An Integrated Analysis of the Response of Colorectal Adenocarcinoma Caco-2 Cells to X-Ray Exposure

Abstract

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 in vitro 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 in vivo 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 G2-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.

Development of Blueberry-Derived Extracellular Nanovesicles for Immunomodulatory Therapy

“…Remarkably, BENVs were found to selectively distribute to the nuclear region of HT-29 cells. Since Caco-2 cells exhibit low cancer phenotypes in comparison with bona fide high grade colorectal adenocarcinoma cells such as HT-29, it would be interesting to explore if BENVs could preferentially target susceptible cells [ 57 ]. The cellular uptake results were also confirmed by flow cytometry in which all human cancer cells contained fluorescence-labelled BENVs after 6 h ( Figure 4 C,D).…”

Chemoresistive Sensors for Cellular Type Discrimination Based on Their Exhalations

“…This was expected for the fibroblasts since they are not tumoral cells and their normal metabolism is expected to emit VOCs at a lower rate in respect to the tumorigenic ones. The CACO-2, derived from human colorectal adenocarcinoma, could also be expected to emit a lower amount of VOCs in respect to more invasive cancer cell lines, since it is a poorly aggressive tumor, and it has been widely adopted as a model of the intestinal epithelial barrier to study the mechanisms implicated in early-stage cancer progression, and to test the radiation therapeutic efficacy [ 50 ]. However, it is not expected, even after 72 h of plating, that the CACO-2 will become differentiated polarized epithelial cells (a stage that is expected to emit low amounts of VOCs), since this happens about after 20 days of plating [ 51 ].…”

Overview of Gas Sensors Focusing on Chemoresistive Ones for Cancer Detection

“…The CACO-2, derived from human colorectal adenocarcinoma, could also be expected to emit a lower amount of VOCs with respect to more invasive cancer cell lines, as was indeed found (Figure 25, lower panel). Indeed, it is a poorly aggressive tumor, and it has been widely adopted as a model of the intestinal epithelial barrier in order to study the mechanisms implicated in early-stage cancer progression and to test radiation therapeutic efficacy [138]. The small responses to human skin fibroblasts were expected since they are healthy; therefore, their metabolism is believed to emit VOCs at a lower rate with respect to the cancerous cells (Figure 25, top panel).…”

“…The CACO-2, derived from human colorectal adenocarcinoma, could also be expected to emit a lower amount of VOCs with respect to more invasive cancer cell lines, as was indeed found (Figure 25, lower panel). Indeed, it is a poorly aggressive tumor, and it has been widely adopted as a model of the intestinal epithelial barrier in order to study the mechanisms implicated in early-stage cancer progression and to test radiation therapeutic efficacy [138]. Different sensors gave different response amplitudes to the same cell line, while the same sensor gave different response amplitudes to different cell lines [95], illuminating the high potential of MOX sensors to discriminate among various cell types when working inside an array [95].…”