br Another characteristic of senescent cells is SASP
Another characteristic of senescent Paxilline is SASP. Senescent cells usually express a persistent pro-inflammatory SASP, which affects the cellular microenvironment, leading to acceleration of aging and the initiation of age-related pathologies such as malignant cancers . Many efforts have been made to minimize the deleterious effects of SASP in aging and cancer treatment. For example, inhibition of mTOR or NF-κB in senescent cells could efficiently suppress the side effects of SASP [44,45]. We also concerned about the regulation of SASP under Mar-C treatment. Firstly, increases in the expression of IL-1α and IL-1β were observed upon Mar-C exposure, but to a much less extent when compared with DOX. More importantly, Mar-C moderately induced other pro-inflammatory factors such as IL-6, and had limited effect on anti-inflammatory cytokines, IL-5, IL-10, IL-13 and TGF-β. This suggests that the induction of SASP by Mar-C was a distinct event. Then, what is the mechanism of SASP induction by Mar-C? Our data clearly showed that Mar-C transcriptionally controlled the SASP components, mainly by activation of p65, a subunit of NF-κB. It appeared that the activation of IKKα/β and IκBα after 5-day treatment, but not one day, indicating that the activation of NF-κB was not an early event responding to DNA damage which was induced at 0.5 h after treatment with Mar-C. Further investigation confirmed the specific role of NF-κB on regulating Mar-C-induced SASP, particularly on the expressions of IL-6, IL-8 and TNF-α, implicating the role of NF-κB on SASP components that may be ne-cessary for maintaining senescence. Surprisingly, we found that
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Fig. 5. Mar-C modulates SASP via different transcriptional factors. (A and B) Conditioned-medium harvested from senescent cells induced by Mar-C (2 μM) were added to the culture of A549 cells and HBE cells and the cell viability were measured by MTT assays. (C) The secretions of different cytokines in the conditioned-medium after Mar-C treatment were measured with protein array. Images of protein array (left). The heatmap showed the secretions of inflammatory cytokines under different treatment (right). The values of concentration are normalized and log2 transformed (D) The mRNA levels of pro-inflammatory cytokines and anti-in-flammatory cytokines were detected with quantitative PCR. (E) The mRNA expression of transcriptional factors STAT1, STAT3, GATA4, and C/EBPβ in A549 cells exposed to Mar-C (2 μM). (F and G) Nuclear translocation of TFE3, TFEB, phospho-STAT1, phospho-STAT3 of A549 cells treated with Mar-C (2 μM). (H and I) Protein expression of phospho-p65, phospho-IKKα/β and phospho-IκBα in A549 cells treated with Mar-C (2 μM) at different time points. (J) Quantitative PCR of the expression of inflammatory cytokines in A549 cells treated with Mar-C (2 μM), in the presence or absence of p65. The efficiency of p65 knocking-down by siRNA treatment was confirmed.
knockdown of p65 did not completely eliminate the transcription of IL-1α and IL-1β, indicating that Mar-C is a selective SASP modulator. Although the involvement of other transcriptional factors or mechan-isms remain further investigation in Mar-C-mediated regulation of SASP, the interventions of pro-inflammatory SASP by Mar-C would al-leviate the deleterious effects of SASP accompanied with the growth inhibition of tumors.
In addition, induction of apoptosis of senescent cells or enhanced immune clearance is also a promising strategy to alleviate the dele-terious effects of SASP in response to chemical agents [46–48]. Curdlan sulfate (CS), a soluble sulfated derivative of curdlan which is an ex-tracellular polysaccharide of bacteria origin, possesses immunological adjuvant properties. Studies on CS had shown that it had im-munopotentiating activity, including activating macrophages, bone marrow derived dendritic cell (BMDC) maturation and inducing sple-nocyte proliferation [15,49,50]. The combination of Mar-C and CS
indeed exhibited anti-tumor effect on tumor-bearing mice without toxicity, though without significant difference between Mar-C and Mar-C + CS. As our first investigation on the combination of Mar-C and CS, it did show a synergistic tendency. These results paced a way for further optimization of combined Mar-C with CS in cancer treatment. More-over, whether Mar-C has immunomodulating activity and if Mar-C has synergistic anti-tumor action when it is used with other im-munomodulators still remain further investigation.
In summary, our results revealed a novel anti-tumor mechanism of Mar-C, selectively promoting cancer cell senescence but with limited cytotoxicity for normal cells and predominantly extending the survival of the tumor-bearing mice. The distinct modulation on SASP by Mar-C may also provide substantial benefits for cancer therapy. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bbagen.2019.05.006.