Cell Cycle Inhibitor P21 Research Articles

Encapsulation of soybean lunasin and amaranth unsaponifiable matter in liposomes induces cell cycle arrest in an allograft melanoma mouse model.

Melanoma is the most aggressive type of skin cancer and can metastasize during primary tumor formation. This research aimed to determine the relationship between the prevention of melanoma development in a mouse model treated with liposomes loaded with soybean lunasin and amaranth unsaponifiable matter (UM + LunLip) and cell cycle arrest. Tumors excised from C57BL/6 mice treated topically or subcutaneously with UM + LunLip were subjected to immunohistochemistry. Markers related to cell cycle inhibition (p16, p21, p27, and p53) and markers involved in cell cycle progression (cyclin-dependent kinase, CDK6, and cyclin D1) were assessed. The results showed that UM + LunLip had antitumor activity in C57BL/6 mice treated either topically or subcutaneously by p16, p21, p27, and p53 overexpression (up to 572-, 134-, 30-, and 57-fold change, FC, respectively) in the tumors of mice treated with 30mg UM + LunLip/kg body weight compared with the tumor-bearing untreated control. However, CDK6 and cyclin D1 expression was not inhibited (up to 1.37 FC and 2.09 FC, respectively), which is a typical behavior of cyclin D in melanoma. Therefore, melanoma tumor development was prevented by the overexpression of cell cycle inhibitors p16, p21, p27, and p53 due to UM + LunLip treatments. Since the topical application was effective, less invasive, and more practical for the user, this application will be recommended for future steps in in vivo studies.

P16Ink4a-induced senescence in cultured mast cells as a model for ageing reveals significant morphological and functional changes.

Mast cells (MCs) are tissue resident cells of the immune system, mainly known for their role in allergy. However, mounting evidence indicates their involvement in the pathology of age-related diseases, such as Alzheimer's disease, Parkinson's disease, and cancer. MC numbers increase in aged tissues, but how ageing affects MCs is poorly understood. We show that MC ageing is associated with the increased expression of the cell cycle inhibitor p16 Ink4a, a marker and inducer of cellular senescence. Relying on this observation and the tight association of ageing with senescence, we developed a model of inducible senescence based on doxycycline-induced expression of p16Ink4a in cultured bone marrow derived MCs (BMMCs). Using this model, we show that senescent MCs upregulate IL-1β, TNF-α and VEGF-A. We also demonstrate that senescence causes marked morphological changes that impact MC function. Senescent MCs are larger, contain a larger number of secretory granules (SGs) and have less membrane protrusions. Particularly striking are the changes in their SGs, reflected in a significant reduction in the number of electron dense SGs with a concomitant increase in lucent SGs containing intraluminal vesicles. The changes in SG morphology are accompanied by changes in MC degranulation, including a significant increase in receptor-triggered release of CD63-positive extracellular vesicles (EVs) and the exteriorisation of proteoglycans, as opposed to a gradual inhibition of the release of β-hexosaminidase. The inducible expression of p16Ink4a imposes MC senescence, providing a model for tracking the autonomous changes that occur in MCs during ageing. These changes include both morphological and functional alterations. In particular, the increased release of small EVs by senescent MCs suggests an enhanced ability to modulate neighbouring cells.

P21Waf1/Cip1 Is a Novel Downstream Target of 40S Ribosomal S6 Kinase 2

Background/Objectives: The ribosomal S6 kinase 2 (S6K2) acts downstream of the mechanistic target of rapamycin complex 1 and is a homolog of S6K1 but little is known about its downstream effectors. The objective of this study was to use an unbiased transcriptome profiling to uncover how S6K2 promotes breast cancer cell survival. Methods: RNA-Seq analysis was performed to identify novel S6K2 targets. Cells were transfected with siRNAs or plasmids containing genes of interest. Western blot analyses were performed to quantify total and phosphorylated proteins. Apoptosis was monitored by treating cells with different concentrations of doxorubicin. Results: Silencing of S6K2, but not S6K1, decreased p21 in MCF-7 and T47D breast cancer cells. Knockdown of Akt1 but not Akt2 decreased p21 in MCF-7 cells whereas both Akt1 and Akt2 knockdown attenuated p21 in T47D cells. While Akt1 overexpression enhanced p21 and partially reversed the effect of S6K2 deficiency on p21 downregulation in MCF-7 cells, it had little effect in T47D cells. S6K2 knockdown increased JUN mRNA and knockdown of cJun enhanced p21. Low concentrations of doxorubicin increased, and high concentrations decreased p21 levels in T47D cells. Silencing of S6K2 or p21 sensitized T47D cells to doxorubicin via c-Jun N-terminal kinase (JNK)-mediated downregulation of Mcl-1. Conclusions: S6K2 knockdown enhanced doxorubicin-induced apoptosis by downregulating the cell cycle inhibitor p21 and the anti-apoptotic protein Mcl-1 via Akt and/or JNK.

Depside and depsidone-rich hydroalcoholic extract, resourced from the lichen Parmelinella wallichiana (Taylor) Elix & Hale selectively restricts Non-Small Cell Lung Cancer by modulating p53, FOXO1 and PALLADIN genes

The non-specificity of contemporary cancer therapeutics has enticed us to develop safer, anticancer alternatives from natural resources. Lichens are unique natural entities which have long been neglected for explorations in cancer therapy, despite their vast potential. Our present study aims to investigate the anti-cancer potential of a wild lichen Parmelinella wallichiana. The anti-proliferative efficacy of the lichen extracts were screened through MTT assay against a panel of cell lines and the potent hydroalcoholic extract was selected for further evaluation against the most sensitive lung-cancer cell line A549 by implementing a wide range of microscopic and flow cytometric applications. The observations suggest that the extract could selectively induce apoptosis by augmenting ROS and disrupting the mitochondrial membrane potentiality. It was also found that the lichen-induced apoptosis was regulated by two crucial tumor suppressor genes, FOXO1, and p53, along with cell cycle inhibitor p21 which ultimately resulted in robust apoptosis through the up-regulation of pro-apoptotic BAX expression. Moreover, the extract also restricted the cancer progression by down-regulating the PALLADIN expression. Further, an LC-MS-based metabolomic profile highlighted a number of depsides, depsidones and dibenzofurans, which included atranorin, physodalic acid, salazinic acid, constictic acid and usnic acid. Then, an in silico docking with these lichen-derived metabolites against the PI3Kα receptor predicted these compounds has a binding affinity close to a standard PI3Kα inhibitor copanlisib. The study concludes that the extract restricts lung cancer possibly through the PI3Kα/FOXO1 axis and thus Parmelinella wallichiana represents a potential resource for anti-lung cancer drug development in future.

Anti-Cancer Potential of Isoflavone-Enriched Fraction from Traditional Thai Fermented Soybean against Hela Cervical Cancer Cells.

Cervical cancer is a leading cause of gynecological malignancies and cancer-related deaths among women worldwide. This study investigates the anti-cancer activity of Thua Nao, a Thai fermented soybean, against HeLa cervical carcinoma cells, and explores its underlying mechanisms. Our findings reveal that the ethyl acetate fraction of Thua Nao (TN-EA) exhibits strong anti-cancer potential against HeLa cells. High-performance liquid chromatography (HPLC) analysis identified genistein and daidzein as the major isoflavones in TN-EA responsible for its anti-cancer activity. TN-EA and genistein reduced cell proliferation and induced G2/M phase arrest, while daidzein induced G1 arrest. These responses were associated with the downregulation of cell cycle regulators, including Cyclin B1, cycle 25C (Cdc25C), and phosphorylated cyclin-dependent kinase 1 (CDK-1), and the upregulation of the cell cycle inhibitor p21. Moreover, TN-EA and its active isoflavones promoted apoptosis in HeLa cells through the intrinsic pathway, evidenced by increased levels of cleaved Poly (ADP-ribose) polymerase (PARP) and caspase-3, loss of mitochondrial membrane potential, and the downregulation of anti-apoptotic proteins B-cell leukemia/lymphoma 2 (Bcl-2), B-cell lymphoma-extra-large (Bcl-xL), cellular inhibitor of apoptosis proteins 1 (cIAP), and survivin. Additionally, TN-EA and its active isoflavones effectively reduced cell invasion and migration by downregulating extracellular matrix degradation enzymes, including Membrane type 1-matrix metalloproteinase (MT1-MMP), urokinase-type plasminogen activator (uPA), and urokinase-type plasminogen activator receptor (uPAR), and reduced the levels of the mesenchymal marker N-cadherin. At the molecular level, TN-EA suppressed STAT3 activation via the regulation of JNK and Erk1/2 signaling pathways, leading to reduced proliferation and invasion of HeLa cells.

Cynara cardunculus L. inhibits adipogenic differentiation of 3T3-L1 cells via activation of AMPK signaling pathway

Promotion of brown adipose tissue phenotype, able to transform energy into heat, is a promising target against obesity development. We investigated the effects of a standardized extract from Cynara cardunculus L. (CCLE) leaves, considered a waste food resource, on adipogenesis inhibition in 3T3-L1 cells, through the thermogenic process involvement. CCLE inhibited adipogenesis (C/EBPβ, PPAR-γ, FABP4, SREBP-1, and FASN) and increased thermogenesis via upregulation of AMPK phosphorylation. Furthermore, CCLE induced FOXO1 pathway as adipogenesis repressive mechanism affecting PPAR-γ activity and induced the expression of cell cycle inhibitor p21. However, pretreatment with the AMPK inhibitor compound C abolished CCLE effects on FOXO1, p21, C/EBPβ, and PPAR-γ. These data confirmed that the antiadipogenic effect of the extract is related to AMPK activation. Therefore, this work provides new perspectives for the use of this extract in obesity prevention and management. Additionally, reuse/recycling of waste food resources provides added value from a circular economy perspective.

Deoxynivalenol induces m6A-mediated upregulation of p21 and growth arrest of mouse hippocampal neuron cells in vitro

Hippocampal neurons maintain the ability of proliferation throughout life to support neurogenesis. Deoxynivalenol (DON) is a mycotoxin that exhibits brain toxicity, yet whether and how DON affects hippocampal neurogenesis remains unknown. Here, we use mouse hippocampal neuron cells (HT-22) as a model to illustrate the effects of DON on neuron proliferation and to explore underlying mechanisms. DON exposure significantly inhibits the proliferation of HT-22 cells, which is associated with an up-regulation of cell cycle inhibitor p21 at both mRNA and protein levels. Global and site-specific m6A methylation levels on the 3’UTR of p21 mRNA are significantly increased in response to DON treatment, whereas inhibition of m6A hypermethylation significantly alleviates DON-induced cell cycle arrest. Further mechanistic studies indicate that the m6A readers YTHDF1 and IGF2BP1 are responsible for m6A-mediated increase in p21 mRNA stability. Meanwhile, 3’UTR of E3 ubiquitin ligase TRIM21 mRNA is also m6A hypermethylated, and another m6A reader YTHDF2 binds to the m6A sites, leading to decreased TRIM21 mRNA stability. Consequently, TRIM21 suppression impairs ubiquitin-mediated p21 protein degradation. Taken together, m6A-mediated upregulation of p21, at both post-transcriptional and post-translational levels, contributes to DON-induced inhibition of hippocampal neuron proliferation. These results may provide new insights for epigenetic therapy of neurodegenerative diseases.Graphical abstractDON inhibits the proliferation of HT-22 cells.RNA m6A hypermethylation on the transcript of p21 enhances the mRNA stability in a YTHDF1- and IGF2BP1-dependent manner, which leads to the upregulation of p21.RNA m6A hypermethylation on the transcript of TRIM21 decreases the mRNA stability in a YTHDF2-dependent manner, which contributes to prevent p21 ubiquitin-mediated degradation.High expression of p21 contributes to inhibit cell proliferation.

Anticancer Activity of Encapsulated Pearl Millet Polyphenol-Rich Extract against Proliferating and Non-Proliferating Breast Cancer Cells In Vitro.

Plant-derived polyphenols are bioactive compounds with potential health-promoting properties including antioxidant, anti-inflammatory, and anticancer activity. However, their beneficial effects and biomedical applications may be limited due to their low bioavailability. In the present study, we have considered a microencapsulation-based drug delivery system to investigate the anticancer effects of polyphenol-rich (apigenin, caffeic acid, and luteolin) fractions, extracted from a cereal crop pearl millet (Pennisetum glaucum), using three phenotypically different cellular models of breast cancer in vitro, namely triple negative HCC1806, ER-positive HCC1428, and HER2-positive AU565 cells. Encapsulated polyphenolic extract induced apoptotic cell death in breast cancer cells with different receptor status, whereas it was ineffective against non-tumorigenic MCF10F cells. Encapsulated polyphenolic extract was also found to be cytotoxic against drug-resistant doxorubicin-induced senescent breast cancer cells that were accompanied by increased levels of apoptotic and necrotic markers, cell cycle inhibitor p21 and proinflammatory cytokine IL8. Furthermore, diverse responses to the stimulation with encapsulated polyphenolic extract in senescent breast cancer cells were observed, as in the encapsulated polyphenolic extract-treated non-proliferating AU565 cells, the autophagic pathway, here cytotoxic autophagy, was also induced, as judged by elevated levels of beclin-1 and LC3b. We show for the first time the anti-breast cancer activity of encapsulated polyphenolic extract of pearl millet and postulate that microencapsulation may be a useful approach for potentiating the anticancer effects of phytochemicals with limited bioavailability.

Diphthamide deficiency promotes association of eEF2 with p53 to induce p21 expression and neural crest defects

Diphthamide is a modified histidine residue unique for eukaryotic translation elongation factor 2 (eEF2), a key ribosomal protein. Loss of this evolutionarily conserved modification causes developmental defects through unknown mechanisms. In a patient with compound heterozygous mutations in Diphthamide Biosynthesis 1 (DPH1) and impaired eEF2 diphthamide modification, we observe multiple defects in neural crest (NC)-derived tissues. Knockin mice harboring the patient’s mutations and Xenopus embryos with Dph1 depleted also display NC defects, which can be attributed to reduced proliferation in the neuroepithelium. DPH1 depletion facilitates dissociation of eEF2 from ribosomes and association with p53 to promote transcription of the cell cycle inhibitor p21, resulting in inhibited proliferation. Knockout of one p21 allele rescues the NC phenotypes in the knockin mice carrying the patient’s mutations. These findings uncover an unexpected role for eEF2 as a transcriptional coactivator for p53 to induce p21 expression and NC defects, which is regulated by diphthamide modification.

Endothelial Cell Flow-Mediated Quiescence Is Temporally Regulated and Utilizes the Cell Cycle Inhibitor p27.

Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time-to-cell cycle reentry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA-seq (single-cell RNA sequencing) analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous flow-exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence misregulation that leads to vascular dysfunction and disease.

Carbon-Coated Iron Oxide Nanoparticles Promote Reductive Stress-Mediated Cytotoxic Autophagy in Drug-Induced Senescent Breast Cancer Cells.

The surface modification of magnetite nanoparticles (Fe3O4 NPs) is a promising approach to obtaining biocompatible and multifunctional nanoplatforms with numerous applications in biomedicine, for example, to fight cancer. However, little is known about the effects of Fe3O4 NP-associated reductive stress against cancer cells, especially against chemotherapy-induced drug-resistant senescent cancer cells. In the present study, Fe3O4 NPs in situ coated by dextran (Fe3O4@Dex) and glucosamine-based amorphous carbon coating (Fe3O4@aC) with potent reductive activity were characterized and tested against drug-induced senescent breast cancer cells (Hs 578T, BT-20, MDA-MB-468, and MDA-MB-175-VII cells). Fe3O4@aC caused a decrease in reactive oxygen species (ROS) production and an increase in the levels of antioxidant proteins FOXO3a, SOD1, and GPX4 that was accompanied by elevated levels of cell cycle inhibitors (p21, p27, and p57), proinflammatory (NFκB, IL-6, and IL-8) and autophagic (BECN1, LC3B) markers, nucleolar stress, and subsequent apoptotic cell death in etoposide-stimulated senescent breast cancer cells. Fe3O4@aC also promoted reductive stress-mediated cytotoxicity in nonsenescent breast cancer cells. We postulate that Fe3O4 NPs, in addition to their well-established hyperthermia and oxidative stress-mediated anticancer effects, can also be considered, if modified using amorphous carbon coating with reductive activity, as stimulators of reductive stress and cytotoxic effects in both senescent and nonsenescent breast cancer cells with different gene mutation statuses.

Embryonic NIPP1 Depletion in Keratinocytes Triggers a Cell Cycle Arrest and Premature Senescence in Adult Mice

NIPP1 is a ubiquitously expressed regulatory subunit of protein phosphatase-1. Its embryonic deletion in keratinocytes causes chronic sterile skin inflammation, epidermal hyperproliferation and resistance to mutagens in adult mice. To explore the primary effects of NIPP1 deletion, we first examined hair-cycle progression of NIPP1 skin knockouts (SKOs). The entry of the 1st hair cycle in the SKOs was delayed due to prolonged quiescence of hair-follicle stem cells (HFSCs). In contrast, the entry of the 2nd hair cycle in the SKOs was advanced as a result of precocious activation of HFSCs. The epidermis of SKOs progressively accumulated senescent cells and this cell-fate switch was accelerated by DNA damage. Primary keratinocytes from SKO neonates and human NIPP1-depleted HaCaT keratinocytes failed to proliferate and showed an increase in the expression of cell-cycle inhibitors (p21, p16/Ink4a and/or p19/Arf) and senescence-associated-secretory-phenotype (SASP) factors as well as in DNA damage (γH2AX and 53bp1). Our data demonstrate that the primary effect of NIPP1 deletion in keratinocytes is a cell-cycle arrest and premature senescence that gradually progresses to chronic senescence and likely contributes to the decreased sensitivity of SKOs to mutagens.

Calotropis procera extract inhibits prostate cancer through regulation of autophagy.

Current treatment options available for prostate cancer (PCa) patients have many adverse side effects and hence, new alternative therapies need to be explored. Anticancer potential of various phytochemicals derived from Calotropis procera has been studied in many cancers but no study has investigated the effect of leaf extract of C. procera on PCa cells. Hence, we investigated the effect of C. procera leaf extract (CPE) on cellular properties of androgen-independent PC-3 and androgen-sensitive 22Rv1 cells. A hydroalcoholic extract of C. procera was prepared and MTT assay was performed to study the effect of CPE on viability of PCa cells. The effect of CPE on cell division ability, migration capability and reactive oxygen species (ROS) production was studied using colony formation assay, wound-healing assay and 2',7'-dichlorodihydrofluorescein diacetate assay, respectively. Caspase activity assay and LDH assay were performed to study the involvement of apoptosis and necrosis in CPE-mediated cell death. Protein levels of cell cycle, antioxidant, autophagy and apoptosis markers were measured by western blot. The composition of CPE was identified using untargeted LC-MS analysis. Results showed that CPE decreased the viability of both the PCa cells, PC-3 and 22Rv1, in a dose- and time-dependent manner. Also, CPE significantly inhibited the colony-forming ability, migration and endogenous ROS production in both the cell lines. Furthermore, CPE significantly decreased NF-κB protein levels and increased the protein levels of the cell cycle inhibitor p27. A significant increase in expression of autophagy markers was observed in CPE-treated PC-3 cells while autophagy markers were downregulated in 22Rv1 cells after CPE exposure. Hence, it can be concluded that CPE inhibits PCa cell viability possibly by regulating the autophagy pathway and/or altering the ROS levels. Thus, CPE can be explored as a possible alternative therapeutic agent for PCa.

FNC (4'-azido-2'-deoxy-2'-fluoro(arbino)cytidine) as an Effective Therapeutic Agent for NHL: ROS Generation, Cell Cycle Arrest, and Mitochondrial-Mediated Apoptosis.

Cytotoxic nucleoside analogs (NAs) hold great promise in cancer therapeutics by mimicking endogenous nucleosides and interfering with crucial cellular processes. Here, we investigate the potential of the novel cytidine analog, 4'-azido-2'-deoxy-2'-fluoro(arbino)cytidine (FNC), as a therapeutic agent for Non-Hodgkin lymphoma (NHL) using Dalton's lymphoma (DL) as a T-cell lymphoma model. FNC demonstrated dose- and time-dependent inhibition of DL cell growth and proliferation. IC-50 values of FNC were measured at 1 µM, 0.5 µM, and 0.1 µM after 24, 48, and 72 h, respectively. Further elucidation of FNC's mechanism of action uncovers its role in inducing apoptosis in DL cells. Notable DNA fragmentation and nuclear condensation point to activated apoptotic pathways. FNC-induced apoptosis was concomitant with changes in cellular membranes, characterized by membrane rupture and altered morphology. The robust anticancer effects of FNC are linked to its capacity to induce reactive oxygen species (ROS) production, prompting oxidative stress-mediated apoptosis. Additionally, FNC disrupted mitochondrial membrane potential (MMP), leading to mitochondrial dysfunction, further promoting apoptosis. Dysregulation of apoptotic genes, with upregulation of Bax and downregulation of Bcl-2 and Bcl-xl, implicates the mitochondrial-mediated apoptosis pathway. Furthermore, FNC-induced G2/M phase cell cycle arrest was mediated through modulation of the cell cycle inhibitor p21. Overall, this study highlights the potential of FNC as a promising therapeutic agent for NHL.

P27 Cell Cycle Inhibitor and Survival in Luminal-Type Breast Cancer: Gene Ontology, Machine Learning, and Drug Screening Analysis.

A widely distributed cell cycle inhibitor, p27, regulates cyclin-dependent kinase-cyclin complexes. Although the prognostic value of p27 has been established for various types of carcinomas, its role in luminal breast cancer remains poorly understood. This study aimed to explore the functional enrichment of p27 and identify potential drug targets in patients with luminal-type breast cancer. Clinicopathological data were collected from 868 patients with luminal-type breast cancer. Additionally, publicly available data from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset (1,500 patients) and the Gene Expression Omnibus database (855 patients) were included in the analysis. Immunohistochemical staining for p27, differential gene expression analysis, disease ontology analysis, survival prediction modeling using machine learning (ML), and in vitro drug screening were also performed. Low p27 expression correlated with younger age, advanced tumor stage, estrogen receptor/progesterone receptor negativity, decreased cluster of differentiation 8+ T cell count, and poorer survival outcomes in luminal-type breast cancer. The METABRIC data revealed that reduced cyclin-dependent kinase inhibitor 1B (CDKN1B) expression (encoding p27) was associated with cell proliferation-related pathways and epigenetic polycomb repressive complex 2. Using ML, p27 emerged as the second most significant survival factor after N stage, thereby enhancing survival model performance. Additionally, luminal-type breast cancer cell lines with low CDKN1B expression demonstrated increased sensitivity to specific anticancer drugs such as voxtalisib and serdemetan, implying a potential therapeutic synergy between CDKN1B-targeted approaches and these drugs. The integration of ML and bioinformatic analyses of p27 has the potential to enhance risk stratification and facilitate personalized treatment strategies for patients with breast cancer.

A Live Tracker of Dormant Disseminated Tumor Cells.

The high prevalence of dormant disseminated tumor cells (DTCs) persisting systemically in patients with metastatic cancer is a major threat to long-lasting cure (Aguirre-Ghiso, Nat Rev Cancer 7:834-846, 2007; Klein, Nat Rev Cancer 20(11):681-694, 2020; Lyden et al. Cancer Cell 40:787-791, 2022). Despite its clinical significance, the study of what drives DTCs in and out of dormancy while they linger in distant sites has been challenged by the lack of tools to find and follow dormant DTCs inside a living organism. Here, leveraging the fact that dormant DTCs are mostly quiescent, we describe a live cell reporter to distinguish dormant from cycling DTCs (Correia, Nat Rev Cancer 22(7):379, 2022; Correia et al. Nature 594(7864):566-571, 2021). Cancer cell lines are engineered to coexpress a luciferase-tdTomato reporter and a fluorescent fusion protein of mVenus with a mutant form of the cell cycle inhibitor p27 (mVenus-p27K-) that identifies quiescent cells. When implanted in animal models or assembled in cocultures in vitro, labeled cells can be imaged longitudinally over time or retrieved alive alongside their surrounding microenvironment for downstream gene, protein, and metabolite profiling, allowing the mapping of tissue-specific determinants of cancer dormancy and metastasis.

Dual-Specificity Phosphatase 6 Deficiency Attenuates Arterial-Injury-Induced Intimal Hyperplasia in Mice.

In response to injury, vascular smooth muscle cells (VSMCs) of the arterial wall dedifferentiate into a proliferative and migratory phenotype, leading to intimal hyperplasia. The ERK1/2 pathway participates in cellular proliferation and migration, while dual-specificity phosphatase 6 (DUSP6, also named MKP3) can dephosphorylate activated ERK1/2. We showed that DUSP6 was expressed in low baseline levels in normal arteries; however, arterial injury significantly increased DUSP6 levels in the vessel wall. Compared with wild-type mice, Dusp6-deficient mice had smaller neointima. In vitro, IL-1β induced DUSP6 expression and increased VSMC proliferation and migration. Lack of DUSP6 reduced IL-1β-induced VSMC proliferation and migration. DUSP6 deficiency did not affect IL-1β-stimulated ERK1/2 activation. Instead, ERK1/2 inhibitor U0126 prevented DUSP6 induction by IL-1β, indicating that ERK1/2 functions upstream of DUSP6 to regulate DUSP6 expression in VSMCs rather than downstream as a DUSP6 substrate. IL-1β decreased the levels of cell cycle inhibitor p27 and cell-cell adhesion molecule N-cadherin in VSMCs, whereas lack of DUSP6 maintained their high levels, revealing novel functions of DUSP6 in regulating these two molecules. Taken together, our results indicate that lack of DUSP6 attenuated neointima formation following arterial injury by reducing VSMC proliferation and migration, which were likely mediated via maintaining p27 and N-cadherin levels.

Periportal hepatocyte proliferation at midgestation governs maternal glucose homeostasis in mice

The maternal liver is challenged by metabolic demands throughout pregnancy. However, hepatocyte dynamics and their physiological significance in pregnancy remain unclear. Here, we show in mice that hepatocyte proliferation is spatiotemporally regulated in each liver lobular zone during pregnancy, with transient proliferation of periportal and pericentral hepatocytes during mid and late gestation, respectively. Using adeno-associated virus (AAV)−8-mediated expression of the cell cycle inhibitor p21 in hepatocytes, we show that inhibition of hepatocyte proliferation during mid, but not late, gestation impairs liver growth. Transcriptionally, genes involved in glucose/glycogen metabolism are downregulated in late pregnancy when midgestational hepatocyte proliferation is attenuated. In addition, hepatic glycogen storage is abolished, with concomitant elevated blood glucose concentrations, glucose intolerance, placental glycogen deposition, and fetal overgrowth. Laser capture microdissection and RNA-seq analysis of each liver lobular zone show zone-specific changes in the transcriptome during pregnancy and identify genes that are periportally expressed at midgestation, including the hyaluronan-mediated motility receptor (Hmmr). Knockdown of Hmmr in hepatocytes by AAV8-shHmmr suppresses periportal hepatocyte proliferation at midgestation and induces impaired hepatic glycogen storage, glucose intolerance, placental glycogen deposition and fetal overgrowth. Our results suggest that periportal hepatocyte proliferation during midgestation is critical for maternal glycogen metabolism and fetal size.

Asiaticoside Prevents Oxidative Stress and Apoptosis in Endothelial Cells by Activating ROS-dependent p53/Bcl-2/Caspase-3 Signaling Pathway.

Asiaticoside (AC) is a triterpenoid saponin found in Centella asiatica (L.) urban extract that has a wide range of pharmacological properties. Our previous study demonstrated that AC could promote angiogenesis in diabetic wounds, but the specific mechanisms remain unknown. This study aimed to examine the effectiveness and mechanism of AC on human umbilical vein endothelial cells (HUVECs) exposed to tert-butyl hydroperoxide (t-BHP) toxicity. Senescence was confirmed using senescence-associated betagalactosidase (SA-β-gal) activity and expression of the cell cycle phase markers p16 and p21. The levels of SOD, NO, MDA, GSH-Px, and ROS were tested. Furthermore, several cell death-related genes and proteins (p53, Bax, Bcl-2 and Caspase-3) were assessed with RT-qPCR and Western blotting. AC significantly reduced SA-β-gal activity, with both the suppression of cellcycle inhibitors p16 and p21. We also found that the induced oxidative stress and apoptosis caused by t-BHP treatment resulted in the decrease of antioxidant enzymes activities, the surge of ROS and MDA, the up-regulation of p53, Bax and caspase-3, and the decrease of SOD, NO, GSH-Px and Bcl-2. These biochemical changes were all reversed by treatment with varying doses of AC. AC alleviates t-BHP-induced oxidative injury and apoptosis in HUVECs through the ROS-dependent p53/Bcl-2/Caspase-3 signaling pathway. It may be a potential antioxidant applied in metabolic disorders and pharmaceutical products.

Skp2 Inhibition Diminishes Essential Cell Survival Markers c-Maf and c-Myc, Enhancing Therapeutic Efficacy Against Drug Resistant Multiple Myeloma

Multiple Myeloma (MM) presents challenges as a hematologic malignancy, with drug resistance leading to limited curative options and disease relapse despite advancements in therapy. Understanding the molecular mechanisms driving MM's development and progression is vital for identifying new treatment approaches. Clinical and experimental evidence supports the role of S-phase kinase-associated protein 2 (Skp2) in driving the progression of several cancers, including MM. We interrogated a public microarray dataset derived from clinical specimens of MM patients and observed a significantly higher expression of Skp2 within the relapsed patient cohort (N=28) compared to the newly diagnosed group (N=63) (P=0.0041). Additionally, we observed elevated Skp2 levels and decreased P27 Kip1 levels in drug-resistant MM cell lines (MM.1R and RPMI 8226.R5) compared to their parental cell lines (MM.1S and RPMI 8226). Hence, a comprehensive understanding of Skp2's molecular functions in drug-resistant MM becomes paramount for facilitating the development of precision-targeted interventions. We investigated Skp2's molecular interactions in MM by performing a targeted knockdown with siRNA in drug-resistant and sensitive cell lines (OPM1, RPMI.8226, MM.1S, and MM.1R). Immunoblot analysis showed reduced STAT3 activation and decreased levels of cell survival markers c-Maf and c-Myc. Treatment with 10uM of the small molecule inhibitor SkpinC1 also inhibited STAT3 activation and led to significant reductions in c-Maf and c-Myc protein levels compared to the vehicle-treated groups. These findings hold immense significance as c-Maf and c-Myc are intricately associated with MM progression and poorer outcomes, although there is limited information on their regulation by the Skp2/P27 axis. Functionally, treatment of the MM cell lines with increasing dosages of SkpinC1 showed a progressive decrease in MM cell line survival, with IC50 values spanning 5-15uM, signifying the broad applicability of the inhibitor among the tested cell lines. Moreover, flow cytometry-based analysis of the surviving cell population demonstrated that SkpinC1 based inhibition of the Skp2/P27 axis considerably decreased the percentage of proliferating M-phase cells, while elevating the proportion of quiescent and apoptotic cells relative to the control group. At the molecular level, targeted inhibition of the Skp2/P27 axis by SkpinC1 caused the accumulation of cell cycle inhibitors P27 Kip1, P21 (Cip1/Waf1), and P57 Kip2, activation of the tumor suppressive TP53 protein with a simultaneous decrease in phospho-MDM2 levels in addition to an increase in the levels of apoptotic markers cleaved PARP and Caspase 3. From a clinical perspective, we also probed the applicability of Skp2 inhibition using SkpinC1 as a potential strategy for managing therapeutic resistance in MM. We treated multiple MM cell lines (OPM1, RPMI.8226, MM.1S and MM.1R) using a combination of Lenalidomide (an immune modulator) or Bortezomib (a proteasomal inhibitor) with SkpinC1 and compared it with single agent treatments. Both Lenalidomide/SkpinC1 as well as Bortezomib/SkpinC1 showed a strong dose dependent decrease in the percentage of surviving cells relative to the single agent treatments, thereby highlighting the remarkable potential of targeting the Skp2/P27 axis for clinical therapy in MM. To gain deeper insights into the molecular roles of Skp2/P27 axis inhibition by SkpinC1, we subjected drug-resistant MM.1R cells to 10uM SkpinC1 treatment for 24 hours, followed by single-cell transcriptome sequencing. This approach illuminated considerable heterogeneity between the drug-treated and control cells, accompanied by global changes in various signaling pathways associated with cell survival. In conclusion, the inhibition of Skp2 in MM cells exerts a significant impact on crucial cell survival pathways, notably c-Maf and c-Myc, thereby promoting apoptosis and offering a promising therapeutic avenue to combat drug resistance in MM. The precise targeting of Skp2 using small molecule inhibitors, such as SkpinC1, demonstrates substantial therapeutic potential, enhancing the efficacy of combinatorial treatment approaches for more effective interventions in MM.