To Study the Effects and Mechanisms of Nm23-H1 on Radio-/Chemo-Sensitivities in Human Head and Neck Squamous Cell Carcinoma: the Role of Glycolysis

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Project title

Code/計畫編號

NSC102-2314-B075-045-MY2

Translated Name/計畫中文名

探討Nm23-H1抑制頭頸鱗狀上皮癌細胞化放療抗性之作用機制---糖解作用之角色

Funding Organization/主管機關

National Science and Technology Council

Co-Investigator(s)/共同執行人

王怡芬(計畫主持人)

Department/Unit

Taipei Veterans General Hospital Department of Otorhinolaryngology-Head and Neck Surgery

Website

https://www.grb.gov.tw/search/planDetail?id=8118494

Year

2014

Start date/計畫起

01-08-2014

Expected Completion/計畫迄

31-07-2015

Co-Investigator(s)

Chun-Ju Chang

Bugetid/研究經費

1100千元

ResearchField/研究領域

臨床醫學
生物技術(醫)

研究背景：頭頸癌的放射線及化學治療抗性及高度轉移能力是其預後不佳的主因。我們以人類口腔鱗狀上皮細胞癌細胞株SAS研究發現：Nm23-H1低度表現會促進細胞轉移及放射化學治療阻抗。我們的結果顯示：Nm23-H1高表現時，能調節化學治療過程中細胞周期蛋白表現及細胞内自由基含量，可能因此抑制頭頸癌細胞增生、促進細胞凋亡、提升放射化學治療敏感性。然而，Nm23-H1低表現之頭頸癌細胞之放射化學治療阻抗及高度轉移能力仍待解決。近年研究證實腫瘤微環境變化(低氧壓、pH值降低、代謝路徑重整...)調節癌症惡化之進行，導致細胞凋亡阻抗、甚至癌症預後不佳。腫瘤微環境的變化可能起因於p53基因突變或功能失常、致癌基因過度表現，進而改變癌細胞的能量需求。另一方面，癌細胞常見粒線體氧化磷酸作用低下、糖解作用旺盛，導致丙酮酸、乳酸等代謝物增加，致使細胞環境酸化及發炎反應前趨因子分泌，進而促進腫瘤附近的發炎反應及腫瘤惡化。因此本研究目的：一、探討Nm23-H1表現對頭頸癌細胞醣類代謝路徑的影響；二、糖解抑制劑對不同Nm23-H1表現量的頭頸癌細胞的放射化學治療敏感性之影響及作用機轉。研究方法：以SAS細胞株所建立的Nm23-H1高度或低度表現之穩定轉殖株(stable clones)共五株，第一年：評估Nm23-H1表現量對細胞能量生成的影響(包括：細胞内ATP含量、耗氧速率、產酸速率、粒線體質量及膜電位...）。第二年：Nm23-H1投以糖解作用酵素活性抑制劑，評估其對放射線及化學治療效果的影響（包括：群落形成能力、48小時存活率及細胞週期）、細胞凋亡現象。第三年：探討Nm23-H1 調節糖解速率並提升化放療敏感性的分子機轉（包括：代謝酵素與粒線體複合體之基因表現及活性）。預期結果：將可呈現Nm23-H1表現對頭頸癌細胞糖解作用的影響、及其在放射化學治療敏感性之調節機轉，期望作為臨床治療策略之參考。 Background: Chemo-resistance and highly metastasis are poorly prognostic factors of head and neck cancer. Our recently studies revealed that knockdown of Nm23-H1 (non-metastatic clone 23 type 1) renders in vitro invasion and chemo-/radio-resistance of head and neck squamous cell carcinoma (HNSCC) SAS cell line. Moreover, overexpression of Nm23-H1 may increase cisplatin-sensitivity via gene expression of cyclins, and accumulation of intracellular peroxide in SAS cells. However, the invasive and apoptosis-resistant properties of Nm23-H1 knockdowned SAS cells remain unsolved. The changes of tumor microenviroment, such as hypoxia, acidosis, and metabolic reprogramming, have reported to regulate chemo-resistance, cell proliferation and cancer progression. These phenomena can lead to a poor prognosis in cancer patients. Many of these changes are caused by malfunction of p53 pathway, overexpression of oncogene in tumor cells. Concurrently, cancer ells are show a shift in energy production from mitochondrial oxidative phosphorylation to aerobic glycolysis. Accumulated pyruvate and lactate may contribute to acidification and induction of pro-inflammatory cytokines. Subsequently, tissue inflammation and tumor progression were raised in neoplastic tissues. Therefore, we aim to investigate whether Nm23-H1 modulates glucose metabolism and bioenergetics in HNSCC cells. Additionally, the effects and mechanisms of glycolytic inhibitor on radio-/chemo-sensitivities of HNSCC cells will be elucidated. Methods: By the established in vitro models of HNSCC cell lines with stable Nm23-H1 overexpression and knockdown, we will research into the effects and mechanisms of Nm23-H1 expression on glucose metabolism and radio-/chemo-sensitivity. Using trypan blue exclusion assay and clonogenic assay, the cytotoxicity of chemothearpy and radiation on these HNSCC cell lines will be assessed, respectively. The changes of glucose metabolic pathways (including celluar ATP content, and oxygen consumption rate, as well as pH of conditioned medium, and mitochondrial mass, mitochondrial membrane potential …)on these HNSCC cell lines will be assessed. Using cell cycle analysis, TUNEL, DNA fragmentation, Annexin V-FITC assays，we will evaluate the effects on cell cycle regulation and classic apoptosis. By Western blot analysis and/or quantitative RT-PCR, we will evaluate the expression levels of metabolic enzymes, cyclins, and DNA repair system. The activity of metabolic enzyme will also be measured and normalized by protein concentration. Anticipated results: The Nm23-H1 -regulated glycolysis and radio-/chemo-sensitivities of HNSCC has will be characterized.

Keyword(s)

低氧壓
酸化
有氧糖解作用
癌症惡化
粒線體
Hypoxia
aerobic glycolysis
acidification
cancer progression
mitochondria

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To Study the Effects and Mechanisms of Nm23-H1 on Radio-/Chemo-Sensitivities in Human Head and Neck Squamous Cell Carcinoma: the Role of Glycolysis

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