Figure 1. Cell senescence signaling pathway
Cell senescence overview
In 1961, American biologist Leonard Hayflick found that in normal cultured fibroblasts, even if the cells were properly grown, the cells would fail when they split to a certain number of algebras, thus making the cell cycle into an irreversible stagnation state (cell senescence). This is the first time that the cell's longevity and proliferative capacity are limited, namely the Hayflick boundary. Cellular senescence means that the normal physiological function and proliferative capacity of cells will gradually decline over time and face the stress of the outside world, thus deviating from the cell cycle. This process is important for tumor, tissue regeneration and body aging. Cellular senescence mainly includes two types of stress-induced senescence caused by telomere shortening and sensation of external stimuli (such as DNA damage, changes in chromatin structure, and overexpression of oncogenes like Ras and Raf). Telomere shortening, or telomere structure disruption is the main cause of cellular senescence. When the cells are senescent, the changes in cell structure mainly include volume enlargement, nuclear sag, nuclear membrane disintegration, chromatin structure changes and mitochondrial decrease (Figure 2); functional degeneration changes are mainly caused by loss of cell replication ability, cells cyclic stagnation, decreased stressor sensitivity, down-regulation of cell cycle gene expression, up-regulation of cell cycle inhibitors and other senescence-related genes. Usually, the body's aging cells and new cells continue to be replaced, thus maintaining normal life activities, and the overall level of aging is generally considered to be the basis of the body's aging. Therefore, studying cell senescence has laid an important foundation for clarifying the body's aging.
Figure 2. Structure changes of cell senescence
Cell senescence family
Many members involved in the family of cellular senescence signaling pathways, including the P53 family, CDKS, pRB, E2F and other signaling pathway families. Here are some major family members: SA-bGa: SA- bGa is one of the most widely used markers of cellular senescence and can be distinguished from cells in quiescent and terminally differentiated states. When the cell is in senescence, its protein expression profile usually changes greatly. The b-galactosidase encoded by GLB1 (galactosidase beta 1) gene is mainly expressed in lysosomes in normal human cells. The suitable pH is 4.0~4.5, while in the case of aging, it expresses high enzyme activity at pH6.0. The substrate of the enzyme mainly includes ganglioside, keratin sulfate and various glycoproteins. When detecting senescent cells, the substrate X-gal and pH 6.0 can be artificially administered. The senescent cells can convert the substrate into a dark blue product under the action of b-galactosidase, which can be detected by ordinary optical microscopy. P16Ink4a: P16Ink4a protein, also known as cyclin-dependent kinase inhibitor 2A (CDKN2A), encoded by the Ink4a locus, has a molecular weight of approximately 16 kDa, and is encoded by three exons. Amino acid residue composition. This protein deletion or mutation is present in about 75% of cancer cell lines and plays a key role in down-regulating CDK4 and CDK6 during cell senescence, and thus can be used as one of the markers of cell senescence. In addition to encoding P16Ink4a, the Ink4a gene locus also encodes a P19Arf with a different upstream promoter (P14Arf is a human homolog), which acts as an upstream gene of P53 and is also involved in the regulation of cellular senescence signaling pathways. P21Cip1: also known as cyclin-dependent kinase inhibitor 1A(CDKN1A), is located on human chromosome 6p21.2 with a molecular weight of 21 kDa and consists of three exons. The encoded 164 amino acid residues are composed. As an important member of the cyclin-dependent kinase inhibitor family downstream of the p53 gene, it can negatively regulate the cell cycle.
Cell senescence signaling pathway
The cell cycle of eukaryotic cells can be divided into four periods: G1, S, G2 and M. There are two main regulatory points, G1-S and G2-M, and the regulation of G1-S is more important. When DNA damage occurs, the cell cycle promoter gene is down-regulated, and the cell cycle inhibitory gene is up-regulated, and cells are induced to block and lead in the G1 phase through different signaling pathways. Cellular senescence provides sufficient time for DNA damage repair to maintain the stability of the cell's genome. Cellular senescence requires signal transduction, and the two most important signaling pathways are the P16Ink4a/Rb (retinoblastoma protein) pathway and the P19Arf/P53/P21Cip1 pathway, which interact but independently regulate the process of the cells cycle. The Ink4 (the inhibitors of cyclindependent kinase 4) belongs to the family of P16Ink4a/Rb pathway and is a CDK (cyclindependent kinases) inhibitor that accumulates intracellularly as the number of cell divisions increases. Usually independent of the P19Arf/P53/P21Cip1 pathway, the P16Ink4a/Rb pathway is thought to be the primary pathway leading to senescence in cells. Cellular senescence caused by stress, especially epithelial cells, is mainly through the P16Ink4a/Rb pathway, while telomere-damaged senescent cells are specific, and function mainly in the mouse through the P19Arf/P53/P21Cip1 pathway. Human cells are co-regulated by two pathways. When cells are stressed, they can cause high expression of P16 protein, but the specific mechanism of this process remains to be elucidated, possibly by reducing the inhibitory protein of Ink4 such as Bmi-1 (B-cell-specific moloney murine leukemia virus insertion site 1) expression. The P16 protein is localized in the nucleus, and its nitrogen terminus has a cyclin homologous structure, which can competitively bind to CDK4/6, inhibiting the phosphorylation of the major substrate retinoblastoma protein Rb, which is in the non-phosphorylated state. Rb and its downstream transcription factors E2F binding prevent E2F from activating, thereby inhibiting the expression of its regulatory site genes. The process of cells entering the S phase from the G1 phase is prevented, inhibiting cell proliferation, and ultimately leading to cell senescence. When the phenotype associated with cell senescence occurs, such as growth arrest, and formation of senescence-associated heterochromatin sites, the maintenance of the P16Ink4a/Rb pathway is no longer required. The P53 protein in the P19Arf/P53/P21Cip1 pathway is a common tumor suppressor protein, which is inactivated in most tumors and up-regulated in senescent cells, whose expression is mainly dependent on the level of post-translational modification. The ubiquitin ligase MDM2 (murine double minute 2, the homologous protein in human body is HDM2), which is overexpressed in some common tumors, can promote the degradation of P53 by related proteases or directly inhibit the activity of P53 protein. The P19Arf protein encoded by the Arf gene locus overlapping with the Ink4a gene locus can bind to and inhibit MDM2 activity, thereby participating in the P53 pathway. When DNA is damaged (such as ionizing radiation and telomere dysfunction), P19Arf protein is up-regulated, inhibits MDM2 activity, and activates P53, and then P53 induces downstream P21Cip1. P21Cip1 acts as a CDK inhibitor to inhibit RB phosphorylation and thus cannot bind to E2F. Blocking in the G1 phase causes cell senescence. When P21Cip1 is stressed, it can also directly cause cell growth arrest, and cells that are only regulated by the P53/P21Cip1 pathway can continue to grow after the P53/P21Cip1 pathway is turned off, while the senescent cells choose to permanently or temporarily grow stagnant. The specific mechanism that plays a decisive role is still unclear.
In theory, the process of cellular senescence can be regulated by various factors and pathways in the cellular senescence signaling pathway. Several common modes of regulation are listed here: Recent studies have found that the activated NOTCH pathway can block cell senescence signaling pathway by increasing the transcriptional block of the target gene HES1. So, it promotes the occurrence of cell senescence escape. However, during the development of melanoma, the regulation of cell senescence by the NOTCH pathway remains poorly understood. Therefore, it is important to fully understand the role of the NOTCH pathway in aging and escape in one of the key steps in the malignant transformation of melanocytes. In addition, recent studies have found that the NOTCH signaling pathway is involved in varying degrees of chemotherapeutic drug resistance in many types of tumors. Among them, the overexpression of NOTCH1 active fragment in melanoma cells caused a significant increase in the GI50 value of the BRAF inhibitor vemurafenib, suggesting that activation of the NOTCH signaling pathway can cause melanoma cells to produce treatment against BRAF inhibitors. Resistance, thereby blocks the transmission of cell senescence signaling pathways, and promotes tumorigenesis and development. Therefore, it is important to explore the role of the NOTCH pathway in melanoma. PI3K: LY294002 significantly inhibits Akt phosphorylation and is a specific inhibitor of the PI3K/Akt signaling pathway. Because of its small molecular weight, it can easily cross the blood-brain barrier, and it has become a research hotspot of the mechanism of this pathway in the development of glioma. It proves that LY294002 can induce cell senescence. The main way is to induce the senescence of tumor cells; second, LY294002 may reverse the senescence state of glioma cells, allowing them to enter the apoptotic pathway, thereby exerting an anti-glioma effect. In the pre-senescence period, tumor cells enter a state of cell cycle arrest, thereby activating the aging pathway induced by p53. Although senescent tumor cells have vitality and are not apoptotic, they cannot undergo mitosis for proliferation. Therefore, cell senescence is similar to the state of cell cycle arrest, and the difference is that senescent tumor cells do not have activation of the apoptotic pathway. The apoptotic index of tumor cells in the LY294002 treatment group was also high. This result strongly suggests that some senescent tumor cells may undergo apoptosis under the sustained action of LY294002, and the confirmation of this conclusion remains to be further studied.
Many literatures and experiments have now proved that the blockage of cellular senescence pathways is closely related to the occurrence and development of cancer, and the symptoms of cancer can be alleviated by using appropriate inhibitors, but the specific mechanisms and clinical promotion of inhibitors need more research.