Via Research Recognition Day 2024 VCOM-Carolinas

Biomedical Studies

Sulforaphane-enhanced Photoprotection Against Squamous Cell Carcinoma in Hydrochlorothiazide-treated Human Skin Cells Madeleine Peck, OMS-II, Rachel Hemsath, OMS-III, Gabriella Fonseca, OMS-II Co-PIs: Stephan Brown, MD, PhD, Tom Lindsey, DO, FACOS, CHSE Edward Via College of Osteopathic Medicine - Carolinas Campus, Spartanburg, SC

Abstract

Results

Conclusions

In 2021, Bigagli et al. conducted a study investigating the effects of HCTZ on human skin cell lines exposed to UV light compared to untreated cells. This project was framed based on the photoreactive results explained in Bigagli et al. An experimental timeline was created, displayed in Figure 1 , to determine the 24-hour short-term effects of treating human skin cell lines with photoreactive HCTZ and UVC light, compared to the same treatment with the addition of sulforaphane. Western blots were utilized in order to visualize the extent of DSBs in cell lines treated with HCTZ and sulforaphane with or without irradiation of UVC. H2A is a core protein in DNA that plays a role in chromatin assembly and is phosphorylated (P-ƔH2A) when DSBs occur in DNA, and therefore was used as a cellular marker to indicate DSBs in the experiment. β-actin, the main component of the cellular cytoskeleton, served as a loading control. Human skin cell lines were first irradiated with varying intensities of UVC light, ranging from 100µJ/cm 2 to 300 µJ/cm 2 , to determine the intensity of UVC light needed to cause DSBs in DNA. An intensity of 200µJ/cm 2 UVC was chosen to move forward with the procedure as part of the control groups. Another Western blot was conducted to demonstrate the requirement of UVC to induce DSBs, ensuring that they are not from HCTZ or sulforaphane. The skin cells were treated acutely for 24 hours and then irradiated with UVC light. The extent of DSBs was compared based on the presence of the P-ƔH2A marker. Squamous cell carcinoma (SCC), the second most common skin cancer, arises from abnormal squamous cell growth due to cumulative ultraviolet (UV) exposure, and can become invasive and metastasize if left untreated. Hydrochlorothiazide (HCTZ) is a diuretic medication that is commonly used to treat hypertension and fluid retention by inhibiting sodium chloride transport in the kidney. However, there is a potential link between HCTZ use and increased incidence of SCC, as it can increase skin cell photosensitivity and cause oxidative DNA/protein damage. Despite this, there is limited literature on this topic, and we hope to contribute to preventive strategies. We aim to investigate whether photoprotective agents, such as sulforaphane, can prevent or slow down the development of SCC in human skin cells that are being treated with HCTZ, as compared to cells without sulforaphane treatment. HCTZ's photoreactivity provides an opportunity to test the efficacy of sulforaphane as a preventive measure. Human skin cell lines were irradiated with UVC light to serve as control groups. The experimental groups included the addition of HCTZ and sulforaphane treated over 24 hours. P-ƔH2A, a cellular marker indicative of double-strand DNA breaks (DSBs), was tested via Western blot following the methodology of Bigagli et al. Data of the experimental groups with 24 hours of acute treatment of HCTZ and sulforaphane showed no visual difference in the amount of DSBs present. Acute treatment demonstrated no visual difference in the amount of DSBs. In an effort to determine the duration of HCTZ required to cause increased DNA damage compared to UVC irradiation alone, the same methodology will be repeated over 30 days. Additionally, the long-term treatment aims to investigate the photoprotective potential of sulforaphane in preventing DNA damage linked to HCTZ effects on human skin cells. Prolonged use of the same methods is beneficial, as most patients take HCTZ for years before an increase in SCC prevalence. Methods

● Figure 2 demonstrated that, as compared to the control, all intensities of UVC light lead to the presence of P-ƔH2A, indicating DSBs. We chose to move forward treating the cells with the middle 200µJ/cm 2 dosage. ● Figure 3 demonstrated the natural tendency of human skin cell DNA to produce DSBs with and without added UVC. HCTZ and sulforaphane alone do not cause DSBs; UVC light is necessary to observe DSBs. ● Figure 4 demonstrated that, despite differing treatment modalities, there was no statistically significant difference between the amount of DSBs the samples produced in the presence of 200µJ/cm 2 UVC. ● Densitometry was conducted based on the data in Figure 4 , which illustrated that the levels of DSBs increased by 15% among the HCTZ and UVC-treated cells compared to the skin cells treated with HCTZ, UVC and sulforaphane. ● We did not necessarily expect changes in a 24 hour treatment, as most patients stay on HCTZ for years before seeing an increase in SCC prevalence. ● We believe the results of the long term study of a 30-day treatment regimen will demonstrate better efficacy of sulforaphane in preventing or decreasing the prevalence of DSBs created in the context of treating human skin cells with HCTZ. Its effects will be observed over the course, modeling Bigagli et al. protocol. ● Our future goals are to understand the role of photoprotective compounds in SCC prevention as a result of HCTZ usage and understand how integrating photoprotective interventions in conjunction with proper sun protection can empower self-prevention of HCTZ-induced SCC.

Figure 2 . The human skin cells were irradiated with UVC light at varying intensities, 100µJ/cm 2 , 200µJ/cm 2 and 300 µJ/cm 2 , respectively.

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Figure 3 . P-ƔH2A, a marker of DSBs, was observed among human skin cells that were subjected to 24 hours of acute treatment of HCTZ and sulforaphane with or without 200µJ/cm 2 of UVC exposure.

References

Acknowledgements

Figure 4 . Human skin cell lines were subjected to 24 hours of acute treatment with the addition of 200µJ/cm 2 UVC to demonstrate the extent of DSBs present due to the presence of the P-ƔH2A complex.

David Eagerton, PhD, F-ABFT Bidyut Mohanty, PhD Daniel Ross, BS, NREMT Cameron Carroll, OMS-II

Figure 1. This demonstrates the suggested experimental timeline based on the Bigagli et al. procedure.

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2024 Research Recognition Day