Jelena Žakula, Lela Korićanac, Otilija Keta, Danijela Todorović, Cirrone Giuseppe A P, Francesco Romano, Giacomo Cuttone, Ivan Petrović, Aleksandra Ristić-Fira
Vinča Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia.
Medical Faculty, University of Kragujevac, Kragujevac, Serbia.
Indian J Med Res. 2016 May;143(Supplement):S120-S128. doi: 10.4103/0971-5916.191811.
BACKGROUND & OBJECTIVES: The main goal when treating malignancies with radiation is to deprive tumour cells of their reproductive potential. One approach is to induce tumour cell apoptosis. This study was conducted to evaluate the ability of carbon ions ( [12] C) to induce apoptosis and cell cycle arrest in human HTB140 melanoma cells.
In this in vitro study, human melanoma HTB140 cells were irradiated with the 62 MeV/n carbon ( [12] C) ion beam, having two different linear energy transfer (LET) values: 197 and 382 keV/μm. The dose range was 2 to 16 Gy. Cell viability was estimated by the sulforhodamine B assay seven days after irradiation. The cell cycle and apoptosis were evaluated 48 h after irradiation using flow cytometry. At the same time point, protein and gene expression of apoptotic regulators were estimated using the Western blot and q-PCR methods, respectively.
Cell viability experiments indicated strong anti-tumour effects of [12] C ions. The analysis of cell cycle showed that [12] C ions blocked HTB140 cells in G2 phase and induced the dose dependent increase of apoptosis. The maximum value of 21.8 per cent was attained after irradiation with LET of 197 keV/μm at the dose level of 16 Gy. Pro-apoptotic effects of [12] C ions were confirmed by changes of key apoptotic molecules: the p53, Bax, Bcl-2, poly ADP ribose polymerase (PARP) as well as nuclear factor kappa B (NFκB). At the level of protein expression, the results indicated significant increases of p53, NFκB and Bax/Bcl-2 ratio and PARP cleavage. The Bax/Bcl-2 mRNA ratio was also increased, while no change was detected in the level of NFκB mRNA.
INTERPRETATION & CONCLUSIONS: The present results indicated that anti-tumour effects of [12] C ions in human melanoma HTB140 cells were accomplished through induction of the mitochondrial apoptotic pathway as well as G2 arrest.
用放射疗法治疗恶性肿瘤的主要目标是剥夺肿瘤细胞的增殖潜能。一种方法是诱导肿瘤细胞凋亡。本研究旨在评估碳离子（¹²C）诱导人HTB140黑色素瘤细胞凋亡和细胞周期阻滞的能力。
在这项体外研究中，人黑色素瘤HTB140细胞用62 MeV/n碳（¹²C）离子束照射，该离子束具有两种不同的线性能量传递（LET）值：197和382 keV/μm。剂量范围为2至16 Gy。照射7天后，通过磺酰罗丹明B测定法评估细胞活力。照射48小时后，使用流式细胞术评估细胞周期和凋亡情况。在同一时间点，分别使用蛋白质印迹法和q-PCR法评估凋亡调节因子的蛋白质和基因表达。
细胞活力实验表明¹²C离子具有强大的抗肿瘤作用。细胞周期分析表明，¹²C离子使HTB140细胞阻滞在G2期，并诱导凋亡呈剂量依赖性增加。在16 Gy剂量水平下，LET为197 keV/μm照射后，凋亡率最高达到21.8%。¹²C离子的促凋亡作用通过关键凋亡分子的变化得到证实：p53、Bax、Bcl-2、聚ADP核糖聚合酶（PARP）以及核因子κB（NFκB）。在蛋白质表达水平上，结果表明p53、NFκB和Bax/Bcl-2比值以及PARP裂解显著增加。Bax/Bcl-2 mRNA比值也增加，而NFκB mRNA水平未检测到变化。
目前的结果表明，¹²C离子对人黑色素瘤HTB140细胞的抗肿瘤作用是通过诱导线粒体凋亡途径以及G2期阻滞实现的。
