROUND: Pheophorbide a (Pa) is a chlorine-based photosensitizer derived from an ethnopharmacological herb, and our group recently synthesized Pa by the removal of a magnesium ion and a phytyl group from chlorophyll-a. In this study, the effect of photodynamic therapy (PDT) with synthesized Pa was examined in a human oral squamous cell carcinoma (OSCC) cells. METHODS: Cells were treated with PDT with Pa, and reactive oxygen species (ROS) and mitochondrial membrane potential [ΔΨ (m)] were examined. Apoptosis was measured using annexin V staining and immunoblot. Autophagy was characterized by the increase in LC3B-II and the formation of autophagosome and acidic vesicular organelles (AVOs). RESULTS: Pa-PDT inhibited the proliferation of OSCC cells in a dose-dependent manner. Pa-PDT increased the number of apoptotic cells by inactivating ERK pathway. Pa-PDT also induced autophagy in OSCC cells evidenced by the increased levels of LC3 type II expression and the accumulation of AVOs. The inhibition of autophagy enhanced Pa-PDT-mediated cytotoxicity through an increase in necrosis. CONCLUSIONS: These results suggest that synthesized Pa-PDT exerts anti-tumor effects by inducing apoptosis and autophagy and provide novel evidence that Pa-PDT induces autophagy, and autophagy inhibition enhances Pa-PDT-mediated necrosis in OSCC cells.

DMP1 and IFITM5 Regulate Osteogenic Differentiation of MC3T3-E1 on PEO-Treated Ti-6Al-4V-Ca2+/Pi surface.

Despite numerous studies on various surface modifications on titanium and its alloys, it remains unclear what kind of titanium-based surface modifications are capable of controlling cell activity. This study aimed to understand the mechanism at the cellular and molecular levels and investigate the in vitro response of osteoblastic MC3T3-E1 cultured on the Ti-6Al-4V surface modified by plasma electrolytic oxidation (PEO) treatment. A Ti-6Al-4V surface was prepared by PEO at 180, 280, and 380 V for 3 or 10 min in an electrolyte containing Ca2+/Pi ions. Our results showed that PEO-treated Ti-6Al-4V-Ca2+/Pi surfaces enhanced the cell attachment and differentiation of MC3T3-E1 compared to the untreated Ti-6Al-4V control but did not affect cytotoxicity as shown by cell proliferation and cell death. Interestingly, on the Ti-6Al-4V-Ca2+/Pi surface treated by PEO at 280 V for 3 or 10 min, MC3T3-E1 showed a higher initial adhesion and mineralization. In addition, the alkaline phosphatase (ALP) activity significantly increased in MC3T3-E1 on the PEO-treated Ti-6Al-4V-Ca2+/Pi (280 V for 3 or 10 min). In RNA-seq analysis, the expression of dentin matrix protein 1 (DMP1), sortilin 1 (Sort1), signal-induced proliferation-associated 1 like 2 (SIPA1L2), and interferon-induced transmembrane protein 5 (IFITM5) was induced during the osteogenic differentiation of MC3T3-E1 on the PEO-treated Ti-6Al-4V-Ca2+/Pi. DMP1 and IFITM5 silencing decreased the expression of bone differentiation-related mRNAs and proteins and ALP activity in MC3T3-E1. These results suggest that the PEO-treated Ti-6Al-4V-Ca2+/Pi surface induces osteoblast differentiation by regulating the expression of DMP1 and IFITM5. Therefore, surface microstructure modification through PEO coatings with Ca2+/Pi ions could be used as a valuable method to improve biocompatibility properties of titanium alloys.

Heat shock factor 4a (HSF4a) represses HSF2 expression and HSF2-mediated transcriptional activity.

Heat shock factors (HSFs) are the main transcriptional regulators of the stress-induced expression of heat shock protein genes. HSF2, which is one of the HSFs, is activated during differentiation and development but it is unclear how they regulate during cellular processes. Here, we examined the role of HSF4a on the regulation of HSF2 in HEK 293 cells. We found that HSF2 levels are negatively correlated with HSF4a expression and that overexpression of HSF4a reduces hemin-induced HSF2 mRNA and protein levels. Moreover, hemin-induced activation of HSF2 was also markedly inhibited in HSF4a expressed cells. Immunoprecipitation assay showed that HSF2 binds to the oligomerization domain of HSF4a. Hemin treatment inhibited their interaction and induced localization of HSF2 and HSF4a in nuclear. In addition, we found that HSF4a or HSF4a DNA binding domain (117 aa) inhibited the activity of hemin-induced HSP70 promoter. Consequently, HSF4a inhibits HSF2 expression or transcriptional activity through negative regulation of HSF2 binding to the HSP70 promoter. In summary, our findings suggest novel mechanisms of HSF2 regulation controlled by HSF4a.

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3ß.

Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3ß (GSK3ß) and 70kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3ß at Ser(9) and, to a lesser extent, Thr(390), the dephosphorylation of p70S6K at Thr(389), and the phosphorylation of p70S6K at Thr(421) and Ser(424). The specific p38 inhibitor SB203080 reduced the p-GSK3ß(Ser9) and autophagy through the phosphorylation of p70S6K(Thr389); however, it augmented the levels of p-ERK, p-GSK3ß(Thr390), and p-70S6K(Thr421/Ser424) induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK(Ser9)/p70S6K(Thr380) and ERK/GSK3ß(Thr390)/p70S6K(Thr421/Ser424) kinase signaling, which is involved in cell survival and cell death.

Homeobox C5 expression is associated with the progression of 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis.

BACKGROUND: Aberrant expression of homeobox genes (HOX), normally required for the differentiation of a particular tissue, has been reported in several types of cancer, but poorly addressed in oral squamous cell carcinoma (OSCC). The present study investigated the expression of HOXC5 in OSCC and identified molecular biomarker whose expression is associated with the multistep oral carcinogenesis. METHODS: The expression of HOXC5, proliferation cell nuclear antigen (PCNA), and Bcl-2 was examined by RT-PCR and Western blot analysis and confirmed by immunohistochemistry and transferase-mediated dUTP nick end-labeling (TUNEL) assay in a 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis model. RESULTS: Homeobox genes C5 was overexpressed in SCC tissues, but not in normal tissues by RT-PCR and Western blot analysis. Along with the progress of multistep carcinogenesis, the levels of HOXC5 expression of mRNA and protein significantly increased during the dysplasia (moderate to severe dysplasia) when compared with normal and hyperplasia. The levels of PCNA and Bcl-2 were sequentially increased from hyperplasia to dysplasia and SCC. By immunohistochemistry, HOXC5 expression was significantly increased in dysplasia, whereas PCNA expression was gradually increased during tongue carcinogenesis. TUNEL-positive cells were increased until dysplasia, but reduced in SCC. CONCLUSIONS: These results indicate that overexpression of HOXC5 is correlated with oral carcinogenesis and strongly contributed to the development of OSCC. HOXC5 may be a useful biomarker and has an emerging therapeutic target of OSCC.