Role of mitochondrial function in the invasiveness of human colon cancer cells.

We investigated the role of mitochondrial function in the invasiveness of human colorectal cancer (CRC) cell lines, using paired primary SW480 and metastatic SW620 cells, and appraised the clinical relevance of the alteration of mtDNA copy number in 33 pairs of CRC specimens after surgical resection. Suppression of mitochondrial function was achieved by the exposure of cells to oligomycin A (OA) or by knockdown of mitochondrial transcriptional factor A (TFAM) to evaluate their effects on energy metabolism, reactive oxygen species, protein expression levels of epithelial-mesenchymal transition (EMT) markers and invasive activity of CRC cells. We found that SW620 cells expressed higher levels of TFAM and mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) and cytochrome c oxidase subunit II (COX-II) and nuclear DNA-encoded NADH ubiquinone oxidoreductase subunit A9 (NDUFA9), iron-sulfur protein subunit B of succinate dehydrogenase (SDHB), ubiquinol‑cytochrome c reductase core protein I/II (UQCRC1/2) and cytochrome c oxidase subunit IV (COX-IV) when compared with the SW480 cells. The mtDNA copy number, ADP-triggered oxygen consumption rate (OCR) and respiratory control ratio (RCR) of succinate-supported respiration in the SW620 cells were higher than those noted in the SW480 cells. The intracellular levels of H2O2 and O2-• in the SW620 cells were lower than levels noted in the SW480 cells. Moreover, SW620 cells displayed lower protein levels of hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI) and lactate dehydrogenase (LDH), and lower lactate production rate, and expressed higher levels of EMT markers N-cadherin, vimentin and Snail, and showed higher Transwell migration and invasion activities as compared with the SW480 cells. After OA treatment, SW620 cells exhibited a decrease in OCR and RCR of succinate-supported respiration, an increase in lactate production rate and intracellular levels of H2O2 and O2-•. Moreover, the level of vimentin and Transwell migration activity of the SW620 cells were decreased. After TFAM knockdown, the protein levels of TFAM, ND6 and COX-II, and mtDNA copy number, OCR and RCR of succinate-supported respiration in the SW620-KD#4 and SW620-KD#5 cells were all lower than those noted in the SW620‑Control cells. By contrast, the protein level of HK-II, lactate production rate, the intracellular levels of H2O2 and O2-• in the SW620-KD#4 and SW620-KD#5 cells were all higher than those noted in the SW620-Control cells. Subsequently, both SW620-KD#4 and SW620-KD#5 cells had lower Transwell invasion activity than did the SW620-Control cells. Furthermore, we found that deeper invasion (P=0.025) and longer tumor length (P=0.069) were associated with higher mtDNA copy ratios in the 33 pairs of CRC specimens obtained from surgical resection. Taken together, we conclude that higher mtDNA copy number and mitochondrial function may confer an invasive advantage to CRCs.

本研究以配对的原代SW480细胞与转移性SW620细胞为模型，探究线粒体功能在人结直肠癌（colorectal cancer, CRC）细胞侵袭性中的作用，并针对33例手术切除的结直肠癌标本，评估线粒体DNA（mtDNA）拷贝数改变的临床相关性。本研究通过将细胞暴露于寡霉素A（oligomycin A, OA）或敲低线粒体转录因子A（TFAM）以抑制线粒体功能，以此评估其对结直肠癌细胞能量代谢、活性氧水平、上皮间质转化（EMT）标志物蛋白表达水平以及侵袭活性的影响。研究结果显示，相较于SW480细胞，SW620细胞的TFAM、线粒体DNA编码的NADH脱氢酶亚基6（ND6）、细胞色素c氧化酶亚基II（COX-II），以及核DNA编码的NADH泛醌氧化还原酶亚基A9（NDUFA9）、琥珀酸脱氢酶铁硫蛋白亚基B（SDHB）、泛醇-细胞色素c还原酶核心蛋白I/II（UQCRC1/2）与细胞色素c氧化酶亚基IV（COX-IV）的表达水平均更高。SW620细胞的mtDNA拷贝数、ADP触发的氧消耗速率（OCR），以及琥珀酸支持的呼吸作用的呼吸控制比（RCR）均高于SW480细胞。SW620细胞内的过氧化氢（H₂O₂）与超氧阴离子（O₂⁻·）水平低于SW480细胞。此外，相较于SW480细胞，SW620细胞的己糖激酶II（HK-II）、葡萄糖-6-磷酸异构酶（GPI）与乳酸脱氢酶（LDH）的蛋白水平更低，乳酸生成速率更慢，且上皮间质转化标志物N-钙粘蛋白、波形蛋白与Snail的表达水平更高，Transwell迁移与侵袭活性也更强。经寡霉素A处理后，SW620细胞的琥珀酸支持的呼吸作用的OCR与RCR均下降，乳酸生成速率以及细胞内H₂O₂与O₂⁻·水平均升高。同时，SW620细胞的波形蛋白水平与Transwell迁移活性均降低。经TFAM敲低后，SW620-KD#4与SW620-KD#5细胞的TFAM、ND6与COX-II的蛋白水平，mtDNA拷贝数、OCR以及琥珀酸支持的呼吸作用的RCR均低于SW620对照组细胞。与之相反，这两株敲低细胞的HK-II蛋白水平、乳酸生成速率以及细胞内H₂O₂与O₂⁻·水平均高于SW620对照组细胞。随后，SW620-KD#4与SW620-KD#5细胞的Transwell侵袭活性均低于SW620对照组细胞。进一步分析显示，在33例手术切除的结直肠癌标本中，更高的mtDNA拷贝数比值与更深的侵袭深度（P=0.025）以及更长的肿瘤长度（P=0.069）具有相关性。综上，本研究认为更高的mtDNA拷贝数与线粒体功能可赋予结直肠癌侵袭优势。