The PI3K/Akt/mTOR axis in head and neck cancer: functions,aberrations, cross‐talk,and therapies

Head and neck squamous cell carcinoma (HNSCC) is one of the most morbid, mortal, and genetically diverse malignancies. Although HNSCC is heterogeneous in nature, alterations in major components of the PI3K/Akt/mTOR pathway are consistently observed throughout the majority of HNSCC cases. These alterations include genetic aberrations, such as mutations or DNA copy number variations, and dysregulation of mRNA or protein expression. In normal physiology, the PI3K/Akt/mTOR axis regulates cell survival, growth, and metabolism. However, alterations in this pathway lead to the malignant phenotype which characterizes HNSCC, among many other cancers. For this reason, both pharmaceutical companies and academic institutions are actively developing and investigating inhibitors of PI3K, Akt, and mTOR in preclinical and clinical studies of HNSCC. Many of these inhibitors have shown promise, while the effects of others are tempered by the mechanisms through which HNSCC can evade therapy. As such, current research aimed at elucidating the interactions between PI3K/Akt/mTOR and other important signaling pathways which may drive resistance in HNSCC, such as p53, NF‐κB, and MAPK, has become a prominent focus toward better understanding how to most effectively treat HNSCC.     

Novel function of Porphyromonas gingivalis gingipains in the PI3K/Akt signaling pathway

Background

Porphyromonas gingivalis is s major oral bacterium closely associated with periodontal diseases including periodontitis and directly affects host cellular signaling. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays multiple roles in various cell functions including cell survival and glucose metabolism. In this review, we describe the effect of gingipains on the PI3K/Akt signaling pathway in P. gingivalis infection.

Histatins,wound healing,and cell migration

Wounds in the oral mucosa heal faster and more efficiently than those in the skin, although the mechanisms underlying these differences are not completely clear. In the last 10 years, a group of salivary peptides, the histatins, has gained attention on behalf of their ability to improve several phases of the wound‐healing process. In addition to their roles as anti‐microbial agents and in enamel maintenance, histatins elicit other biological effects, namely by promoting the migration of different cell types contained in the oral mucosa and in non‐oral tissues. Histatins, and specifically histatin‐1, promote cell adhesion and migration in oral keratinocytes, gingival and dermal fibroblasts, non‐oral epithelial cells, and endothelial cells. This is particularly relevant, as histatin‐1 promotes the re‐epithelialization phase and the angiogenic responses by increasing epithelial and endothelial cell migration. Although the molecular mechanisms associated with histatin‐dependent cell migration remain poorly understood, recent studies have pointed to the control of signaling endosomes and the balance of small GTPases. This review aimed to update the literature on the effects of histatins in cell migration, with a focus on wound healing. We will also discuss the consequences that this increasing field will have in disease and therapy design.     

PI3K/Akt/mTOR通路在头颈部鳞状细胞癌中的靶向治疗研究进展

头颈部鳞状细胞癌（HNSCC）在全球最常见的恶性肿瘤中位居第六,晚期患者转移并复发率高,预后较差,为患者家庭和社会经济带来严重损失。靶向药物结合经典放化疗的个性化方案有望提高治疗功效并延长生存期。磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/Akt/mTOR）在HNSCC中普遍存在过度激活,是控制肿瘤发生、发展及研发靶向药物的重要通路。本文就PI3K/Akt/mTOR通路应用于HNSCC靶向治疗的个体性差异发生的潜在机制,以及临床试验取得的进展与目前所面临的困境进行探讨,拟为HNSCC的临床靶向治疗提供参考思路,从而提高患者的生存质量。     

Keratinocyte integrins in wound healing and chronic inflammation of the human periodontiurn

Periodontal epithelium plays a critical role in protection, destruction and repair of human periodontium. During optimal repair, epithelium migrates and covers the wound surface to prevent infection and damage of the vulnerable underlying connective tissue. During periodontal destruction, junctional epithelium undergoes transformation to pocket epithelium that has quite different characteristics from junctional epithelium. In the course of periodontal disease the epithelial attachment to the tooth surface is lost and the epithelium proliferates and extends pseudo-rete ridges deep into the inflamed connective tissue. Both scenarios, repair and destruction, involve active epithelial migration either in the wound provisional matrix or in the inflamed connective tissue matrix, respectively. This review covers recent research data on cellular receptors, integrins, that mediate epithelial cell migration during wound healing and destruction of human periodontiurn.     

Role of myofibroblasts in normal and pathological periodontal wound healing

Myofibroblasts represent specific subpopulations of cells with important roles in tissue remodeling in both health and disease. They are not usually found in resting healthy tissues. However, they increase in number during the proliferative phase of wound healing. In these conditions, myofibroblasts secrete and organize different molecular components of the extracellular matrix that with time will reconstitute and hopefully regenerate the damaged tissue. Importantly, these cell populations must be eliminated after wound healing has been completed. However, deficiencies in their differentiation or the persistence of this cell population has been associated with the development of delayed wound healing and fibrosis, respectively. In the present review, we analyze the involvement of myofibroblasts in periodontal wound healing and their potential contribution to tissue homeostasis and disease.     

Actinobacillus actinomycetemcomitans lipopolysaccharide stimulates collagen phagocytosis by human gingival fibroblasts

Introduction:  Collagen phagocytosis by fibroblasts is involved in the intracellular pathway related to collagen breakdown in soft connective tissues. The possible role of lipopolysaccharide (LPS) in regulating this fibroblast function has not been elucidated so we investigated the effect of LPS from Actinobacillus actinomycetemcomitans , a periodontopathic bacterium, on collagen phagocytic activity in human gingival fibroblasts and associated regulatory mechanisms.
Methods:  LPS pretreatment stimulated binding of collagen-coated beads to cells and, subsequently, their internalization.
Results:  The LPS-activated collagen phagocytic process was enhanced in the presence of the soluble form of CD14 (sCD14) or LPS-binding protein (LBP), while the LPS/LBP treatment activated Akt and induced actin reorganization. Furthermore, these LPS/LBP-induced effects were partially suppressed by adding phosphatidyl-inositol-3 kinase (PI3K) inhibitors.
Conclusion:  These results suggest that A. actinomycetemcomitans LPS disturbs the homeostasis of collagen metabolism within gingival tissue by facilitating collagen phagocytosis by gingival fibroblasts, and serum sCD14 and LBP positively regulate the action of LPS. In addition, the PI3K/Akt signaling is thought to partially mediate the LPS/LBP-stimulated collagen phagocytic pathway, which may be dependent on actin cytoskeletal rearrangement.     

Immunohistochemical detection of phosphorylated Akt, PI3K, and PTEN in ameloblastic tumors

OBJECTIVE: To evaluate roles of the Akt signaling pathway in oncogenesis and cytodifferentiation of odontogenic tumors, expression of phosphorylated Akt (pAkt), PI3K, and PTEN was analyzed in ameloblastic tumors as well as in tooth germs. METHODS: 11 tooth germs, 40 ameloblastomas, and 5 malignant ameloblastic tumors were examined immunohistochemically with antibodies against pAkt, PI3K, and PTEN. RESULTS: Immunoreactivity for pAkt, PI3K, and PTEN was detected predominantly in odontogenic epithelial cells near the basement membrane in tooth germs and ameloblastic tumors. The levels of immunoreactivity for pAkt and PI3K were slightly higher in ameloblastic tumors than in tooth germs. Plexiform ameloblastomas showed significantly higher expression of PI3K than follicular ameloblastomas, and PI3K immunoreactivity in ameloblastomas without cellular variation was significantly higher than that in acanthomatous ameloblastomas. The level of PTEN immunoreactivity was significantly lower in ameloblastomas than in tooth germs. CONCLUSION: Expression of pAkt, PI3K, and PTEN in tooth germs and ameloblastic tumors suggests that these signaling molecules regulate cell survival and growth in normal and neoplastic odontogenic tissues by mediating growth factor signals. Increased expression of pAkt and PI3K and decreased expression of PTEN in ameloblastic tumors may participate in oncogenesis of odontogenic epithelium by activating the Akt signaling pathway.     

PI3K/AKT信号通路在2型糖尿病患者种植体骨结合中作用机制的研究进展

口腔种植治疗目前被认为是缺牙患者的首选治疗方案,但糖尿病仍然是种植治疗的相对禁忌证。糖尿病患者的高血糖环境以及微血管病变等可能会影响成骨细胞及破骨细胞的功能,影响骨代谢,导致种植失败。目前,对于糖尿病影响种植体骨结合的确切机制尚未阐明。PI3K/AKT信号通路在骨组织代谢中起重要作用,可促进成骨细胞的增殖和分化;此外,PI3K/AKT/mTOR也是响应胰岛素信号传导的经典途径。该文分别对PI3K/AKT信号通路在2型糖尿病和种植体骨结合中作用机制的研究进展进行综述,为今后促进糖尿病患者种植体周围骨再生等研究奠定基础。     

PI3K/AKT/mTOR/p70S6K通路在人舌鳞癌细胞耐药中的作用

目的:通过抑制舌鳞癌细胞中PI3K/AKT/mTOR/p70S6K信号通路的表达,检测细胞凋亡与自噬的变化,探讨舌鳞癌耐药的机制。方法:以舌鳞癌细胞Cal27与舌鳞癌耐顺铂细胞Cal27/CDDP为研究对象。分别用PI3K/AKT抑制剂LY294002、mTOR抑制剂Rapamycin、p70S6K抑制剂LY2584702作用该通路各个环节。Western blot检测通路抑制剂作用后相关蛋白的变化。Cyto-ID荧光染色检测自噬体的形成。流式细胞术检测细胞凋亡水平。结果:Western blot结果显示加入抑制剂后舌鳞癌细胞Beclin1表达分别高于对照组,LC3II与LC3I以及Bax与Bcl-2的比值均升高,该通路的p-AKT、p-mTOR、p-p70S6K等蛋白均有下降(P<0.05)。流式结果显示加入抑制剂后的舌鳞癌细胞凋亡率分别较对照组升高(P<0.05)。Cyto-ID荧光染色后结果显示加入抑制剂后的舌鳞癌细胞自噬小体的数目明显高于对照组(P<0.05)。对比两组细胞显示加入抑制剂后的Cal27细胞发生凋亡与自噬的水平高于Cal27/CDDP(P<0.05)。结论:抑制PI3K/AKT/mTOR/p70S6K信号通路可诱导舌鳞癌细胞Cal27与舌鳞癌耐顺铂细胞Cal27/CDDP发生凋亡与自噬,激活的PI3K/AKT/mTOR/p70S6K通路抑制细胞凋亡与自噬是Cal27/CDDP细胞产生顺铂耐药的原因之一。     

PI3K/Akt mediates expression of TNF‐α mRNA and activation of NF‐κB in calyculin A‐treated primary osteoblasts

Objective: The effect of calyculin A (CA), a serine/threonine protein phosphatase inhibitor, on tumor necrosis factor‐α (TNF‐α) in primary osteoblasts was investigated to determine whether protein phosphatases could affect primary osteoblasts and if so which signaling pathways would be involved. Materials and methods: Primary osteoblasts were prepared from newborn rat calvaria. Cells were treated with 1 nM CA for different time periods. The expressions of TNF‐α and GAPDH mRNA were determined by RT‐PCR. Cell extracts were subjected to SDS‐PAGE and the activation of Akt and NF‐κB were analyzed by western blotting. Results: Calyculin A‐treatment markedly increased the expression of TNF‐α mRNA and enhanced the phosphorylation level of Akt (Ser473) in these cells. Pretreatment with the PI3K inhibitor LY294002 suppressed the increase in TNF‐α mRNA expression and the phosphorylation of Akt in response to CA. Western blot analysis showed that CA stimulated the phosphorylation and nuclear translocation of NF‐κB in primary osteoblasts, and these responses were blocked by pretreatment with LY294002. Conclusion: Calyculin A elicits activation of PI3K/Akt pathway which leads to expression of TNF‐α mRNA and activation of NF‐κB. This NF‐κB activation involves both phosphorylation and nuclear translocation of NF‐κB.     

PRF促进小鼠全层皮肤损伤修复的实验研究

目的:探讨富血小板纤维蛋白(PRF)对小鼠皮肤损伤的修复作用。方法:采用8~10周的Balb/C小鼠21只,将其分为3组,分别为皮肤缺损空白对照组,上市产品对照组(AD甲壳素),冻干异种PRF颗粒组,在背部制备1个1.5 cm圆形全层皮肤损伤创面。通过大体观察,测定伤口愈合时间和愈合率,并在第21天,取皮肤组织做病理切片,观察组织愈合情况。结果:伤后21 d,各组创面逐渐缩小,冻干异种PRF颗粒组创面面积明显小于其余各组(P＜0.01),并且愈合时间较其余各组短。结论:PRF可以明显促进创面愈合速度,提高愈合质量,为临床皮肤损伤修复提供新的治疗方法。     

Mammalian target of rapamycin (mTOR) is involved in the survival of cells mediated by chemokine receptor 7 through PI3K/Akt in metastatic squamous cell carcinoma of the head and neck

Metastatic squamous cell carcinoma (SCC) of the head and neck expresses chemokine receptor 7 (CCR7), which activates phosphoinositide-3 kinase (PI3K) to promote invasion and survival of SCC cells in the head and neck. We hypothesised that mammalian target of rapamycin (mTOR) may be the downstream molecule of the CCR7-PI3K pathway. Results have shown that interaction between CCR7 and its ligand CCL19 induces the phosphorylation of mTOR and its target p70s6k. This phosphorylation is abolished by inhibition of CCR7 and PI3K/Akt, indicating that mTOR is involved in the CCR7-PI3K cascade. The inhibitors of mTOR and CCR7-PI3K also lead to a significant increase in CCL19-induced death, apoptosis, and cell-cycle arrest of metastatic SCC cells in the head and neck. Taken together, our data indicate the important part played by mTOR in CCR7-induced survival of such SCC cells.     

Inhibition of phosphatidylinositol 3-kinase amplifies TEGDMA-induced apoptosis in primary human pulp cells

Cytotoxicity of triethylene glycol dimethacrylate (TEGDMA), a co-monomer of dental resinous restorative materials, is firmly established in vitro, but the molecular mechanisms are unknown. Here we examined apoptosis and necrosis induced by TEGDMA in human primary pulp cells. The levels of apoptotic and necrotic cell populations differentially increased after exposure to increasing concentrations of TEGDMA. A two-fold increase in the percentage of apoptotic cells was induced by 1 mmol/L TEGDMA. However, a population shift among cells in apoptosis and necrosis was detected when cell cultures were exposed to 2 mmol/L TEGDMA. Inhibition of the MAP Kinase/ERK pathway had no influence on cell survival, but inhibition of phosphatidylinositol 3 kinase (PI3-Kinase; Akt/protein kinase B) by LY294002 amplified TEGDMA-induced apoptosis. Moreover, Akt phosphorylation was inhibited in the presence of TEGDMA. These results suggest that depression of PI3K signaling may be a primary target in TEGDMA-induced apoptosis.     

人参皂苷Rg1通过Akt/eNOS信号调控尼古丁胁迫下人牙周膜细胞的增殖和迁移

目的:探讨人参皂苷Rg1对尼古丁胁迫下的人牙周膜细胞(human periodontal ligament cells,HPDLCs)增殖和迁移的影响及分子机制.方法:采用组织块法分离培养HPDLCs,尼古丁(500 ng/mL)胁迫培养细胞7d.第3天分别进行人参皂苷Rg1 (0.01 μmol/L)处理、人参皂苷Rg1与PI3K抑制剂LY294002 (0.5 μmol/L)共处理、人参皂苷Rg1与Akt抑制剂Tricirbine(5μmol/L)共处理、人参皂苷Rg1与eNOS抑制剂L-NAME(1 mmol/L)共处理(Rg1+L-NAME组),持续处理至第7天.采用MTT法和Transwell法检测各处理组HPDLCs活力变化和细胞迁移率,并使用实时定量PCR与Western印迹法检测PI3K/Akt/eNOS信号蛋白变化,采用SPSS20.0软件包对数据进行统计学分析.结果:尼古丁抑制HPDLCs的增殖和迁移并显著上调PI3K表达,抑制Akt和eNOS表达;人参皂苷Rg1减缓尼古丁对细胞增殖和迁移抑制作用以及尼古丁对Akt和eNOS表达的抑制作用;Trieirbine与L-NAME衰减人参皂苷Rg1对尼古丁的抑制作用.结论:人参皂苷Rg1通过Akt/eNOS信号调控尼古丁胁迫下HPDLCs的增殖和迁移.     

Enamel matrix derivative exhibits angiogenic effect in vitro and in a murine model

OBJECTIVES: Angiogenesis is one of the most critical events in the wound healing process. Any increase in angiogenesis could result in more rapid and complete healing. A recent study found that enamel matrix derivative (EMD) could accelerate early periodontal wound healing. We wanted to clarify whether EMD caused an angiogenic effect and, thus, possibly enhanced wound healing. METHODS: We performed in vitro proliferation and chemotaxis assays on human umbilical vein endothelial cell (HUVEC) cultures, and a tissue culture assay using blood vessel fragments in fibrin gel. Collagen membranes soaked with EMD were implanted subcutaneously in mice to test the in vivo angiogenic effect. RESULTS: While there were no significant differences between the negative control and EMD groups in the proliferation assay, EMD treatment did exhibit a significantly greater dose-dependent chemotactic effect on HUVEC than control group treatments. The tissue culture in fibrin gel showed new blood vessel outgrowths in the EMD groups, but none in the negative control group. In the animal studies, significantly more endothelial cells were detected in the EMD group of mice. CONCLUSIONS: Our findings show that EMD does exhibit some angiogenic effects. However, the underlying molecules and mechanisms are still unidentified. We discuss several possibilities.     

Tissue regeneration in dentistry: Can salamanders provide insight?

The ability to regenerate damaged tissues would be of tremendous benefit for medicine and dentistry. Unfortunately, humans are unable to regenerate tissues such as teeth and fingers or to repair injured spinal cord. With an aging population, health problems are more prominent and dentistry is no exception as loss of bone tissue in the orofacial sphere from periodontal disease is on the rise. Humans can repair oral soft tissues exceptionally well; however, hard tissues, such as bone and teeth, are devoid of the ability to repair well or at all. Fortunately, Mother Nature has solved nearly every problem that we would like to solve for our own benefit and tissue regeneration is no exception. By studying animals that can regenerate, like Axolotls (Mexican salamander), we hope to find ways to stimulate regeneration in humans. We will discuss the role of the transforming growth factor beta cytokines as they are central to wound healing in humans and regeneration in Axolotls. We will also compare wound healing in humans (skin and oral mucosa) to Axolotl skin wound healing and limb regeneration. Finally, we will address the problem of bone regeneration and present results in salamanders which indicate that in order to regenerate bone you need to recruit non‐bone cells. Fundamental research, such as the work being performed in animals that can regenerate, offers insight to help understand why some treatments are successful while others fail when it comes to specific tissues such as bones.     

骨桥蛋白在新生大鼠背部皮肤创伤愈合过程中的表达

目的：检测骨桥蛋白（osteopontin,OPN）在新生大鼠皮肤及创伤愈合过程中的表达,探讨OPN参与皮肤创伤愈合的可能机制。方法：在7d龄SD大鼠背部皮肤做直径为1ClTI的圆形全层皮肤切口,运用免疫组织化学方法检测OPN阳性细胞在皮肤及创伤愈合过程中的分布情况,并分别运用RT—PCR及Westernblot检测OPN在创伤愈合不同时间点（1、3、5及7d）的表达情况。结果：在大鼠正常皮肤,免疫组织化学结果显示OPN阳性细胞分布在毛囊、皮脂腺及汗腺,表皮则无表达;RT—PCR及Westernblot均未检测到OPN在正常皮肤中的表达。从创伤后第1天起,OPN阳性细胞开始集中分布在创伤边缘的真皮部位,且在第3天达到高峰,第、5天表达开始减弱。RT—PCR及Westemblot结果一致．OPN相对表达量在第3天达到高峰。结论：研究结果表明OPN参与了皮肤创伤愈合,尤其在创伤修复的炎症期及细胞外基质沉积期。