Abstract
BackgroundThe integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt microtubules. However, due to associated side effects on healthy cells, there is a need to develop safer drug regimens that still retain clinical efficacy. Currently, many questions remain open regarding the extent of effects on cellular physiology of microtubule-interacting drugs at clinically relevant and low doses. Here, we use super-resolution microscopies (single-molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid.ResultsWe identify previously undetected microtubule (MT) damage caused by clinically relevant doses of colcemid. Short exposure to 30–80 nM colcemid results in aberrant microtubule curvature, with a trend of increased curvature associated to increased doses, and curvatures greater than 2 rad/μm, a value associated with MT breakage. Microtubule fragmentation was detected upon treatment with ≥ 100 nM colcemid. Remarkably, lower doses (< 20 nM after 5 h) led to subtle but significant microtubule architecture remodelling characterized by increased curvature and suppression of microtubule dynamics.ConclusionsOur results support the emerging hypothesis that microtubule-interacting drugs induce non-mitotic effects in cells, and establish a multi-modal imaging assay for detecting and measuring nanoscale microtubule dysfunction. The sub-diffraction visualization of these less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs offers potential for improved understanding and design of anticancer agents.
Highlights
The integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer
Low doses of colcemid cause remodelling of microtubule architecture dSTORM was used to detect sub-diffraction effects of colcemid by immunolabelling tubulin in fixed HeLa cells treated with 0–200 nM colcemid for 5 h (Fig. 1)
At 50 nM colcemid, while filaments were still mostly linear, some filament sections had become visibly more curved; 65 nM and 80 nM colcemid treatments resulted in more pronounced filament curvature throughout the cell
Summary
The integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. The use of colchicine, is limited by its toxicity and poorly defined dosing thresholds between non-toxic, toxic and lethal [13,14,15] It can cause neurotoxicity [16] and is associated with renal and liver failure in cases of colchicine poisoning [13]. Despite these outcomes, colchicine still presents some potential as an anticancer compound where low concentration doses reduce the proliferation of cholangiocarcinoma cell lines, as well as decrease tumour size in mouse models [17]. Several synthetic derivatives of colchicine are being clinically tested and show promise to treat cancers in the future [18]
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