KDM1A inhibition increases UVA toxicity and enhances photodynamic therapy efficacy.

BACKGROUND: Lysine-specific histone demethylase 1 (KDM1A/LSD1) regulates multiple cellular functions, including cellular proliferation, differentiation, and DNA repair. KDM1A is overexpressed in squamous cell carcinoma of the skin and inhibition of KDM1A can suppress cutaneous carcinogenesis. Despite the role of KDM1A in skin and DNA repair, the effect of KDM1A inhibition on cellular ultraviolet (UV) response has not been studied. METHODS: The ability of KDM1A inhibitor bizine to modify cell death after UVA and UVB exposure was tested in normal human keratinocytes and melanocytes, HaCaT, and FaDu cell lines. KDM1A was also downregulated using shRNA and inhibited by phenelzine in HaCaT and FaDu cells to confirm the role of KDM1A in UVA response. In addition, cellular reactive oxygen species (ROS) changes were assessed by a lipid-soluble fluorescent indicator of lipid oxidation, and ROS-related gene regulation using qPCR. During photodynamic therapy (PDT) studies HaCaT and FaDu cells were treated with aminolaevulinic acid (5-ALA) or HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a) sodium and irradiated with 0-8 J/cm2 red LED light. RESULTS: KDM1A inhibition sensitized cells to UVA radiation-induced cell death but not to UVB. KDM1A inhibition increased ROS generation as detected by increased lipid peroxidation and the upregulation of ROS-responsive genes. The effectiveness of both ALA and HPPH PDT significantly improved in vitro in HaCaT and FaDu cells after KDM1A inhibition. CONCLUSION: KDM1A is a regulator of cellular UV response and KDM1A inhibition can improve PDT efficacy.

Draft Genome Sequences of Lacticaseibacillus rhamnosus cek-R1, Lacticaseibacillus paracasei cek-R2, and Lentilactobacillus otakiensis cek-R3, Isolated from a Beetroot Product.

Lactic acid bacteria (LAB) participate in fermentation processes and have probiotic potential. The genomes of three LAB strains, Lacticaseibacillus rhamnosus cek-R1, Lacticaseibacillus paracasei subsp. paracasei cek-R2, and Lentilactobacillus otakiensis cek-R3, isolated from a beetroot product, were characterized. The results contribute to our understanding of the beneficial properties of LAB.

Inhibitors of Nucleotide Excision Repair Decrease UVB-Induced Mutagenesis-An In Vitro Study.

The high incidence of skin cancers in the Caucasian population is primarily due to the accumulation of DNA damage in epidermal cells induced by chronic ultraviolet B (UVB) exposure. UVB-induced DNA photolesions, including cyclobutane-pyrimidine dimers (CPDs), promote mutations in skin cancer driver genes. In humans, CPDs are repaired by nucleotide excision repair (NER). Several commonly used and investigational medications negatively influence NER in experimental systems. Despite these molecules' ability to decrease NER activity in vitro, the role of these drugs in enhancing skin cancer risk is unclear. In this study, we investigated four molecules (veliparib, resveratrol, spironolactone, and arsenic trioxide) with well-known NER-inhibitory potential in vitro, using UVB-irradiated CHO epithelial and HaCaT immortalized keratinocyte cell lines. Relative CPD levels, hypoxanthine phosphoribosyltransferase gene mutation frequency, cell viability, cell cycle progression, and protein expression were assessed. All four molecules significantly elevated CPD levels in the genome 24 h after UVB irradiation. However, veliparib, spironolactone, and arsenic trioxide reduced the mutagenic potential of UVB, while resveratrol did not alter UVB-induced mutation formation. UVB-induced apoptosis was enhanced by spironolactone and arsenic-trioxide treatment, while veliparib caused significantly prolonged cell cycle arrest and increased autophagy. Spironolactone also enhanced the phosphorylation level of mammalian target of rapamycin (mTOR), while arsenic trioxide modified UVB-driven mitochondrial fission. Resveratrol induced only mild changes in the cellular UVB response. Our results show that chemically inhibited NER does not result in increased mutagenic effects. Furthermore, the UVB-induced mutagenic potential can be paradoxically mitigated by NER-inhibitor molecules. We identified molecular changes in the cellular UVB response after NER-inhibitor treatment, which may compensate for the mitigated DNA repair. Our findings show that metabolic cellular response pathways are essential to consider in evaluating the skin cancer risk-modifying effects of pharmacological compounds.

Cyclobutane pyrimidine dimers from UVB exposure induce a hypermetabolic state in keratinocytes via mitochondrial oxidative stress.

Ultraviolet B radiation (UVB) is an environmental complete carcinogen, which induces and promotes keratinocyte carcinomas, the most common human malignancies. UVB induces the formation of cyclobutane pyrimidine dimers (CPDs). Repairing CPDs through nucleotide excision repair is slow and error-prone in placental mammals. In addition to the mutagenic and malignancy-inducing effects, UVB also elicits poorly understood complex metabolic changes in keratinocytes, possibly through CPDs. To determine the effects of CPDs, CPD-photolyase was overexpressed in keratinocytes using an N1-methyl pseudouridine-containing in vitro-transcribed mRNA. CPD-photolyase, which is normally not present in placental mammals, can efficiently and rapidly repair CPDs to block signaling pathways elicited by CPDs. Keratinocytes surviving UVB irradiation turn hypermetabolic. We show that CPD-evoked mitochondrial reactive oxygen species production, followed by the activation of several energy sensor enzymes, including sirtuins, AMPK, mTORC1, mTORC2, p53, and ATM, is responsible for the compensatory metabolic adaptations in keratinocytes surviving UVB irradiation. Compensatory metabolic changes consist of enhanced glycolytic flux, Szent-Györgyi-Krebs cycle, and terminal oxidation. Furthermore, mitochondrial fusion, mitochondrial biogenesis, and lipophagy characterize compensatory hypermetabolism in UVB-exposed keratinocytes. These properties not only support the survival of keratinocytes, but also contribute to UVB-induced differentiation of keratinocytes. Our results indicate that CPD-dependent signaling acutely maintains skin integrity by supporting cellular energy metabolism.

PARP1 Inhibition Augments UVB-Mediated Mitochondrial Changes-Implications for UV-Induced DNA Repair and Photocarcinogenesis.

Keratinocytes provide the first line of defense of the human body against carcinogenic ultraviolet (UV) radiation. Acute and chronic UVB-mediated cellular responses were widely studied. However, little is known about the role of mitochondrial regulation in UVB-induced DNA damage. Here, we show that poly (ADP-ribose) polymerase 1 (PARP1) and ataxia-telangiectasia-mutated (ATM) kinase, two tumor suppressors, are important regulators in mitochondrial alterations induced by UVB. Our study demonstrates that PARP inhibition by ABT-888 upon UVB treatment exacerbated cyclobutane pyrimidine dimers (CPD) accumulation, cell cycle block and cell death and reduced cell proliferation in premalignant skin keratinocytes. Furthermore, in human keratinocytes UVB enhanced oxidative phosphorylation (OXPHOS) and autophagy which were further induced upon PARP inhibition. Immunoblot analysis showed that these cellular responses to PARP inhibition upon UVB irradiation strongly alter the phosphorylation level of ATM, adenosine monophosphate-activated kinase (AMPK), p53, protein kinase B (AKT), and mammalian target of rapamycin (mTOR) proteins. Furthermore, chemical inhibition of ATM led to significant reduction in AMPK, p53, AKT, and mTOR activation suggesting the central role of ATM in the UVB-mediated mitochondrial changes. Our results suggest a possible link between UVB-induced DNA damage and metabolic adaptations of mitochondria and reveal the OXPHOS-regulating role of autophagy which is dependent on key metabolic and DNA damage regulators downstream of PARP1 and ATM.

Silymarin: Friend or Foe of UV Exposed Keratinocytes?

The application of natural plant extracts in UV-protection is popular and intensively studied. Silymarin (from Silibum marianum), a naturally occurring polyphenol, has recently received attention due to its antioxidant, anti-inflammatory and anti-apoptotic effects. However, its role in the UV-mediated keratinocyte cell response is still controversial. In this study, we investigated the effects of Silibum marianum extracts with different origins and formulations on UVA-exposed HaCaT keratinocytes in vitro. Our results show, that silymarin treatment caused an inverse dose-dependent photosensitivity relationship (at higher doses, a decrease in cell viability and ROS production) after UVA exposure. The attenuation of the UVA-induced ROS generation after silymarin treatment was also observed. Moreover, silymarin pre-treatment increased the cyclobutane pyrimidine dimer photolesions in keratinocytes after UVA exposure. These results indicated the dual role of silymarin in UVA-exposed keratinocytes. It scavenges ROS but still induces phototoxicity. Based on our results dermatological applications of silymarin and related compounds should be considered very carefully.

5-Aminolevulinic acid photodynamic therapy with and without Er:YAG laser for actinic keratosis: Changes in immune infiltration.

INTRODUCTION: Ultraviolet light induced DNA damage, combined with immunosuppression and inflammation are involved in the pathogenesis of actinic keratosis. Photodynamic therapy not only destroys dysplastic cells via tissue destruction and vascular shutdown, but also induces an acute local inflammatory response and activates both the innate and adaptive immune system. In our current work we aimed to compare immunohistochemistry features of inflammatory infiltrate of actinic keratoses after 5-aminolevulinic acid photodynamic therapy with or without Er:YAG laser resurfacing. METHODS: Eleven patients with multiple actinic keratosis on the scalp, face, hands or forearms were treated by conventional and Er:YAG laser assisted 5-aminolevulinic acid PDT in split-site manner. Biopsies of AKs were taken before, 48 h and 3 months after the treatment. CD3, CD4, CD8, CD1a, Ki67 and p53 expressions were analyzed by immunohistochemical methods. RESULTS: The number of p53 and Ki67 positive cells decreased significantly 3 months after treatment, but the abnormal cells were not eliminated totally. The number of CD1a+ Langerhans cells significantly decreased 48 h after both treatments, while CD8+ T cell count was significantly lower 3 months after Er:YAG laser assisted photodynamic therapy. However, the number of CD3+ and CD4+ T cells were not changed significantly 48 h and 3 months later. CONCLUSIONS: One session of 5-aminolevulinic acid photodynamic therapy even with Er:YAG laser pretreatment could not terminate actinic damage totally. Photodynamic therapy induced immunological changes. However further investigations are needed to answer how the composition of actinic keratosis' immune infiltrate influence the effect of photodynamic therapy.

Immunological effects of photodynamic therapy in the treatment of actinic keratosis and squamous cell carcinoma.

The use of photodynamic therapy is extensive, due to its antitumoral, antibacterial and photorejuvenation effects. It destroys tumor via direct cell destruction and indirectly via vascular shutdown, induction of acute local inflammatory response and activation of the immune system. Both innate and adaptive immune cells are involved in the immunological effects of photodynamic therapy. In addition to UV-induced DNA damage, inflammation and immunosuppression are also essential elements in the pathogenesis of actinic keratosis. Both immunosuppression induced by UV and defective immune response to dysplastic keratinocytes may be the target of photodynamic therapy to eliminate actinic keratosis. These elements are discussed in the present review, highlighting the possible mechanism of photodynamic therapy to effectively treat actinic keratosis.

Photodynamic therapy does not induce cyclobutane pyrimidine dimers in the presence of melanin.

Photodynamic therapy (PDT) is an office-based treatment for precancerous and early cancerous skin changes. PDT induces cell death through the production of reactive oxygen species (ROS). Cyclobutane pyrimidine dimers (CPDs) are the most important DNA changes responsible for ultraviolet (UV) carcinogenesis. Recently ROS induced by UVA were shown to generate CPDs via activating melanin. This raised the possibility that PDT induced ROS may also induce CPDs and mutagenesis in melanin containing cells. Previously the effect of PDT on CPDs in melanin containing cells has not been assessed. Our current work aimed to compare the generation of CPDs in melanin containing cells subjected to UVA treatment and porfimer sodium red light PDT. We used ELISA to detect CPDs. After UVA we found a dose dependent increase in CPDs in melanoma cells (B16-F10, MNT-1) with CPD levels peaking hours after discontinuation of UVA treatment. This indicated the generation of UVA induced dark-CPDs in the model. Nevertheless, PDT in biologically relevant doses was unable to induce CPDs. Our work provides evidence for the lack of CPD generation by PDT in melanin containing cells.

Mycoplasma infection followed by time-lapse microscopy.

Early detection of mycoplasma infection is crucial for saving precious often irreplaceable data from the tissues of patients. Mycoplasma infections cause diseases in the upper and lower respiratory tracts, urethritis in men resulting in painful dysuria, urgency and urethral discharge. Cough, fever, headache, urethritis may persist for several weeks and convalescence is slow. The symptoms of these diseases are aggravated by the detection of mycoplasma infections, that takes either a long time, besides being expensive or is specific and restricted to only a limited number of contaminant strains. Mycoplasmas are hard to detect visually but could be seen and followed by time-lapse microscopy. Our hypothesis is that one can detect mycoplasma infection irrespective of its origin and type of mycoplasma. Main lines of supporting evidence are provided by the time-lapse microscopy showing dynamic morphological alterations caused by mycoplasmas before changes in human cell cultures become visible. Morphometric measurements of mycoplasma infections revealed four subphases: i) detachment of infected cells, ii) aggregation, iii) biofilm formation and iv) shrinkage of infected cells. The applicability of time-lapse microscopy for the detection of mycoplasma infection was validated by a mycoplasma test Kit. Most important implications related to morphometric parameters include the observation of mycoplasma infected cultures for an extended period of time instead of applying static snap-shot microscopy. A reliable method is offered to estimate the time of mycoplasma exposure that elapsed during the cell growth. This microphotometric approach served a more economical detection of mycoplasma contamination at its early stage of cell growth and spread, irrespective of the origin of contaminated serum, without defining the type of mycoplasma.

Chemically Induced Cell Cycle Arrest in Perfusion Cell Culture.

In contrast to most present methods, continuous imaging of live cells would require full automation in each processing step. As an integrated system that would meet all requirements does not exist, we have established a long-term scanning-perfusion platform that: (a) replaces old medium with fresh one, (b) bypasses physical contact with the cell culture during continuous cell growth, (c) provides uninterrupted photomicrography of single cells, and (d) secures near physiological conditions and sterility up to several weeks. The system was validated by synchronizing cells using serum starvation and butyrate-induced cell cycle arrest of HaCaT cells.