Photoaging mainly occurs due to ultraviolet (UV) radiation, and is accompanied by increased secretion of matrix metalloproteinases (MMPs) and degradation of collagen. UV radiation induces cell senescence in the skin; however, the role of senescent cells in photoaging remains unclear. Therefore, to elucidate the role of senescent cells in photoaging, we evaluated the effect of senolytics in a photoaging mouse model and investigated the underlying mechanism of their antiaging effect. Both UV-induced senescent human dermal fibroblasts and a photoaging mouse model, ABT-263 and ABT-737, demonstrated senolytic effects on senescent fibroblasts. Moreover, we found that several senescence-associated secretory phenotype factors, such as IL-6, CCL5, CCL7, CXCL12, and SCF, induced MMP-1 expression in dermal fibroblasts, which decreased after treatment with ABT-263 and ABT-737 in vivo and in vitro. Both senolytic drugs attenuated the induction of MMPs and decreased collagen density in the photoaging mouse model. Our data suggest that senolytic agents reduce UV-induced photoaging, making strategies for targeting senescent dermal fibroblasts promising options for the treatment of photoaging.
Calreticulin (CRT) is a highly conserved and abundant multifunctional protein that is encoded by a small gene family and is often associated with abiotic/biotic stress responses in plants. However, the roles played by this protein in salt stress responses in wheat (Triticum aestivum) remain obscure. In this study, three TaCRT genes were identified in wheat and named TaCRT1, TaCRT2 and TaCRT3-1 based on their sequence characteristics and their high homology to other known CRT genes. Quantitative real-time PCR expression data revealed that these three genes exhibit different expression patterns in different tissues and are strongly induced under salt stress in wheat. The calcium-binding properties of the purified recombinant TaCRT1 protein were determined using a PIPES/Arsenazo III analysis. TaCRT1 gene overexpression in Nicotiana tabacum decreased salt stress damage in transgenic tobacco plants. Physiological measurements indicated that transgenic tobacco plants showed higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than non-transgenic tobacco under normal growth conditions. Interestingly, overexpression of the entire TaCRT1 gene or of partial TaCRT1 segments resulted in significantly higher tolerance to salt stress in transgenic plants compared with their WT counterparts, thus revealing the essential role of the C-domain of TaCRT1 in countering salt stress in plants.
Histone deacetylases (HDACs) remove acetyl groups from lysine chains on histones and other proteins and play a crucial role in epigenetic regulation and aging. Previously, we demonstrated that HDAC4 is consistently downregulated in aged and ultraviolet (UV)-irradiated human skin in vivo. Cellular senescence is a permanent cell cycle arrest induced by various stressors. To elucidate the potential role of HDAC4 in the regulation of cellular senescence and skin aging, we established oxidative stress- and UV-induced cellular senescence models using primary human dermal fibroblasts (HDFs). RNA sequencing after overexpression or knockdown of HDAC4 in primary HDFs identified candidate molecular targets of HDAC4. Integrative analyses of our current and public mRNA expression profiles identified DNA damage-inducible transcript 4 (DDIT4) as a critical senescence-associated factor regulated by HDAC4. Indeed, DDIT4 and HDAC4 expressions were downregulated during oxidative stress- and UV-induced senescence. HDAC4 overexpression rescued the senescence-induced decrease in DDIT4 and senescence phenotype, which were prevented by DDIT4 knockdown. In addition, DDIT4 overexpression reversed changes in senescence-associated secretory phenotypes and aging-related genes, suggesting that DDIT4 mediates the reversal of cellular senescence via HDAC4. Collectively, our results identify DDIT4 as a promising target regulated by HDAC4 associated with cellular senescence and epigenetic skin aging.
Abstract Recently, our proteomics study on breast cancer (BC) reported a possible association of RHBDF1 with triple-negative breast cancer (TNBC). The overall survival of TNBC patients on METABRIC data set implicated that the RHBDF1 high-expression group has a shorter life expectancy than the RHBDF1 low-expression group. Although many TNBC patients have high expression levels of RHBDF1 and poor prognosis, the mechanism of RHBDF1 in the pathogenesis of TNBC remained unclear. Rhomboid 5 homolog 1 (RHBDF1) is a catalytically inactive pseudoprotease on the Golgi and endoplasmic reticulum membrane. Despite its inactive status of peptidase, it regulates the activity of ADAM17 protease that cleaves the extracellular portion of EGFR ligands such as TGF alpha and EGF. Eventually, RHBDF1 and ADAM17 complex activate the EGFR signaling pathway, thereby increasing cell proliferation, migration, and tumorigenesis. First of all, we recruited the clinical data of the overall survival (OS) and disease-free survival (DFS) in the hospital of Seoul National University (SNUH cohort, n= 203) and compared IHC score 0 or 1 with survival rate. To investigate the underlying biological mechanism of RHBDF1 in TNBC, the effect of knockdown or overexpression of RHBDF1 was observed in TNBC cell lines (MDA-MB-468, HCC1143 and BT20) using siRNA (small interfering RNA) and lentiviral systems. Cell proliferation assay was performed to clarify whether RHBDF1 affects cell growth. Then, we estimated the ability of migration, invasion, and sphere formation to confirm the characteristics of cancer cells including cancer stemness and metastasis. SNUH cohort with TNBC patients showed that the RHBDF1 high-expression group has a poorer disease-free survival (DFS) than the low-expression group (p<0.05). In vitro assay, RHBDF1 knockdown inhibited proliferation in MDA-MB-468 and HCC1143. RHBDF1 downregulation also decreased the ability of migration and invasion. We found that RHBDF1 activates specific cell growth signaling pathways and increases cell mobility in TNBC. The sphere formation assay was performed to measure cancer stemness characteristics. As a result, RHBDF1 knockdown TNBC cell lines couldn’t form spheres relative to RHBDF1 overexpressed TNBC cell lines. In conclusion, we demonstrated that RHBDF1, a potential triple-negative breast cancer biomarker, is highly amplified in TNBC patients and TNBC cell lines. Its functions of accelerating cell proliferation, metastasis and cancer stemness were unveiled. But we need to study ultimately how RHBDF1 regulates ADAM17 activity at Golgi and endoplasmic reticulum membrane and which transcription factor activates RHBDF1 expression. Citation Format: Hayeon Kim, Soo Young Park, Da Sol Kim, Cheng Hyun Lee, Min Ji Song, Han Suk Ryu. RHBDF1 is a potential modulator of EGFR activation and oncogenic biomarker in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6990.
This is a book about a crisis that creates conditions under which further crises become inevitable.Such malevolent inertia has come to define the contemporary moment in even wider vistas than are explored in this study.In times like these our care networks become ever more crucial, so I am particularly grateful to all those that provided guidance, interlocution, and camaraderie as
Carnosol is a phenolic antioxidant present in rosemary (Rosmarinus officinalis).It is known for anti-inflammatory effects, analgesic activity and anti-cancer effects.However, no study has been dedicated yet to its effect on atopic dermatitis (AD).Here, we show that carnosol effectively inhibited LPS-induced nitric oxide (NO) generation and expression of inflammatory marker proteins (iNOS and COX-2) in RAW 264.7 cells.In addition, carnosol effectively inhibits the phosphorylation of STAT3 and DNA binding activity in RAW 264.7 cells.Pull down assay and docking model analysis showed that carnosol directly binds to the DNA binding domain (DBD) of STAT3.We next examined the anti-atopic activity of carnosol (0.05 μg/cm 2 ) using 5% Phthalic anhydride (PA)induced AD model in HR1 mice.Carnosol treatment significantly reduced 5% PA-induced AD like skin inflammation in skin tissues compared with control mice.Moreover, carnosol treatment inhibits the expression of iNOS and COX-2 in skin tissue.In addition, the levels of TNF-a, IL-1β, and Immunoglobulin-E in blood serum was significantly decreased in carnosol treated mice compared with those of 5% PA treated group.Furthermore, the activation of STAT3 in skin tissue was decreased in carnosol treated mice compared with control mice.In conclusion, these findings suggest that carnosol exhibited a potential anti-AD activity by inhibiting pro-inflammatory mediators through suppression of STAT3 activation via direct binding to DBD of STAT3.
Abstract The HER2 diagnostics is necessary for selection of patients harboring HER2 gene amplification or protein overexpression who will benefit from anti-HER2 therapies in breast cancer. However, HER2 testing is still challenging due to the subjective natures of immunohistochemistry (IHC) and in situ hybridization (ISH), standard methods for determining HER2 status. Thus, a new method is needed to accurately quantify HER2 levels. Here, we developed a clinically reliable HER2 testing method enabling ultra-fast detection of HER2 gene amplification with high accuracy by using the digital real-time PCR (drPCR) system, a potential new diagnostic platform with improved performance by integrating both real-time and digital PCR technologies. For drPCR-based HER2 copy number (CN) measurement, primer-probe sets specific to HER2 gene and a genomic region adjacent to chromosome 17 centromere (CEP17) were designed, and the optimal drPCR condition was determined in clinical breast tumor specimens. To test the clinical validity and standardize procedures of drPCR-based HER2 status evaluation, three independent breast cancer cohorts from different institutions were enrolled, which assigned as a training (SCHU hospital, n = 103) and two validation sets (SNU hospital, n = 170; CNUH hospital, n = 45), and the drPCR assay was compared with current standard HER2 testing methods. In the training cohort, the HER2/CEP17 ratio values from FISH and drPCR tests were highly correlated (r2 = 0.81; P < 0.001), and the drPCR results displayed 98.1% concordance to HER2 status defined by IHC and/or FISH with 92.6% sensitivity and 100% specificity. Eight samples further verified by targeted NGS showed 100% concordance of dPCR to NGS. Consistently, two validation cohorts also showed high concordance of drPCR to IHC and/or ISH results (accuracy = 97.1% and 97.8% in SNU and CNUH cohorts, respectively). The optimal cutoff for HER2 positivity in the drPCR assay was set as a HER2/CEP17 ratio ≥ 1.9 with AUC of 0.963 based on the results from training cohort, and the same cut-off for drPCR was applicable to two independent validation cohorts, supporting the clinical validity of our drPCR-based HER2 assessment. In some discordant cases, low tumor purity (≤ 25%) was observed and microdissection partly improved the drPCR results. The discordance between drPCR and ISH results was also found in marginal HER2+ cases with HER2/CEP17 ratio 2-3, but these cases showed inter-observer variability when re-evaluating the ISH/IHC data due to intratumoral HER2 heterogeneity. Of note, in HER2 IHC3+ cases with negative drPCR results, re-evaluation of IHC using an artificial intelligence (AI)-based HER2 scoring system revised the HER2 IHC 3+ score to 2+, and ISH assessment also confirmed that these cases are indeed HER2-negative, proving the high accuracy of HER2 CN drPCR assay. In conclusion, given the advantages of drPCR-based HER2 assessment with high accuracy, sensitivity, and simplicity, the drPCR assay could be a complementary or alternative method to IHC and ISH to greatly improve current HER2 testing. Citation Format: Jin hyuk Chang, YoonSik Kim, Hee-Joo Choi, Soo Young Park, Ji-Hye Park, Hee-Young Won, Min Ji Song, Da Sol Kim, Hayeon Kim, Sohyeon Yang, Nam Hun Heo, Minsik Song, Seung-Shick Shin, Do Young Lee, Han Suk Ryu, Si-Hyong Jang, Jeong-Yeon Lee. Ultra-rapid and precise measurement of HER2 copy number alteration by next-generation digital PCR capable of real-time analysis in patients with breast cancer: A multicenter retrospective study [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A005.
Skin ageing is caused by numerous factors that result in structural and functional changes in cutaneous components. Research has shown that senescent cells are known to accumulate in skin ageing, however, the role of senescent cells in skin ageing has not been defined.To elucidate the role of the senescent cell in skin ageing, we evaluated the effect of known senolytic drugs on senescent dermal fibroblasts.Primary human dermal fibroblasts (HDFs) were induced to senescence by long-term passaging, UV irradiation, and H2 O2 treatment. Cell viability was measured after treatment of ABT-263 and ABT-737 on HDFs. Young and aged hairless mice were intradermally injected with drugs or vehicle on the dorsal skin for 10 days. Skin specimens were obtained and reverse-transcription quantitative PCR, western blotting, and histological analysis were performed.We found that ABT-263 and ABT-737 induced selective clearance of senescent dermal fibroblasts, regardless of the method of senescence induction. Aged mouse skin treated with ABT-263 or ABT-737 showed increased collagen density, epidermal thickness, and proliferation of keratinocytes, as well as decreased senescence-associated secretory phenotypes, such as MMP-1 and IL-6.Taken together, our results indicate that selective clearance of senescent skin cells can attenuate and improve skin ageing phenotypes and that senolytic drugs may be of potential use as new therapeutic agents for treating ageing of the skin.
Abstract Aging is accompanied by impaired mitochondrial function and accumulation of senescent cells. Mitochondrial dysfunction contributes to senescence by increasing the levels of reactive oxygen species and compromising energy metabolism. Senescent cells secrete a senescence‐associated secretory phenotype (SASP) and stimulate chronic low‐grade inflammation, ultimately inducing inflammaging. Mitochondrial dysfunction and cellular senescence are two closely related hallmarks of aging; however, the key driver genes that link mitochondrial dysfunction and cellular senescence remain unclear. Here, we aimed to elucidate a novel role of carnitine acetyltransferase (CRAT) in the development of mitochondrial dysfunction and cellular senescence in dermal fibroblasts. Transcriptomic analysis of skin tissues from young and aged participants showed significantly decreased CRAT expression in intrinsically aged skin. CRAT downregulation in human dermal fibroblasts recapitulated mitochondrial changes in senescent cells and induced SASP secretion. Specifically, CRAT knockdown caused mitochondrial dysfunction, as indicated by increased oxidative stress, disruption of mitochondrial morphology, and a metabolic shift from oxidative phosphorylation to glycolysis. Mitochondrial damage induced the release of mitochondrial DNA into the cytosol, which activated the cyclic GMP‐AMP synthase (cGAS)‐stimulator of interferon genes (STING) and NF‐ĸB pathways to induce SASPs. Consistently, fibroblast‐specific CRAT‐knockout mice showed increased skin aging phenotypes in vivo, including decreased cell proliferation, increased SASP expression, increased inflammation, and decreased collagen density. Our results suggest that CRAT deficiency contributes to aging by mediating mitochondrial dysfunction‐induced senescence.
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