Ultraviolet (UV) Damage and Protection: How HaCaT Cells Aid in Understanding Photoaging and Skin Cancer Risk

Introduction

Ultraviolet (UV) radiation is the primary external factor that causes skin damage, photoaging, and skin cancer. To understand its mechanisms and develop effective protective strategies, scientists require controllable in vitro models. The HaCaT cell line, an immortalized human keratinocyte line, serves as an ideal tool for simulating the response of skin cells to UV radiation due to its stable biological characteristics. It provides an important platform for photobiology research.

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Simulating Solar Damage

In a laboratory setting, researchers irradiate cultured HaCaT cells with specific UV sources to mimic the exposure of human skin to sunlight. This process is precisely controlled. Researchers can use either UVA (320-400 nm) or UVB (290-320 nm) lamps and set an accurate dose using a radiometer, with the unit typically measured in Joules per square centimeter (J/cm²). By adjusting the irradiation dose, it is possible to simulate varying degrees of UV exposure, from daily sun exposure to excessive sunburn, thereby establishing a quantifiable and reproducible cell damage model.

Investigating UV-Induced Cellular Responses

UV radiation triggers a series of complex biological events within keratinocytes. The HaCaT cell model makes it possible to study these events in detail.

1. Cell Apoptosis and Survival

UV radiation is a cellular stressor, and high doses can induce cell death. Researchers can quantify the survival rate of HaCaT cells after UV exposure using cell viability assays like MTT or CCK-8 to assess the cytotoxicity of different UV doses. Furthermore, Annexin V/PI double staining combined with flow cytometry allows for the precise quantification of early and late apoptotic (or necrotic) cell populations, providing deeper insight into the specific modes of cell death.

2. DNA Damage and Repair

UVB is the main cause of direct DNA damage. Its energy can be absorbed by DNA, leading to the formation of photoproducts like cyclobutane pyrimidine dimers (CPDs), which are initial lesions in skin carcinogenesis. In UV-irradiated HaCaT cells, researchers can detect CPD formation using specific antibodies via immunofluorescence. In response, cells activate mechanisms like nucleotide excision repair (NER) to remove this damage. By detecting the phosphorylation levels of repair proteins such as γ-H2AX (a marker of DNA double-strand breaks), the activation status of the cellular DNA damage response pathway can be evaluated.

3. Oxidative Stress and Inflammation

UVA is less likely to damage DNA directly but can penetrate deeper into the skin, causing oxidative stress through the production of reactive oxygen species (ROS). Using fluorescent probes like DCFH-DA, researchers can detect the sharp increase in intracellular ROS levels in HaCaT cells after UV exposure. Excessive ROS can damage proteins, lipids, and DNA, and activate inflammatory signaling pathways. By measuring the release of inflammatory cytokines (e.g., IL-6, TNF-α) in the cell culture supernatant using methods like ELISA, the UV-induced skin inflammatory response can be assessed.

Evaluating Sunscreen Products and Photoprotective Agents

The HaCaT cell model provides an efficient in vitro platform for screening and evaluating photoprotective strategies. Before UV irradiation, researchers can pre-treat HaCaT cells with medium containing specific sunscreen ingredients or antioxidants (e.g., Vitamin C, Vitamin E, resveratrol). After irradiation, by measuring the aforementioned endpoints (cell viability, DNA damage, ROS levels, inflammatory cytokine release), the protective effects of these active compounds against UV damage can be quantitatively evaluated. This method allows for the rapid screening of effective protective ingredients and provides preliminary cell-based evidence for their mechanisms of action.

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Conclusion

The HaCaT cell line is a key in vitro tool for investigating the effects of ultraviolet radiation on human keratinocytes. It enables researchers to simulate solar damage under controlled conditions and to dissect core molecular mechanisms, including apoptosis, DNA damage repair, oxidative stress, and inflammation. The model also plays a significant role in the early-stage screening and efficacy evaluation of photoprotective ingredients, providing a solid scientific foundation for developing more effective sunscreen products and strategies to prevent photo-related skin diseases.

References:

[1]Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A., & Fusenig, N. E. (1988). Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. The Journal of Cell Biology, 106(3), 761–771.

[2]Lehmann, J., Seve, M., Wulff, M., & Bohn, S. (2006). The impact of ultraviolet irradiation on cell death, mutation load and signalling in human HaCaT keratinocytes. Apoptosis, 11(11), 1939–1951.

[3]Bosch, R., Philips, N., Suárez-Pérez, J. A., Juarranz, A., Devmurari, A., & Chalensouk-Khaosaat, J. (2015). Mechanisms of photoaging and chronological skin aging. Biomolecules, 5(2), 536–565. 

[4]Svobodová, A., Zdarilová, A., & Vostálová, J. (2009). Lonicera caerulea and its flavonoids inhibit UVA-induced damage in human keratinocytes. Journal of Photochemistry and Photobiology B: Biology, 97(1), 1-7.