Effects of beta-lapachone on wound healing

beta-lapachone，是由lapacho樹(Tabebuia avellanedae)樹皮中萃取出來的天然聚合物，目前已證實：在不同的濃度和條件下，beta-lapachone對癌症、發炎、病毒和寄生蟲有很好的抵抗性。但beta-lapachone對傷口癒合的影響則仍懸而未解，尚需要進ㄧ步的研究證實。本論文的研究目的就是探討 beta-lapachone是否能刺激不同的細胞生長及移行能力的效應、並且探究 beta-lapachone促進傷口癒合的過程機轉，進而討論 beta-lapachone作為傷口癒合促進藥劑的可能性。 傷口癒合是個相當複雜的過程，需要許多不同種類細胞的合作才能完成。在這樣的過程中，內皮細胞和纖維母細胞佔有非常重要的角色，他們負責產生新的細胞外基質，讓其他的細胞得以移動到傷口處生長，使傷口復原。在我們的實驗中，證實：(1) 低濃度的beta-lapachone確實可以增加細胞增生，包括有人類新生兒角質細胞(HEKn)、老鼠角質細胞(XB-2)、人類纖維母細胞(HS68)、老鼠纖維母細胞(3T3)、人類臍帶靜脈內皮細胞(HUVEC)及人類內皮細胞(EAhy926)；(2) beta-lapachone透過MAPK訊息傳遞路徑促進老鼠纖維母細胞(3T3)和人類內皮細胞(EAhy926)的增生和移行能力；(3) 不論在正常(C57BL/6)老鼠或是糖尿病(db/db)鼠身上，beta-lapachone都可以透過ERK 訊息傳遞路徑加速傷口的癒合。另外，beta-lapachone還可以刺激巨噬細胞分泌血管內皮生長因子(VEGF)和表皮生長因子(EGF)，這兩種生長因子對於許多種類細胞的生長都是非常有助益的。球糖尿病盛行率正以驚人的速度攀升，糖尿病儼然成為醫界之最大挑戰。糖尿病患者常受神經病變、傷口不易癒合、和全身感染之苦。對糖尿病患者來說，傷口癒合的延遲是ㄧ個很嚴重的問題，若能加速傷口癒合可以免除截肢的危險。本論文中，我們確認 beta-lapachone可以增加角質細胞、纖維母細胞和內皮細胞的增生；促進纖維母細胞和內皮細胞的移動能力；並且加速正常和糖尿病老鼠傷口癒合的過程。因此，beta-lapachone具有成為傷口癒合治療藥物的潛力，也許能成為臨床用藥。Impaired wound healing is a serious problem for diabetic patients. Wound healing is a complex process that requires the cooperation of many cell types, including keratinocytes, fibroblasts, endothelial cells, and macrophages. beta-Lapachone, a natural compound extracted from the bark of the lapacho tree (Tabebuia avellanedae), is well known for its anti-tumor, anti-inflammatory, and anti-neoplastic effects at different concentrations and conditions, but its effects on wound-healing have not been studied. The purpose of the present study was to investigate the effects of beta-lapachone on the wound healing and its underlying mechanism.n this study, we demonstrated that a low dose of beta-lapachone enhanced the proliferation in several types of cells, including keratinocytes, fibroblasts, and endothelial cells, facilitated the migration of mouse 3T3 fibroblasts and human endothelial EAhy926 cells through different MAPK signaling pathways, and accelerated scrape-wound healing in vitro. Application of ointment with or without beta-lapachone to a punched wound in normal and diabetic (db/db) mice showed that the healing process was faster in beta-lapachone-treated animals than in those treated with vehicle only. In addition, beta-lapachone induced macrophages to release VEGF and EGF that are beneficial for growth of many cells. We also proved that ERK signal is indeed involved in the beta-lapachone-facilitated wound healing in vivo.ur results showed that beta-lapachone can increase the cell proliferation including keratinocytes, fibroblasts and endothelial cells and the migration of fibroblasts and endothelial cells and thus accelerate wound healing in vitro and in vivo. Therefore, we suggest that beta-lapachone may have potential for therapeutic use for wound healing.目錄謝 -------------------------------------------- 1寫表------------------------------------------- 3文摘要----------------------------------------- 4bstract ------------------------------------------ 6art I: beta-lapachone promotes cell proliferation and migration through MAPK signaling. ----------------------------------------- 8 Abstract --------------------------------------------------------- 9 Introduction ----------------------------------------------------- 11 Materials and Methods --------------------------------------- 16 Results ---------------------------------------------------------- 22 Discussion ------------------------------------------------------ 27art II: beta-lapachone facilitates wound healing in vivo. ------- 32 Abstract --------------------------------------------------------- 33 Introduction ----------------------------------------------------- 34 Materials and Methods ---------------------------------------- 40 Results ----------------------------------------------------------- 44 Discussions ----------------------------------------------------- 47art III: Conclusion ------------------------------------------------ 50 Conclusion ------------------------------------------------------ 51art IV Figures ---------------------------------------------------- 52eferences --------------------------------------------------------- 81ppendix ----------------------------------------------------------- 103 Perspective I --------------------------------------------------- 104 Perspective II -------------------------------------------------- 106 Perspective III ------------------------------------------------- 108 Papers-----------------------------------------------------------------11

Analysis of Tumor Environmental Response and Oncogenic Pathway Activation Identifies Distinct Basal and Luminal Features in Her2-Related Breast Tumor Subtypes

Introduction: Breast cancer heterogeneity occurs as a consequence of the dysregulation of numerous oncogenic pathways as well as many non-genetic factors, including tumor microenvironmental stresses such as hypoxia, lactic acidosis, and glucose deprivation. Although the importance of these non-genetic factors is well recognized, it is not clear how to integrate these factors within the genetic framework of cancer as the next logical step in understanding tumor heterogeneity. Methods: We report here the development of a series of gene expression signatures to measure the influences of microenvironmental stresses. The pathway activities of hypoxia, lactic acidosis, acidosis and glucose deprivation were investigated in a collection of 1, 143 breast tumors, which have been separated into 17 breast tumor subgroups defined by their distinct patterns of oncogenic pathways. A validation dataset comprised of 547 breast tumors was also used to confirm the major findings, and representative breast cancer cell lines were utilized to validate in silico results and mechanistic studies. Results : Through the integrative pathway analysis of microenvironmental stresses and oncogenic events in breast tumors, we identified many known and novel correlations between these two sources of tumor heterogeneity. Focusing on differences between two human epidermal growth factor receptor 2 (HER2)-related subgroups, previously identified based on patterns of oncogenic pathway activity, we determined that these subgroups differ with regards to tumor microenvironmental signatures, including hypoxia. We further demonstrate that each of these subgroups have features consistent with basal and luminal breast tumors including patterns of oncogenic signaling pathways, expression of subtype specific genes, and cellular mechanisms that regulate the hypoxia response. Importantly, we also demonstrate that the correlated pattern of hypoxia- related gene expression and basal- associated gene expression are consistent across HER2-related tumors whether we analyze the tumors as a function of our pathway-based classification scheme, using the intrinsic gene list (ERBB2+ ), or based on HER2 IHC status. Our results demonstrate a cell lineage-specific phenomenon in which basal-like tumors, HER2-related tumors with high hypoxia, as well as normal basal epithelial cells express increased mRNA levels of HIF- 1 alpha compared to luminal types and silencing of HIF-1 alpha results in decreased expression of hypoxia-induced genes. Conclusions: This study demonstrates differences in microenvironmental conditions in HER2-related subgroups defined by distinct oncogenic pathway activities, and provides a mechanistic explanation for differences in the observed hypoxia response between these subgroups. Collectively, these data demonstrate the potential of a pathway-based classification strategy as a framework to integrate genetic and non-genetic factors to investigate the basis of tumor heterogeneity

Glutamine Synthetase Is a Genetic Determinant of Cell Type-Specific Glutamine Independence in Breast Epithelia

Although significant variations in the metabolic profiles exist among different cells, little is understood in terms of genetic regulations of such cell type-specific metabolic phenotypes and nutrient requirements. While many cancer cells depend on exogenous glutamine for survival to justify the therapeutic targeting of glutamine metabolism, the mechanisms of glutamine dependence and likely response and resistance of such glutamine-targeting strategies among cancers are largely unknown. In this study, we have found a systematic variation in the glutamine dependence among breast tumor subtypes associated with mammary differentiation: basal - but not luminal-type breast cells are more glutamine-dependent and may be susceptible to glutamine-targeting therapeutics. Glutamine independence of luminal-type cells is associated mechanistically with lineage-specific expression of glutamine synthetase (GS). Luminal cells can also rescue basal cells in co-culture without glutamine, indicating a potential for glutamine symbiosis within breast ducts. The luminal-specific expression of GS is directly induced by GATA3 and represses glutaminase expression. Such distinct glutamine dependency and metabolic symbiosis is coupled with the acquisition of the GS and glutamine independence during the mammary differentiation program. Understanding the genetic circuitry governing distinct metabolic patterns is relevant to many symbiotic relationships among different cells and organisms. In addition, the ability of GS to predict patterns of glutamine metabolism and dependency among tumors is also crucial in the rational design and application of glutamine and other metabolic pathway targeted therapies

Nrf2 is the key to chemotherapy resistance in MCF7 breast cancer cells under hypoxia

Hypoxia leads to reactive oxygen species (ROS) imbalance, which is proposed to associate with drug resistance and oncogenesis. Inhibition of enzymes of antioxidant balancing system in tumor cells was shown to reduce chemoresistance under hypoxia. However, the underlying mechanism remains unknown. The key regulator of antioxidant balancing system is nuclear factor erythroid 2-related factor 2 (NFE2L2, Nrf2). In this study, we showed that hypoxia induced ROS production and increased the Nrf2 activity. Nrf2 activation increased levels of its downstream target antioxidant enzymes, including GCLC and GCLM. The Nrf2-overexpressing also confers chemo-resistant MCF7 cells under normoxia. The in vivo mouse model also demonstrated that the chemical inhibition of Nrf2 can increase cisplatin (CDDP) cytotoxicity. Together, these results showed that Nrf2 serves as a key regulator in chemotherapeutic resistance under hypoxia through ROS-Nrf2-GCLC-GSH pathway. Therefore, targeting Nrf2 can be a potential treatment for hypoxia-induced drug resistance in breast cancer cells

The lamellae-free-type pseudobranch of the euryhaline milkfish (Chanos chanos) is a Na+, K+-ATPase-abundant organ involved in hypoosmoregulation

In teleosts, the pseudobranch is hemibranchial, with a gill-like structure located near the first gill. We hypothesized that the pseudobranch of the milkfish might exhibit osmoregulatory ability similar to that of the gills. In this study, the obtained Na+, K+-ATPase (NKA) activity and protein abundance profiles showed that these parameters were higher in the pseudobranchs of the seawater (SW)- than the freshwater (FW)-acclimated milkfish, opposite the situation in the gills. The pseudobranch of the milkfish contained two types of NKA-immunoreactive cells, chloride cells (CCs) and pseudobranch-type cells (PSCs). To further clarify the roles of CCs and PSCs in the pseudobranch, we investigated the distributions of two ion transporters: the Na+, K+, 2Cl(-) cotransporter (NKCC) and the cystic fibrosis transmembrane conductance regulator (CFTR). NKCC on the basolateral membrane and CFTR on the apical membrane were found only in pseudobranchial CCs of SW-acclimated individuals. Taken together, the results distinguished NKA-IR CCs and PSCs in the pseudobranch of milkfish using antibodies against NKCC and CFTR as markers. In addition, increases in the numbers and sizes of CCs as well as in NKA expression observed upon salinity challenge indicated the potential roles of pseudobranchs in hypoosmoregulation in this euryhaline teleost. (C) 2014 Elsevier Inc. All rights reserved

In Vitro and in Vivo Wound Healing-Promoting Activities of Beta-Lapachone

Impaired wound healing is a serious problem for diabetic patients. Wound healing is a complex process that requires the cooperation of many cell types, including keratinocytes, fibroblasts, endothelial cells, and macrophages. beta- Lapachone, a natural compound extracted from the bark of the lapacho tree (Tabebuia avellanedae), is well known for its antitumor, antiinflammatory, and antineoplastic effects at different concentrations and conditions, but its effects on wound healing have not been studied. The purpose of the present study was to investigate the effects of beta- lapachone on wound healing and its underlying mechanism. In the present study, we demonstrated that a low dose of beta- lapachone enhanced the proliferation in several cells, facilitated the migration of mouse 3T3 fibroblasts and human endothelial EAhy926 cells through different MAPK signaling pathways, and accelerated scrape-wound healing in vitro. Application of ointment with or without beta-lapachone to a punched wound in normal and diabetic (db/db) mice showed that the healing process was faster in beta-lapachone- treated animals than in those treated with vehicle only. In addition, beta-lapachone induced macrophages to release VEGF and EGF, which are beneficial for growth of many cells. Our results showed that beta-lapachone can increase cell proliferation, including keratinocytes, fibroblasts, and endothelial cells, and migration of fibroblasts and endothelial cells and thus accelerate wound healing. Therefore, we suggest that beta-lapachone may have potential for therapeutic use for wound healing