Guardamagna Isabella, Lonati Leonardo, Savio Monica, Stivala Lucia A, Ottolenghi Andrea, Baiocco Giorgio
Laboratory of Radiation Biophysics and Radiobiology, Department of Physics, University of Pavia, Pavia, Italy.
Immunology and General Pathology Unit, Department of Molecular Medicine, University of Pavia, Pavia, Italy.
Front Oncol. 2021 Jun 3;11:688919. doi: 10.3389/fonc.2021.688919. eCollection 2021.
Colorectal cancer is among the three top cancer types for incidence and the second in terms of mortality, usually managed with surgery, chemotherapy and radiotherapy. In particular, radiotherapeutic concepts are crucial for the management of advanced rectal cancer, but patients' survival remains poor, despite advances in treatment modalities. The use of well-characterized cell culture systems offers an important preclinical strategy to study mechanisms at the basis of cell response to therapeutic agents, including ionizing radiation, possibly leading to a better understanding of the response to the treatment. In this context, we present an integrated analysis of results obtained in an extensive measurement campaign of radiation effects on Caco-2 cells, derived from human colorectal adenocarcinoma. Cells were exposed to X-rays with doses up to 10 Gy from a radiotherapy accelerator. We measured a variety of endpoints at different post-irradiation times: clonogenic survival after ~ 2 weeks; cell cycle distribution, cell death, frequency of micronucleated cells and atypical mitoses, activation of matrix metalloproteases (MMPs) and of different proteins involved in DNA damage response and cell cycle regulation at earlier time points, up to 48 h post-exposure. Combined techniques of flow cytometry, immunofluorescence microscopy, gelatin zymography and western blotting were used. For selected endpoints, we also addressed the impact of the irradiation protocol, comparing results obtained when cells are plated before irradiation or first-irradiated and then re-plated. Caco-2 resistance to radiation, previously assessed up to 72 h post exposure in terms of cell viability, does not translate into a high clonogenic survival. Survival is not affected by the irradiation protocol, while endpoints measured on a shorter time frame are. Radiation mainly induces a G-phase arrest, confirmed by associated molecular markers. The activation of death pathways is dose- and time-dependent, and correlates with a dose-dependent inhibition of MMPs. Genomic aberrations are also found to be dose-dependent. The phosphorylated forms of several proteins involved in cell cycle regulation increase following exposure; the key regulator FoxM1 appears to be downregulated, also leading to inhibition of MMP-2. A unified molecular model of the chain of events initiated by radiation is proposed to interpret all experimental results.
结直肠癌是发病率排名前三的癌症类型之一，死亡率位居第二，通常采用手术、化疗和放疗进行治疗。特别是，放射治疗理念对于晚期直肠癌的治疗至关重要，但尽管治疗方式有所进步，患者的生存率仍然很低。使用特征明确的细胞培养系统为研究细胞对治疗药物（包括电离辐射）反应机制提供了重要的临床前策略，这可能有助于更好地理解对治疗的反应。在此背景下，我们对在广泛的辐射对源自人结肠腺癌的Caco-2细胞影响的测量活动中获得的结果进行了综合分析。细胞用放疗加速器产生的高达10 Gy的X射线进行照射。我们在不同的照射后时间测量了多种终点指标：约2周后的克隆形成存活率；细胞周期分布、细胞死亡、微核细胞和非典型有丝分裂的频率，以及在更早时间点（直至照射后48小时）基质金属蛋白酶（MMPs）和参与DNA损伤反应及细胞周期调控的不同蛋白质的激活情况。使用了流式细胞术、免疫荧光显微镜、明胶酶谱和蛋白质印迹等联合技术。对于选定的终点指标，我们还探讨了照射方案的影响，比较了细胞在照射前接种或先照射然后再接种时获得的结果。Caco-2细胞对辐射的抗性，此前在照射后72小时内根据细胞活力进行评估，并未转化为高克隆形成存活率。存活率不受照射方案的影响，而在较短时间框架内测量的终点指标则受影响。辐射主要诱导G期停滞，相关分子标志物证实了这一点。死亡途径的激活是剂量和时间依赖性的，并且与MMPs的剂量依赖性抑制相关。还发现基因组畸变是剂量依赖性的。照射后，参与细胞周期调控的几种蛋白质的磷酸化形式增加；关键调节因子FoxM1似乎下调，这也导致MMP-2的抑制。提出了一个由辐射引发的事件链的统一分子模型来解释所有实验结果。
