P53 activation suppresses irinotecan metabolite SN-38-induced cell damage in non-malignant but not malignant epithelial colonic cells

doi:10.1016/j.tiv.2020.104908

Toxicology in Vitro

Volume 67, September 2020, 104908

https://doi.org/10.1016/j.tiv.2020.104908 Get rights and content

Highlights

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p53 inhibitor Nutlin-3a may protect intestinal epithelial cells during chemotherapy.
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Nutlin-3a reduced proliferation in p53 wild-type but not p53 aberrant cells.
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SN-38 induced more potent cytotoxicity in colorectal cancer cells than FHs-74 cells.
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Nutlin-3a protected FHs-74 cells, but not WT p53 HCT cells from SN-38 toxicity.
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Nutlin-3a may provide protection from chemotherapy-related intestinal toxicity.

Abstract

Nutlin-3a is a p53 activator and potential cyclotherapy approach that may also mitigate side effects of chemotherapeutic drugs in the treatment of colorectal cancer. We investigated cell proliferation in a panel of colorectal cancer (CRC) cell lines with wild-type or mutant p53, as well as a non-tumorigenic fetal intestinal cell line following Nutlin-3a treatment (10 μM). We then assessed apoptosis at 24 and 48 h following administration of the active irinotecan metabolite, SN-38 (0.001 μM - 1 μM), alone or following pre-treatment with Nutlin-3a (10 μM).

Nutlin-3a treatment (10 μM) significantly reduced proliferation in wild-type p53 expressing cell lines (FHS 74 and HCT116^+/+) at 72 and 96 h, but was without effect in cell lines with mutated or deleted p53 (Caco-2, SW480, and HCT 116^−/−). SN-38 treatment induced significant apoptosis in all cell lines after 48 h. Nutlin-3a unexpectedly increased cell death in the p53 wild-type CRC cell line, HCT116^+/+, while Nutlin-3a pre-treatment provided protection from SN-38 in the p53 wild-type normal cell line, FHs 74. These results demonstrate Nutlin-3a's selective growth-arresting efficacy in p53 wild-type non-malignant intestinal cell lines, enabling the selective targeting of malignant cells with chemotherapy drugs. These studies highlight the potential of Nutlin-3a to minimise intestinal mucosal damage following chemotherapy.

Introduction

Colorectal cancer (CRC) is one of the most common causes of cancer globally, commonly arising from an aberrant activation of an oncogene and/or inactivation of tumour suppression genes such as p53 (Li et al., 2015). These alterations result in a loss of regulation in essential processes such as cellular differentiation, apoptosis and proliferation (Chen et al., 2010). p53 maintains an essential role in the prevention of oncogenic transformation, where cellular signals from injury such as DNA damage or abnormal oncogene expression engage the p53 pathway and stabilize p53 protein levels (Bunz et al., 1998).

Chemotherapy in the treatment of CRC involves the use of highly cytotoxic agents such as the topoisomerase inhibitor irinotecan, which also induces apoptosis in normal proliferating cells in the intestinal mucosa (Ribeiro et al., 2016). This relatively non-specific targeting inflicts epithelial damage, inflammation and ulceration, commonly termed mucositis associated with chemotherapy-induced gastrointestinal toxicity (CIGT). The gastrointestinal mucosa and submucosa respond to the damage through cell death, leading to crypt ablation in the intestinal tract (Mayo et al., 2017). Mucosal loss can lead to vomiting and nausea, lowered food intake and appetite, weight loss, increased infection risk and a reduction in the tolerated chemotherapy dose (Thorpe et al., 2013). As such it represents a significant risk to the clinical management of such affected patients.

Cyclotherapy is a novel approach eliciting a transient cell cycle arrest by targeting wild-type p53 (Blagosklonny and Darzynkiewicz, 2002; Rao et al., 2013). Nutlin-3a is a selective MDM2 antagonist that induces a temporary, reversible growth arrest in cells that retain wild-type p53 (Korotchkina et al., 2009). As the effects of Nutlin-3a are highly dependent on the presence of wild-type p53, it may be useful to selectively induce p53-dependent cell cycle arrest in normal cells (Carvajal et al., 2005; van Leeuwen et al., 2012). As Nutlin-3a has been shown to have negligible effect on tumour cell proliferation (Pishas et al., 2011), this strategy potentially offers the gastrointestinal mucosa protection from the injurious effects of chemotherapy and the development of CIGT. However, some studies have found effects of Nutlin-3a that may occur independently of p53 expression (Candido et al., 2019), requiring further studies in both p53 wild-type and deficient conditions.

In this study we characterised the comparative effects of Nutlin-3a in a panel of p53 wild-type and deficient intestinal epithelial cell lines against toxicity induced by the active metabolite of irinotecan, SN-38. In this way we aimed to determine the effectiveness and selectivity of Nutlin-3a as a potential adjunctive, intestinal-sparing cyclotherapy treatment during chemotherapy.

Section snippets

Reagents and chemicals

All reagents and chemicals were provided by Sigma-Aldrich (Sydney, Australia) unless otherwise stated, including 7-ethyl-10-hydroxycamptothecin (SN-38). Nutlin-3a was obtained through Cayman Biochemicals, (Ann-Arbor, MI, USA). 7-Aminoactinomycin D (7-AAD) was obtained via Invitrogen (Thermo-Fisher Scientific, Waltham, MA, USA).

Cell culture

The non-malignant fetal small intestinal cell line FHs 74 was provided by Dr. Joanne Bowen (University of Adelaide) and maintained in DMEM supplemented with 1 mmol/L

TP53 Gene sequencing in gastrointestinal epithelial cell lines

Gene sequencing of all cell lines validated their recognised p53 status (Table 2). FHs 74 and HCT116^+/+ are p53 wild-type cell lines, while HCT116^−/− is a p53 knockout cell line.

Caco-2 produces a non-functional truncated p53 (Ahmed et al., 2013) while SW480 is a p53 mutated cell line with up to 20-fold increased p53 expression compared to wild-type gut epithelial cell lines (Ahmed et al., 2013) (Rochette et al., 2005). This was supported by Western blot results clearly showing increased

Discussion

Toxicity to normal proliferating cells during conventional chemotherapy is unavoidable unless drugs are highly specific to cancer cells. While immunotherapies are advancing targeted efficacy and mitigating side effects associated with chemotherapy, additional strategies that exploit factors involved in the dysregulation of normal growth control by cancer cells to selectively protect normal cells may be desirable, enabling a further decrease in the occurrence and severity of side effects (

Declaration of Competing Interest

None.

Acknowledgements

This project was supported through general internal research funding via The Faculty of Health and Medical Sciences, The University of Adelaide. The authors would like to thank Professor David Callen, Benjamin Harvey, Alaknanda Adwal, Kathleen Pishas, Reshma Shakya and Renee Schulz.

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