Studying the Mechanisms of Chemotherapy-Induced Alopecia and the Effect of Cooling using in Vitro Human Keratinocyte Models

Chemotherapy-induced alopecia (CIA) is widely regarded as the most traumatic side effect associated with cancer treatment and the associated stress can be detrimental to overall outcomes. Yet there has been little research into its pathobiology and no pharmaceutical intervention is available. CIA is caused by chemotherapy-mediated damage of the rapidly dividing cells of the hair follicle and although it is normally reversible, on regrowth the hair is often different in colour and/or texture and only grows gradually. The only effective treatment for CIA currently available is scalp cooling. It has been hypothesised that scalp cooling works by a combination of vasoconstriction, a reduction in the metabolic rate and/or reduced drug uptake by cells in the hair bulb.

The ability of cooling to protect from CIA has been clinically demonstrated for years yet, to date, no cell biology is available to support its cytoprotective effects. The overall aim of this work was to for the first time provide a systematic investigation of the effects of cooling on chemotherapy-induced toxicity in human cells. The work established cellular models to determine the efficacy of cooling in rescuing from toxicity, investigate the temperature conditions providing maximal rescue and understand not only the mechanisms responsible for drug-mediated cytotoxicity, but also the way in which cooling regulates such mechanisms. Various human keratinocyte models were established, including normal (epidermal, NHEK, and follicular, HHFK) cells and adapted HaCaT (HaCaTa) cells. Viability, cell cycle and apoptosis assays were used, alongside Reactive Oxygen Species (ROS) detection, mitochondrial integrity assays and Western blotting, as well as functional pharmacological inhibition experiments. A panel of chemotherapy drugs commonly used in the clinic were employed, including doxorubicin, docetaxel and active metabolite of cyclophosphamide, 4-hydroxy-cyclophosphamide (4-OH-CP) and 5-FU, whilst a series of temperature conditions were tested, including 22°C as well as more severe cooling, particularly 18°C and 14°C (and even extreme cooling at 10°C).

This study showed that cooling dramatically reduces or completely prevents the cytotoxic effects of docetaxel (T), doxorubicin (A), 5-FU (F) and particularly 4-OH-CP (C); however, optimal rescue was observed in conjunction with mono-therapy treatments (and substantial rescue with dual therapies, e.g. AC), whereas combinatorial treatment (TAC) showed relatively poor response to cooling, in agreement with clinical observations. Importantly, the work demonstrated that lowering the temperature below the widely accepted 22C threshold, even by a small number of degrees (e.g. 18C), resulted in significantly improved or even complete cytoprotection, a striking observation strongly suggesting that the scalp temperature achieved clinically is of critical importance in dictating the success of head cooling in CIA prevention.

The panel of chemotherapy drugs tested caused differential effects on keratinocyte cell cycle progression and drug-mediated cell cycle arrest was significantly attenuated by cooling. Notably, cooling alone appeared to decelerate cell cycle progression, providing evidence for metabolic effects. More importantly, protective pre-conditioning (PPC) achieved either by growth factor removal or pharmacological inhibition of EGFR activation enhanced the cytoprotective effects of cooling and significantly reduced the effects of the chemotherapy drugs. As the ability of PPC to enhance protection from drug cytotoxicity could be attributed to its propensity to regulate the cell cycle progression, the work provided evidence that one mechanism via which cooling cytoprotects might be due to its ability to decelerate cell cycle progression.

Disruption of mitochondrial membrane potential and elevation of ROS indicated the activation of an apoptotic pathway, which was confirmed by cell death-specific assays that confirmed a mitochondrial apoptotic pathway, as evident by plasma membrane disruption, caspase activation and DNA fragmentation. Importantly, cooling at a variety of temperatures (but mainly at or below 18C) attenuated drug-mediated apoptosis. To further investigate the precise mechanisms of growth arrest and/or cytotoxicity, activation/regulation of critical intracellular signalling mediators was investigated at the protein level. The majority of the drugs used induced activation of p53 and subsequent induction of p53-inducible mediators such as p21, as well as pro-apoptotic mediators associated with the mitochondrial pathway, such as Bak, PUMA and Noxa, whilst induction of pro-apoptotic FasL and Bid cleavage was detected, suggesting possible cross-talk with the extrinsic apoptotic pathway. Strikingly, cooling attenuated or blocked in a time- and, more importantly, temperature-dependent fashion induction of these pro-apoptotic mediators (an effect that became more marked as the temperature was reduced from 37C, to 22C, 18C and 14C); these results have provided for the first time a more detailed mechanistic explanation for the cytoprotective effects of cooling.

As ROS appeared to be important in cytotoxicity, the hypothesis raised was that the cytoprotective effect of cooling might be enhanced via co-treatment with an antioxidant (e.g. NAC), aimed at enhancing the cytoprotective capacity of cooling at sub-optimal temperatures (such as 26°C). The findings presented here suggested that cooling plus topical treatment with antioxidants might represent a promising approach to improve the cytoprotective effects without compromising the anticancer effects of chemotherapy.

Overall, despite their reductive nature, these in vitro models have provided experimental evidence for the ability of cooling to rescue from chemotherapy drug-mediated toxicity and shown that the choice of temperature may be critical in determining the efficacy of cooling in the clinic. This, whilst generating a novel combinatorial approach that has the potential to significantly enhance the ability of scalp cooling to protect against CIA in the clinic.