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 22C threshold, even by a small number of degrees (e.g. 18C), 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 18C) 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 37C, to 22C, 18C and 14C); 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

Hypoxia-Modified Cancer Cell Metabolism

While oxygen is critical to the continued existence of complex organisms, extreme levels of oxygen within a system, known as hypoxia (low levels of oxygen) and hyperoxia (excessive levels of oxygen), potentially promote stress within a defined biological environment. The consequences of tissue hypoxia, a result of a defective oxygen supply, vary in response to the gravity, extent and environment of the malfunction. Persistent pathological hypoxia is incompatible with normal biological functions, and as a result, multicellular organisms have been compelled to develop both organism-wide and cellular-level hypoxia solutions. Both direct, including oxidative phosphorylation down-regulation and inhibition of fatty-acid desaturation, and indirect processes, including altered hypoxia-sensitive transcription factor expression, facilitate the metabolic modifications that occur in response to hypoxia. Due to the dysfunctional vasculature associated with large areas of some cancers, sections of these tumors continue to develop in hypoxic environments. Crucial to drug development, a robust understanding of the significance of these metabolism changes will facilitate our understanding of cancer cell survival. This review defines our current knowledge base of several of the hypoxia-instigated modifications in cancer cell metabolism and exemplifies the correlation between metabolic change and its support of the hypoxic-adapted malignancy

Use of in vitro human keratinocyte models to study the effect of cooling on chemotherapy drug-induced cytotoxicity

A highly distressing side-effect of cancer chemotherapy is chemotherapy-induced alopecia (CIA). Scalp cooling remains the only treatment for CIA, yet there is no experimental evidence to support the cytoprotective capacity of cooling. We have established a series of in vitro models for the culture of human keratinocytes under conditions where they adopt a basal, highly-proliferative phenotype thus resembling the rapidly-dividing sub-population of native hair-matrix keratinocytes. Using a panel of chemotherapy drugs routinely used clinically (docetaxel, doxorubicin and the active metabolite of cyclophosphamide 4-OH-CP), we demonstrate that although these drugs are highly-cytotoxic, cooling can markedly reduce or completely inhibit drug cytotoxicity, in agreement with clinical observations. By contrast, we show that cytotoxicity caused by specific combinatorial drug treatments cannot be adequately attenuated by cooling, supporting data showing that such treatments do not always respond well to cooling clinically. Importantly, we provide evidence that the choice of temperature may be critical in determining the efficacy of cooling in rescuing cells from drug-mediated toxicity. Therefore, despite their reductive nature, these in vitro models have provided experimental evidence for the clinically-reported cytoprotective role of cooling and represent useful tools for future studies on the molecular mechanisms of cooling-mediated cytoprotection

A Clinical and Biological Guide for Understanding Chemotherapy-Induced Alopecia and its Prevention

Chemotherapy-induced alopecia (CIA) is the most visibly distressing side effect of commonly administered chemotherapeutic agents. As psychological health has huge relevance on lifestyle, diet and self-esteem, it is important for clinicians to fully appreciate the psychological burden that CIA can place on patients. Here, for the first time, we provide a comprehensive review encompassing the molecular characteristics of the human hair follicle (HF), how different anticancer agents damage the HF to cause CIA, subsequent HF pathophysiology and we assess known and emerging prevention modalities that have aimed to reduce or prevent CIA. We argue that, at present, scalp cooling is the only safe and FDA-cleared modality available, and we highlight the extensive available clinical and experimental (biological) evidence for its efficacy. The likelihood of a patient that uses scalp cooling during chemotherapy maintaining enough hair to not require a wig is approximately 50%. This is despite different types of chemotherapy regimens, patient-specific differences and possible lack of staff experience in effectively delivering scalp cooling. The increased use of scalp cooling and an understanding of how to deliver it most effectively to patients has enormous potential to ease the psychological burden of CIA, until other, more efficacious, equally safe treatments become available