Senescence-associated Β-galactosidase Staining Research Articles

Effects of triggers of senescence and senolysis in murine pancreatic cancer cells

BackgroundThe combination of senescence triggers with senolytic drugs is considered a promising new approach to cancer therapy. Here, we studied the efficacy of the genotoxic agent etoposide (Eto) and irradiation in inducing senescence of Panc02 pancreatic cancer cells, and the capability of the Bcl-2 inhibitor navitoclax (ABT-263; Nav) to trigger senolysis. MethodsPanc02 cells were treated with Eto or irradiated with 5–20 Gy before exposure to Nav. Cell survival, proliferation, and senescence were assessed by trypan blue staining, quantification of DNA synthesis, and staining of senescence-associated β-galactosidase (SA-β-Gal)-positive cells, respectively. Levels of mRNA were determined by real-time polymerase chain reaction, and protein expression was analyzed by immunoblotting. Panc02 cells were also grown as pancreatic tumors in mice, which were subsequently treated with Eto and Nav. ResultsEto and irradiation had an antiproliferative effect on Panc02 cells that was significantly or tendentially enhanced by Nav. In vivo, Eto and Nav together, but not Eto alone, significantly reduced the proportion of proliferating cells. The expression of the senescence marker γH2AX and tumor infiltration with T-cells were not affected by the treatment. In vitro, almost all Eto-exposed cells and a significant proportion of cells irradiated with 20 Gy were SA-β-Gal-positive. Application of Nav reduced the percentage of SA-β-Gal-positive cells after irradiation but not after pretreatment with Eto. In response to triggers of senescence, cultured Panc02 cells showed increased protein levels of γH2AX and the autophagy marker LC3B-II, and higher mRNA levels of Cdkn1a, Mdm2, and PAI-1, while the effects of Nav were variable. ConclusionsIn vitro and in vivo, the combination of senescence triggers with Nav inhibited tumor cell growth more effectively than the triggers alone. Our data also provide some evidence for senolytic effects of Nav in vitro.

Non-targeted renal metabolomics reveals multi-target effects of Dahuang Zhechong Pills on renal aging in rats

This study aims to observe the effects of the traditional Chinese medicine prescription Dahuang Zhechong Pills(DHZCP on renal aging and explore its potential multi-target effects. Rats were assigned into the normal, model, DHZCP, and vitamin E(VE)groups. Firstly, the rat model of D-galactose(D-gal)-induced renal aging was established. During the modeling period, the rats in the 4 groups were administrated with double distilled water, double distilled water, DHZCP suspension, and VE suspension, respectively,by gavage every day. On day 60 of intervention, the indicators of renal aging and injury in rats were measured, including the function,histopathological characteristics, senescence-associated β-galactosidase( SA-β-gal) staining, and expression levels of Klotho and proteins associated with cell cycle arrest and senescence-associated secretory phenotype(SASP) in the renal tissue. Moreover, nontargeted metabolomic analysis of the renal tissue was performed for the 4 groups of rats to screen out the potential biomarkers and metabolic pathways. Finally, the signaling pathways of key targets were preliminarily validated. The results showed that DHZCP and VE significantly improved the renal function, histopathological features of renal tubular/interstitial tissue, and degree of SA-β-gal staining, up-regulated the expression level of Klotho, and down-regulated the expression levels of proteins associated with cell cycle arrest and SASP in the renal tissue of the aging rats. In addition, DHZCP and VE regulated the metabolites in the renal tissue of the aging rats. There were 21 common differential metabolites. Among them, 5 differential metabolites were significantly increased in the aging rats and recovered after DHZCP or VE treatment, and they were involved in the lipid metabolism and energy metabolism pathways. The areas under the curves of the groups in comparison varied within the range of 0. 88-1. DHZCP regulated multiple signaling pathways, such as the adenosine monophosphate-activated protein kinase(AMPK), cyclic guanosine monophosphate-protein kinase G( c GMP-PKG), cyclic adenylic acid( c AMP), phosphatidylinositol-3-kinase-protein kinase B( PI3K-Akt), mammalian target of rapamycin(mTOR), and autophagy signaling pathways. In addition, it affected the multiple metabolic pathways, such as renin secretion, longevity regulation pathway, diabetic cardiomyopathy, and niacin and nicotinamide metabolism. DHZCP and VE significantly up-regulated the expression level of the key proteins in the AMPK signaling pathway in the renal tissue of the aging rats. In all, DHZCP and VE could mitigate renal aging and injury. DHZCP exerted multi-target effects via multiple signaling pathways and metabolic pathways in the kidney, in which the AMPK signaling pathway may be one of the key targets for action.

Nesfatin-1 attenuated lipopolysaccharide-induced inflammatory response and senescence in human dental pulp cells

Lipopolysaccharide (LPS)-triggered damage in human dental pulp cells (hDPCs) is associated with the progression of gingivitis, which is inflammation of the gingival tissue. Nesfatin-1 is a peptide secreted by neurons and peripheral tissues. Here, we report a novel property of Nesfatin-1 in ameliorating LPS-induced inflammatory response and senescence in hDPCs. First, we demonstrate that Nesfatin-1 repressed LPS-triggered expression of inflammatory factors. Secondly, Nesfatin-1 restored telomerase activity and the expression of human telomerase reverse transcriptase (hTERT) and telomeric repeat binding factor 2 (TERF2) against LPS. Senescence-associated β-galactosidase (SA-β-gal) staining assay revealed that Nesfatin-1 attenuated LPS-induced cellular senescence in hDPCs. We also found that Nesfatin-1 increased telomerase activity in LPS-challenged hDPCs. It is also shown that Nesfatin-1 reduced the expression of plasminogen activator inhibitor-1 (PAI-1) and p16. Additionally, LPS stimulation reduced the expression of SIRT1, which was rescued by Nesfatin-1. However, the silencing of sirtuin1 (SIRT1) abrogated the protective property of Nesfatin-1 in preventing cellular senescence, implying that the function of Nesfatin-1 is regulated by SIRT1. Taken together, our findings suggest that Nesfatin-1 might possess a protective effect against gingivitis.

The protective effects of orexin-A in alleviating cell senescence against interleukin-1β (IL-1β) in chondrocytes.

Osteoarthritis (OA) is one of the most important causes of global disability, and dysfunction of chondrocytes is an important risk factor. The treatment of OA is still a challenge. Orexin-A is a hypothalamic peptide, and its effects in OA are unknown. In this study, we found that exposure to interleukin-1β (IL-1β) reduced the expression of orexin-2R, the receptor of orexin-A in TC-28a2 chondrocytes. Importantly, the senescence-associated β-galactosidase (SA-β-gal) staining assay demonstrated that orexin-A treatment ameliorates IL-1β-induced cellular senescence. Importantly, the presence of IL-1β significantly reduced the telomerase activity of TC-28a2 chondrocytes, which was rescued by orexin-A. We also found that orexin-A prevented IL-1β-induced increase in the levels of Acetyl-p53 and the expression of p21. It is shown that orexin-A mitigates IL-1β-induced reduction of sirtuin 3 (SIRT3). Silencing of SIRT3 abolished the protective effects of orexin-A against IL-1β-induced cellular senescence. These results imply that orexin-A might serve as a promising therapeutic agent for OA.

The protective effects of Chromofungin in oligomeric amyloid β42 (Aβ42)-induced toxicity in neurons in Alzheimer's disease.

Oligomeric Aβ42 is considered to play a harmful role in the pathophysiology of Alzheimer's disease (AD). Prolonged exposure to oligomeric Aβ42 could induce neuronal damage including cellular senescence. Amelioration of Aβ42-induced cellular senescence has been considered as a promising strategy for the treatment of AD. Chromofungin, a chromogranin A-derived peptide, has displayed various biological functions in different types of cells and tissues. However, the effects of Chromofungin on oligomeric Aβ42-induced cellular senescence have not been previously reported. In the current study, we report a novel function of Chromofungin by showing that treatment with Chromofungin could ameliorate Aβ42-induced neurotoxicity in M17 neuronal cells. The Cell Counting Kit-8 (CCK-8) assay and the lactate dehydrogenase (LDH) release experiments revealed that 0.5 and 1 mM are the optimal concentrations of Chromofungin for cell culture in M17 cells. Challenging with oligomeric Aβ42 (5 μM) for 7 and 14 days led to a significant decrease in telomerase activity, which was rescued by Chromofungin dose-dependently. Additionally, the senescence-associated β-galactosidase (SA-β-gal) staining assay demonstrated that Chromofungin mitigated oligomeric Aβ42-induced cellular senescence. Correspondingly, treatment with Chromofungin reversed the gene expression of human telomerase reverse transcriptase (hTERT), telomeric repeat-binding factor 2 (TERF2), and p21 against oligomeric Aβ42 in M17 neurons. Interestingly, Chromofungin attenuated oligomeric Aβ42-induced oxidative stress (OS) in M17 cells by reducing the production of intracellular reactive oxygen species (ROS) but increasing the levels of intracellular superoxide dismutase (SOD). Importantly, the presence of Chromofungin reduced the expression of cyclooxygenase2 (COX-2) as well as the generation of prostaglandin E2 (PGE2). Transduction with Ad-COX-2 impaired the effects of Chromofungin on telomerase activity and the profile of cellular senescence. Our findings suggest that Chromofungin might act as a potential agent for the treatment of AD.

Enhancing chronic wound healing with Thai indigenous rice variety, Kaab Dum: Exploring ER stress and senescence inhibition in HaCaT keratinocyte cell line.

Kaab Dum, a prominent indigenous rice variety cultivated in the Pak Phanang Basin of Nakhon Si Thammarat, Thailand, is the focus of our study. We investigate the therapeutic potential of indigenous Kaab Dum rice extract in the context of chronic wounds. Our research encompasses an examination of the nutritional compositions and chemical profiles of Kaab Dum rice extract. Additionally, we assess how the extract affects chronic wounds in TGF-β-induced HaCaT cells. Our evaluation methods include the detection of cellular oxidative stress, the examination of endoplasmic reticulum (ER) stress, wound healing assays, analysis of cell cycle arrest and the study of cellular senescence through senescence-associated β-galactosidase (SA-β-gal) staining. Our research findings demonstrate that TGF-β induces oxidative stress in HaCaT cells, which subsequently triggers ER stress, confirmed by the expression of the PERK protein. This ER stress results in cell cycle arrest in HaCaT cells, characterized by an increase in p21 protein, a cyclin-dependent kinase inhibitor (CDKI). Ultimately, this leads to cellular senescence, as confirmed by SA-β-gal staining. Importantly, our study reveals the effectiveness of Kaab Dum rice extract in promoting wound healing in the chronic wound model. The extract reduces ER stress and senescent cells. These beneficial effects are potentially linked to the antioxidant and anti-inflammatory properties of the rice extract. The findings of our study have the potential to make significant contributions to the development of enhanced products for both the prevention and treatment of chronic wounds.

MiRNA-142-3p aggravates hydrogen peroxide-induced human umbilical vein endothelial cell premature senescence by targeting SIRT1.

Vascular endothelial cell premature senescence plays an important part in stroke. Many microRNAs (miRNAs) are known to be involved in the pathological process of vascular endothelial cell premature senescence. The present study aimed to investigate the mechanism of hydrogen peroxide (H2O2)-induced premature senescence in human umbilical vein endothelial cells (HUVECs) and effect of miR-142-3p on hydrogen peroxide (H2O2)-induced premature senescence. HUVECs were exposed to H2O2 to establish a model premature senescence in endothelial cells. CCK-8 assay was performed to detect cell viability. Senescence-associated β-galactosidase staining assay and senescence-related proteins p16 and p21 were used to detect changes in the degree of cell senescence. RT-qPCR and Western blot were conducted to measure mRNA and protein levels, respectively. The scratch wound-healing assay, transwell assay, and EdU assay were performed to evaluate the ability of migration and proliferation, respectively. miRNA-142-3p and silencing information regulator 2 related enzyme 1 (SIRT1) binding was verified using Targetscan software and a dual-luciferase assay. We found that miRNA-142-3p is abnormally up-regulated in HUVECs treated with H2O2. Functionally, miRNA-142-3p inhibition may mitigate the degree of HUVEC senescence and improve HUVEC migration and proliferation. Mechanistically, SIRT1 was validated to be targeted by miRNA-142-3p in HUVECs. Moreover, SIRT1 inhibition reversed the effects of miRNA-142-3p inhibition on senescent HUVECs exposed to H2O2. To our knowledge, this is the first study to show that miRNA-142-3p ameliorates H2O2-induced HUVECs premature senescence by targeting SIRT1 and may shed light on the role of the miR-142-3p/SIRT1 axis in stroke treatment.

A novel mechanism of major ginsenosides from Panax ginseng against multiple organ aging in middle-aged mice: Phosphatidylcholine-myo-inositol metabolism based on metabolomic analysis

Aging is a complex, degenerative process associated with various metabolic abnormalities. Ginsenosides (GS) is the main active components of Panax ginseng, which has anti-aging effects and improves metabolism. However, the anti-aging effect and the mechanism of GS in middle-aged mice has not been elucidated. In this study, GS after 3-month treatment significantly improved the grip strength, fatigue resistance, cognitive indices, and cardiac function of 15-month-old mice. Meanwhile, GS treatment reduced the fat content and obviously inhibited histone H2AX phosphorylation at Ser 139 (γ-H2AX), a marker of DNA damage in major organs, especially in the heart and liver. Further, the correlation analysis of serum metabolomics combined with aging phenotype suggested that myo-inositol (MI) upregulated by GS was positively correlated with left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), the main indicators of cardiac function. More importantly, liver tissue metabolomic analysis showed that GS increased MI content by promoting the synthesis pathway from phosphatidylcholine (PC) to MI for the inhibition of liver aging. Finally, we proved that MI reduced the percentage of senescence-associated β-galactosidase staining, γ-H2AX immunofluorescence staining, p21 expression, and the production of reactive oxygen species in H2O2-induced cardiomyocytes. These results suggest that GS can enhance multiple organ functions, especially cardiac function for promoting the healthspan of aging mice, which is mediated by the conversion of PC to MI in the liver and the increase of MI level in the serum. Our study might provide new insights into the potential mechanisms of ginsenosides for prolonging the healthspan of natural aging mice.

Senolytic effects on dental pulp stem cell's proliferation and differentiation during long-term expansion

Objective: To investigate the impact of intermittent senescent cell clearance on the proliferation and differentiation of dental pulp stem cells (DPSC) in long-term, large-scale expansion, and to explore strategies for maintaining the youthful state of DPSC in vitro. Methods: Human-derived dental pulp stem cells were isolated from healthy permanent teeth extracted for orthodontic or impeding eruption reasons, provided by the Department of Oral and Maxillofacial Surgery at West China Hospital of Stomatology, Sichuan University. Long-term, large-scale in vitro expansion of DPSC was conducted. The study compared young DPSC (passage 5) with aged DPSC (passage 25) using cellular senescence-associated β-galactosidase staining, colony formation assay, and Alizarin Red S staining for osteogenic differentiation induction. To assess the differences between the two cell populations in terms of senescence and amplification and differentiation ability. Medicine screening for the most effective senolytic was compared among 5 common senolytics [Navitoclax (ABT-263), curcumin, dasatinib, fisetin, and quercetin]. The clearance efficacy was compared using cellular senescence-associated β-galactosidase staining to reflect the changes in senescent cell ratio. The senolytic with the highest efficacy was chosen for further experiments. The passage at which the proportion of senescent cells significantly increased was identified, and the selected senolytic was administered three times at three-generation intervals from that passage to remove senescent cells. Both the control and senolytic-treated groups were estimated by fluorescence cellular senescence-associated β-galactosidase staining, real-time fluorescence quantitative PCR (RT-qPCR), colony formation assay, wound healing assay, and Alizarin Red S staining for osteogenic differentiation induction. Subcutaneous heterotopic osteogenesis was performed in nude mice and the grafts were analyzed by HE staining and alkaline phosphatase (ALP) immunohistochemical staining. Results: The proportion of senescent cells increased as the expansion extended, leading to decreased proliferation and osteogenic differentiation ability of senescent DPSC compared to young DPSC (P<0.05). Senescent DPSC exhibited altered mRNA expression levels of senescence-related genes, including p21, p16INK4a, IL-6, and Ki67 (P<0.001). Among the five senolytics, ABT-263 had the biggest decreases in the proportion of senescent cells. After intermittent ABT-263 treatment during expansion, the proportion of senescent cells in the senolytic-treated group [(6.72±2.34)%] was significantly lower than that in the control group [(31.82±0.57)%] (P<0.001). RT-qPCR confirmed that compared with the control group, mRNA expressions of p21, p16INK4a, and IL-6 in the senolytic-treated group were significantly decreased (P<0.05), while mRNA expressions of Ki67 were significantly increased (P<0.01). Furthermore, the cell healing ability and osteogenic differentiation ability of the senolytic-treated group were higher than those of the control group (P<0.05). In vivo experimental results indicated that the relative new bone area [(2.36±0.48)%] after DPSC transplantation in the senolytic-treated group was greater than that in the control group [(1.00±0.46)%] (P<0.05), and the expression of ALP was higher than that in the control group (P<0.01). Conclusions: ABT-263 can effectively eliminate senescent cells in long-term large-scale DPSC expansion. Continuous treatment with ABT-263 during cultivation can maintain the proliferation and differentiation ability of DPSC both in vivo and in vitro.

Intermittent oral supplementation with fisetin improves arterial function in doxorubicin chemotherapy-treated mice by suppressing cellular senescence

Introduction. Arterial dysfunction (endothelial dysfunction and aortic stiffening) is a key antecedent to the development of cardiovascular diseases. Cellular senescence, a state of nearly irreversible cell cycle arrest, develops in arteries following doxorubicin (Doxo) chemotherapy treatment and induces arterial dysfunction. Fisetin is a natural senolytic (senescent cell clearing compound) that improves arterial function in old mice. However, it is unknown if fisetin supplementation can decrease cellular senescence to reverse Doxo-induced arterial dysfunction. Purpose. To determine if fisetin supplementation can improve arterial function following Doxo treatment by decreasing cellular senescence. Methods &amp; Results. Cell culture. Human aortic endothelial cells were treated with fisetin for 48 hr (0.25, 0.5, 1 uM) following Doxo-induced cellular senescence (200 nm Doxo for 48 hr). 1 uM fisetin lowered senescence burden (senescence-associated β-galactosidase staining) by ~50% ( P = 0.0003). Animals. Young adult (4 mo) male/female p16-3MR mice received Doxo (10 mg/kg) or sham (saline) intraperitoneally. Four weeks later (established time-point of Doxo-induced arterial dysfunction), animals were treated daily with vehicle (V; 10% EtOH, 30% PEG400, 60% Phosal 50 PG) or fisetin (F; 100 mg/kg) via oral gavage using an intermittent 1 week on - 2 weeks off - 1 week on dosing paradigm, yielding 4 groups (Sham-V; Sham-F; Doxo-V; Doxo-F). Data from Sham-V and Sham-F groups were similar for all outcomes. Data are combined due to no sex differences. Endothelial function. Endothelial function (carotid artery endothelium dependent dilation [EDD] to acetylcholine [ACh]) and nitric oxide (NO)-mediated EDD (± L-NAME) were measured following fisetin (or vehicle) supplementation. Doxo-V mice had lower peak EDD relative to Sham-V (73 ± 2 v. 89 ± 2 %; P = 0.003), and this difference was ameliorated in Doxo-F animals (88 ± 3 %). Likewise, Doxo-V mice had lower NO-mediated EDD compared to Sham-V (39 ± 7% v. 55 ± 8%, P = 0.047), and this difference was ameliorated in Doxo-F mice (66 ± 7%). Incubation of carotid arteries ex vivo with ganciclovir (GCV) to clear p16+ senescent cells in p16-3MR mice normalized peak EDD (Sham-V, 93 ± 1; Doxo-V, 93 ± 2; Doxo-F, 91 ± 2%), suggesting that reducing cellular senescence is the mechanism by which fisetin supplementation improved endothelial function. Aortic stiffness. The Doxo-induced increase in aortic stiffness (aortic pulse wave velocity [PWV]) (Doxo-V, 426 ± 20 vs. Sham-V, 332 ± 5 cm/sec; P &lt; 0.0001) was reversed following fisetin supplementation in Doxo-F mice (pre-fisetin: 426 ± 10 v. post-fisetin: 345 ± 5 cm/sec; P = 0.001). The differences in PWV were associated with corresponding differences in ex vivo aortic intrinsic mechanical wall stiffness (Doxo-V, 5991 ± 362 v. Doxo-F, 3996 ± 415 vs. Sham-V, 4813 ± 133 kPa; P = 0.010). Ex vivo incubation of aorta rings with GCV for 48h ameliorated group differences in aortic intrinsic mechanical wall stiffness, suggesting a causal role for suppression of cellular senescence in the response. Conclusion: Intermittent fisetin supplementation may be a novel strategy to favorably modulate cellular senescence, a key cellular signaling pathway mediating arterial dysfunction with Doxo chemotherapy. R21 AG078408 &amp; K99 HL159241. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ganoderma spore lipid ameliorates docetaxel, cisplatin, and 5-fluorouracil chemotherapy-induced damage to bone marrow mesenchymal stem cells and hematopoiesis

BackgroundA triplet chemotherapy regimen of docetaxel, cisplatin, and 5-fluorouracil (TPF) is used to treat head and neck squamous cell carcinoma; however, it is toxic to bone marrow mesenchymal stem cells (BMSCs). We previously demonstrated that Ganoderma spore lipid (GSL) protect BMSCs against cyclophosphamide toxicity. In this study, we investigated the protective effects of GSL against TPF-induced BMSCs and hematopoietic damage.MethodsBMSCs and C57BL/6 mice were divided into control, TPF, co-treatment (simultaneously treated with GSL and TPF for 2 days), and pre-treatment (treated with GSL for 7 days before 2 days of TPF treatment) groups. In vitro, morphology, phenotype, proliferation, senescence, apoptosis, reactive oxygen species (ROS), and differentiation of BMSCs were evaluated. In vivo, peripheral platelets (PLTs) and white blood cells (WBCs) from mouse venous blood were quantified. Bone marrow cells were isolated for hematopoietic colony-forming examination.ResultsIn vitro, GSL significantly alleviated TPF-induced damage to BMSCs compared with the TPF group, recovering their morphology, phenotype, proliferation, and differentiation capacity (p < 0.05). Annexin V/PI and senescence-associated β-galactosidase staining showed that GSL inhibited apoptosis and delayed senescence in TPF-treated BMSCs (p < 0.05). GSL downregulated the expression of caspase-3 and reduced ROS formation (p < 0.05). In vivo, GSL restored the number of peripheral PLTs and WBCs and protected the colony-forming capacity of bone marrow cells (p < 0.05).ConclusionsGSL efficiently protected BMSCs from damage caused by TPF and recovered hematopoiesis.

Co-targeting SKP2 and KDM5B inhibits prostate cancer progression by abrogating AKT signaling with induction of senescence and apoptosis.

Prostate cancer (PCa) is the second-leading cause of cancer mortalities in the United States and is the most commonly diagnosed malignancy in men. While androgen deprivation therapy (ADT) is the first-line treatment option to initial responses, most PCa patients invariably develop castration-resistant PCa (CRPC). Therefore, novel and effective treatment strategies are needed. The goal of this study was to evaluate the anticancer effects of the combination of two small molecule inhibitors, SZL-P1-41 (SKP2 inhibitor) and PBIT (KDM5B inhibitor), on PCa suppression and to delineate the underlying molecular mechanisms. Human CRPC cell lines, C4-2B and PC3 cells, were treated with small molecular inhibitors alone or in combination, to assess effects on cell proliferation, migration, senescence, and apoptosis. SKP2 and KDM5B showed an inverse regulation at the translational level in PCa cells. Cells deficient in SKP2 showed an increase in KDM5B protein level, compared to that in cells expressing SKP2. By contrast, cells deficient in KDM5B showed an increase in SKP2 protein level, compared to that in cells with KDM5B intact. The stability of SKP2 protein was prolonged in KDM5B depleted cells as measured by cycloheximide chase assay. Cells deficient in KDM5B were more vulnerable to SKP2 inhibition, showing a twofold greater reduction in proliferation compared to cells with KDM5B intact (p < 0.05). More importantly, combined inhibition of KDM5B and SKP2 significantly decreased proliferation and migration of PCa cells as compared to untreated controls (p < 0.005). Mechanistically, combined inhibition of KDM5B and SKP2 in PCa cells abrogated AKT activation, resulting in an induction of both cellular senescence and apoptosis, which was measured via Western blot analysis and senescence-associated β-galactosidase (SA-β-Gal) staining. Combined inhibition of KDM5B and SKP2 was more effective at inhibiting proliferation and migration of CRPC cells, and this regimen would be an ideal therapeutic approach of controlling CRPC malignancy.

Pazopanib stimulates senescence of renal carcinoma cells through targeting nuclear factor E2-related factor 2 (Nrf2).

Renal cell carcinoma (RCC) is the most common kidney cancer with high mortality rate. Pazopanib has been approved for the treatment of RCC. However, the underlying mechanism is not clear. Here, we report a novel finding by showing that treatment with Pazopanib could promote cellular senescence of the human RCC cell line ACHN. Cells were stimulated with 5, 10, and 20 μM Pazopanib, respectively. Cellular senescence was measured using senescence-associated β-galactosidase (SA-β-Gal) staining. Western blot analysis and real-time polymerase chain reactionwere used to measure the mRNA and protein expression of nuclear factor E2-related factor 2 (Nrf2), γH2AX, human telomerase reverse transcriptase (hTERT), telomeric repeat binding factor 2 (TERF2), p53 and plasminogen activator inhibitor (PAI). First, we found that exposure to Pazopanib reduced the cell viability of ACHN cells. Additionally, Pazopanib induced oxidative stress by increasing the production of reactive oxygen species,reducing the levels of glutathione peroxidase,and promoting nuclear translocation of Nrf2. Interestingly, Pazopanib exposure resulted in DNA damage by increasing the expression of γH2AX. Importantly, Pazopanib increased cellular senescence and reduced telomerase activity. Pazopanib also reduced the gene expression of hTERT but increased the gene expression of TERF2. Correspondingly, we found that Pazopanib increased the expression of p53 and PAI at both the mRNA and protein levels. To elucidate the underlying mechanism, the expression of Nrf2 was knocked down by transduction with Ad- Nrf2 shRNA. Results indicate that silencing of Nrf2 in ACHN cells abolished the effects of Pazopanib in stimulating cellular senescence and reducing telomerase activity. Consistently, knockdown of Nrf2 restored the expression of p53 and PAI in ACHN cells. Based on these results, we explored a novel mechanism whereby which Pazopanib displays a cytotoxicity effect in RCC cells through promoting cellular senescence mediated by Nrf2.

Cytotoxic Activity and Senescence Modulatory Effect of Hesperetin on Triple-Negative Breast Cancer Cells and Kidney Cells Co-Treatment with Cisplatin

Cisplatin (Cisp) is a non-specific chemotherapeutic agent for breast cancer. Hesperetin (HST), a flavanone found in various citrus fruits, exhibits bioactive properties, functioning as an antioxidant, anti-inflammatory, and anticancer agent. The objective of this research was to investigate the potential of HST as a co-chemotherapeutic agent in conjunction with Cisp, specifically focusing on its cytotoxic effects against 4T1 triple-negative breast cancer cells and senescence modulatory effect on Vero normal kidney cells. The cytotoxic effect and viability cell of HST were evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. In addition, the effect of cellular senescence inhibition on the Vero cell line was measured using senescence-associated β-galactosidase (SA-β-gal) staining. In the MTT assay, both HST and cisplatin demonstrated a reduction in the viability of 4T1 cells in a dose-dependent manner, yielding IC50 values of 498 μM and 2 μM, respectively. The co-treatment of HST and cisplatin showed an increase in sensitivity of the 4T1 cells with a combination index of &lt;1. HST showed low cytotoxic activity against Vero cells, with IC50 values of over 500 μM. HST decreased cellular senescence induced by cisplatin exposure on Vero cells. These results indicated that HST in co-treatment with cisplatin decreased 4T1 cell viability synergistically. HST independently reduces the cellular senescence of normal cells. Consequently, HST holds promise for potential development as a co-treatment agent in combination with cisplatin for breast cancer cells, and it may also serve as an alternative for counteracting senescence in healthy tissues.Keywords: cytotoxic, senescence, hesperetin, cisplatin, breast cancer.

Abstract 4568: Rational targeting of BCL-xL and/or mTOR enhance the efficacy of KRASG12D inhibitor in pancreatic cancer

Abstract Background: Pancreatic cancer (PaCa) is a highly aggressive malignancy with a very low five-year survival rate secondary to late diagnosis, limited treatment options and resistance to existing therapies. While the emergence of mutant-selective KRASG12D (G12D) inhibitors brings promise for treating PaCa patients carrying these mutations, their effectiveness as single agents is expected to be limited as evident from the clinical efficacy of KRASG12C inhibitors. Experimental Design: First, multiple G12D-mutated PaCa cell lines were treated with a G12D inhibitor (MRTX1133) and subjected to immunoblot analysis for the expression of KRAS effectors and BCL-2 family proteins. Next, a proteolysis-targeting chimera (PROTAC)-based platelet-sparing BCL-xL degrader (DT2216) and/or an mTOR inhibitor (everolimus) were evaluated in combination with MRTX1133 for viability, clonogenicity and apoptosis. Subsequently, mechanistic investigations were carried out using immunoblotting, senescence-associated β-galactosidase staining and qPCR. Finally, therapeutic efficacy and safety of the combinations were evaluated in cell-derived and patient-derived xenograft models. Results: MRTX1133 treatment creates apoptotic vulnerabilities by inducing aberrations in BCL-2 family proteins, with elevated BIM levels and decreased NOXA levels, in G12D-mutated PaCa cells. Furthermore, DT2216 and everolimus were shown to release BIM and stabilize NOXA, respectively, and their combination with MRTX1133 synergistically killed G12D-mutated PaCa cells in vitro and caused significant tumor growth inhibition or regression compared to single agents in vivo. Additionally, MRTX1133 treatment led to induction of senescence in a fraction of PaCa cells. These senescent cells were found to be dependent on BCL-xL and MCL-1 for their survival and were effectively killed with a combination of DT2216 and everolimus through BCL-xL degradation and NOXA-mediated MCL-1 inhibition, respectively. Conclusions: Our findings suggest that the single agent efficacy of MRTX1133 is limited due to apoptosis inhibition and complimentary senescence induction, and therefore its combination with DT2216 and/or everolimus is synergistic, which can potentially overcome resistance, by enhanced apoptosis and clearance of senescent cells. Citation Format: Sajid Khan, Lin Cao, Vignesh Vudhata, Yang Yang, Peiyi Zhang, Guangrong Zheng, Jose G. Trevino, Daohong Zhou. Rational targeting of BCL-xL and/or mTOR enhance the efficacy of KRASG12D inhibitor in pancreatic 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 4568.

Abstract 7270: Combination targeting of SKP2 and KDM5B induces senescence in castration-resistant prostate cancer cells via AKT signaling

Abstract Prostate cancer (PCa) is the second-leading cause of cancer mortalities and morbidities in the United States and is the most commonly diagnosed malignancy in men. Despite the fact that androgen deprivation therapy is the first-line option to initial responses, most PCa patients invariably develop castration-resistant prostate cancer (CRPC). CRPC is a lethal type of malignancy, therefore, novel and effective treatment strategies are needed. The goal of this study was to evaluate the combination treatment of small molecule inhibitors, SZL-P1-41, a SKP2 target and PBIT, a KDM5B target on PCa growth and progression; as well as to delineate the underlying mechanisms of suppressing CRPC. Literature reports that S-Phase Kinase Associated Protein 2 (SKP2) is upregulated in PCa and Lysine-specific demethylase 5B (KDM5B) serves as a histone demethylase with a crucial role in cancers. Studies showed that KDM5B is increased in human PCa and KDM5B knockout decreases carcinogenic properties of PCa cells. We previously reported that SKP2 loss partially decreases the growth of prostate tumors and that KDM5B levels are reversely regulated by SKP2 in PCa cells. However, the mechanisms of KDM5B and SKP2 interplay on PCa malignancy is unknown. We hypothesize that combination of PBIT and SZL-P1-41 treatment will enhance anti-cancer effects on PCa progression by inducing cellular senescence. Here, we showed that inhibition of KDM5B and SKP2 decreased the proliferation of PCa cells, and KDM5B KO cells were more vulnerable to SKP2 inhibition. More importantly, a combined inhibition of KDM5B and SKP2 significantly blocked malignant progression and migration of PCa cells. Mechanistically, combined inhibition of KDM5B and SKP2 in PCa cells abrogated AKT activation, resulting in an induction of cellular senescence, which was measured via Western blot analysis and senescence-associated β-galactosidase (SA-β-Gal) staining. Taken together, our results show that combined inhibition of KDM5B and SKP2 is more efficacious in inhibiting proliferation and migration in CRPC cells, and this regimen would be an ideal therapeutic approach for treating CRPC. Citation Format: LaKendria K. Brown, Thanigaivelan Kanagasabai, Guoliang Li, Sherly I. Celada, Zhenbang Chen. Combination targeting of SKP2 and KDM5B induces senescence in castration-resistant prostate cancer cells via AKT signaling [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 7270.

Uric acid–driven NLRP3 inflammasome activation triggers lens epithelial cell senescence and cataract formation

Excessive uric acid (UA) is associated with age-related cataract. A previous study showed that a high UA level in the aqueous humor stimulated the senescence of lens epithelial cells (LECs), leading to cataract progression. To better understand the underlying mechanisms, we investigated UA-driven senescence in human lens tissue samples obtained during surgery, rat lens organ cultures, and in vivo experiments, using senescence-associated β-galactosidase (SA-β-gal) staining, electronic microscopy, Western blotting, and histological analyses. Initially, we identified markedly higher expressions of NLRP3 and caspase-1 in the lens capsules of hyper-uricemic patients compared to normo-uricemic patients. This increase was accompanied by a significant rise in the SA-β-gal positive rate. We next built a cataract model in which rat lenses in an organ culture system were treated with an increasing dosage of UA. Notably, opacification was apparent in the lenses treated with 800 μM of UA starting on the fifth day. Mechanistically, UA treatment not only significantly induced the expression of NLRP3, caspase-1, and IL-1β, but also upregulated the levels of SA-β-gal and the senescence regulators p53 and p21. These effects were fully reversed, and lens opacification was ameliorated by the addition of MCC950, a selective NLRP3 antagonist. Moreover, an in vivo model showed that intravitreal UA injection rapidly induced cataract phenotypes within 21 days, an effect significantly mitigated by co-injection with MCC950. Together, our findings suggest that targeting the UA-induced NLRP3 inflammasome with MCC950 could be a promising strategy for preventing cataract formation associated with inflammageing.

Phloroglucinol Enhances Anagen Signaling and Alleviates H2O2-Induced Oxidative Stress in Human Dermal Papilla Cells.

Phloroglucinol (PG) is one of the abundant isomeric benzenetriols in brown algae. Due to its polyphenolic structure, PG exhibits various biological activities. However, the impact of PG on anagen signaling and oxidative stress in human dermal papilla cells (HDPCs) is unknown. In this study, we investigated the therapeutic potential of PG for improving hair loss. A non-cytotoxic concentration of PG increased anagen-inductive genes and transcriptional activities of β-Catenin. Since several anagen-inductive genes are regulated by β-Catenin, further experiments were performed to elucidate the molecular mechanism by which PG upregulates anagen signaling. Various biochemical analyses revealed that PG upregulated β-Catenin signaling without affecting the expression of Wnt. In particular, PG elevated the phosphorylation of protein kinase B (AKT), leading to an increase in the inhibitory phosphorylation of glycogen synthase kinase 3 beta (GSK3β) at serine 9. Treatment with the selective phosphoinositide 3-kinase/AKT inhibitor, LY294002, restored the increased AKT/GSK3β/β-Catenin signaling and anagen-inductive proteins induced by PG. Moreover, conditioned medium from PG-treated HDPCs promoted the proliferation and migration of human epidermal keratinocytes via the AKT signaling pathway. Subsequently, we assessed the antioxidant activities of PG. PG ameliorated the elevated oxidative stress markers and improved the decreased anagen signaling in hydrogen peroxide (H2O2)-induced HDPCs. The senescence-associated β-galactosidase staining assay also demonstrated that the antioxidant abilities of PG effectively mitigated H2O2-induced senescence. Overall, these results indicate that PG potentially enhances anagen signaling and improves oxidative stress-induced cellular damage in HDPCs. Therefore, PG can be employed as a novel therapeutic component to ameliorate hair loss symptoms.

Early Pulmonary Fibrosis-like Changes in the Setting of Heat Exposure: DNA Damage and Cell Senescence.

It is well known that extreme heat events happen frequently due to climate change. However, studies examining the direct health impacts of increased temperature and heat waves are lacking. Previous reports revealed that heatstroke induced acute lung injury and pulmonary dysfunction. This study aimed to investigate whether heat exposure induced lung fibrosis and to explore the underlying mechanisms. Male C57BL/6 mice were exposed to an ambient temperature of 39.5 ± 0.5 °C until their core temperature reached the maximum or heat exhaustion state. Lung fibrosis was observed in the lungs of heat-exposed mice, with extensive collagen deposition and the elevated expression of fibrosis molecules, including transforming growth factor-β1 (TGF-β1) and Fibronectin (Fn1) (p < 0.05). Moreover, epithelial-mesenchymal transition (EMT) occurred in response to heat exposure, evidenced by E-cadherin, an epithelial marker, which was downregulated, whereas markers of EMT, such as connective tissue growth factor (CTGF) and the zinc finger transcriptional repressor protein Slug, were upregulated in the heat-exposed lung tissues of mice (p < 0.05). Subsequently, cell senescence examination revealed that the levels of both senescence-associated β-galactosidase (SA-β-gal) staining and the cell cycle protein kinase inhibitor p21 were significantly elevated (p < 0.05). Mechanistically, the cGAS-STING signaling pathway evoked by DNA damage was activated in response to heat exposure (p < 0.05). In summary, we reported a new finding that heat exposure contributed to the development of early pulmonary fibrosis-like changes through the DNA damage-activated cGAS-STING pathway followed by cellular senescence.

FTO Stabilizes MIS12 to Inhibit Vascular Smooth Muscle Cell Senescence in Atherosclerotic Plaque.

Atherosclerosis is the main cause of atherosclerotic cardiovascular disease (CVD). Here, we aimed to uncover the role and mechanisms of fat mass and obesity-associated genes (FTO) in the regulation of vascular smooth muscle cell (VSMC) senescence in atherosclerotic plaques. ApoE-/- mice fed a high-fat diet (HFD) were used to establish an atherosclerotic animal model. Immunohistochemistry, and the staining of hematoxylin-eosin, Oil Red O, Sirius red, and Masson were performed to confirm the role of FTO in atherosclerosis in vivo. Subsequently, FTO expression in primary VSMCs is either upregulated or downregulated. Oxidized low-density lipoprotein (ox-LDL) was used to treat VSMCs, followed by EdU staining, flow cytometry, senescence-associated β-galactosidase (SA-β-gal) staining, immunofluorescence, telomere detection, RT-qPCR, and Western blotting to determine the molecular mechanisms by which FTO inhibits VSMC senescence. Decreased FTO expression was observed in progressive atherosclerotic plaques of ApoE-/- mice fed with HFD. FTO upregulation inhibits atherosclerotic lesions in mice. FTO inhibits VSMC aging in atherosclerotic plaques by helping VSMC withstand ox-LDL-induced cell cycle arrest and senescence. This process is achieved by stabilizing the MIS12 protein in VSMC through a proteasome-mediated pathway. FTO inhibits VSMC senescence and subsequently slows the progression of atherosclerotic plaques by stabilizing the MIS12 protein.