根據實驗室之前的研究發現，在口腔癌細胞中粒線體DNA 4977 bp斷裂程度與粒線體DNA套數都相對於周圍正常的基質細胞來得高。其中，粒線體DNA套數與腫瘤侵襲性程度都和病人癒後狀況有相當大的關聯性；粒線體DNA突變的累積更是與腫瘤的癌化發展有關。另外，近來受到廣泛研究的腫瘤幹細胞是一具有複製、分化的能力且可以極少量的細胞於免疫缺陷老鼠中引發腫瘤的生長。然而，粒線體的功能於腫瘤幹細胞中尚未被了解得很透徹。所以本篇研究以兩組不同癌化程度的細胞株依其腫瘤幹細胞CD44 marker來分群，以此比較粒線體在其中扮演的角色是否會有不同。其中，CD44+的細胞群相對於CD44-的細胞群，在兩株細胞中粒線體DNA套數都較低，且有較高的且氧氣消耗量，此結果意味著CD44+細胞群依賴氧化磷酸化產生ATP。另外，HSC3的CD44+細胞群含有較高的細胞外酸化程度與質子產生率，代表在較為癌化的細胞株中CD44+細胞群除了依賴氧化磷酸化之外也同時依賴糖解產生ATP。因此我們進一步探討超氧陰離子（O2-）、過氧化氫（H2O2），和錳超氧化物歧化酶(MnSOD)的表現。結果發現在兩株細胞中，CD44+細胞群相對於CD44-細胞群都含有較少量活性氧自由基。由於文獻指出活性氧自由基與粒線體DNA大片段缺失有關係，我們也進一步觀察到DOK的CD44+細胞群有較高程度的粒線體DNA 4977 bp缺失。總結研究結果，在不同癌化程度的DOK和HSC3細胞株，其含有CD44+腫瘤幹細胞標記的細胞群中相對於CD44-細胞群，皆會有較低套數的粒線體DNA，並且有較高的氧氣消耗量與較低的活性氧自由基的含量。

Accumulation of mitochondrial DNA mutation is an important contributing factor to the carcinogenesis progression. Mitochondrial DNA 4977 bp deletion (ΔmtDNA4977) is the most frequently noted mtDNA deletion occurring in various types of human cancers. Our previous study discovered that mtDNA 4977 bp is common deletion in oral cancer cell lines, and cancer cells may have reduced mitochondrial DNA copy numbers which is associated with tumor aggressiveness and poor prognosis. High proportion of ΔmtDNA4977 results in the damage of membrane potential, rate of ATP synthesis, and cellular ATP/ADP ratio. Cancer stem cells (CSCs) are a subpopulation which possesses the ability of self-renewal, differentiation, and regenerate a tumor when implanted into the NOD-SCID mouse with small number cells. However, the role of mitochondria in CSCs is still unclear. In this study, we use two different carcinogenesis progression cancer cell models to compare cancer cell subpopulations expressing CD44 as a stem cell marker for their mitochondrial DNA copy numbers, metabolic way, ROS level, and mitochondrial DNA large scale deletion. We found that mitochondrial DNA copy numbers are both decreased in the CD44+ subpopulation comparing to CD44- subpopulation. Oxygen consumption rate (OCR) is increased in the CD44+ subpopulation in both cell lines and it means that CD44+ subpopulation depended on oxidative phosphorylation as metabolic way. Extracellular acidification rate (ECAR) and proton production rate (PPR) are increased in CD44+ subpopulation in HSC3 which is more aggressive cell line and it means that CD44+ subpopulation in HSC3 depended on glycolysis as well. We further investigated the amount of H2O2, O2-, and anti-oxidant enzyme-Manganese superoxide dismutase (MnSOD). CD44+ subpopulation is maintained lower content of ROS than CD44- subpopulation. It has been suggested that the correlation between the level of ROS and mitochondrial DNA large scale deletion. Mitochondrial DNA 4977 bp deletion (ΔmtDNA4977) increased in CD44+ subpopulation in the DOK pre-cancer cell line. In conclusion, CD44+ subpopulation is presented lower mtDNA copy numbers, higher oxygen consumption rate, and lower ROS content than CD44- subpopulation no matter in pre-cancer cell line DOK or aggressive cell line HSC3.

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