• 2019-10
  • 2019-11
  • 2020-03
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  • 2020-08
  • br a step to explore the effect


    a step to explore the effect of Mar-C on transcriptional factors, including NF-κB, TFEB, TFE3, STAT1, STAT3, GATA4 and C/EBPβ, which are able to modulate SASP [28–31]. The mRNA level of STAT1, STAT3, GATA4 and C/EBPβ were not significantly altered upon Mar-C treatment (Fig. 5E). Nuclear translocation of TFEB, TFE3, phospho-STAT1 and phospho-STAT3 was also unaffected in response to Mar-C compared to the control (Fig. 5F and G). However, phosphorylation of p65, a subunit of NF-κB that is a downstream gene of GATA4 and controls many pro-inflammatory gene expressions [32,33], was notably increased in re-sponse to Mar-C (Fig. 5H). Phospho-IKKα/β and phospho-IκBα, which would result in the releasing and translocation of NF-κB into the nuclei, were elevated after 5-day rather than 1-day treatment with Mar-C (Fig. 5I), implicating the involvement of IKK/NF-κB in the regulation of senescent-associated gene transcription [34]. To verify the importance of NF-κB in regulating SASP, we analyzed the changes of secretory factors in Rapamycin via the suppression of p65. As expected, p65 depletion almost completely blocked the expressions of IL-6, IL-8 and TNF-α, to some extent, reversed Mar-C-mediated regulatory effect on these genes (Fig. 5J), supporting the role of NF-κB in regulating SASP related gene expressions. However, knockdown of p65 did not completely affect the expressions of IL-1α and IL-1β in the presence of Mar-C (Fig. 5J), suggesting that other transcription factors were involved in the Mar-C-mediated regulation on these factors. Taken together, these findings suggested that Mar-C was a selective SASP modulator, and NF-κB, at least in part, participated in the regulation of SASP following the challenge with Mar-C.
    3.6. Antitumor activity of Mar-C in combination with immunomodulator curdlan sulfate in vivo
    C57BL/6 homograft animals were employed for further confirming the effect of Mar-C on tumor growth. Mice were randomly assigned to five group (n = 8). After subcutaneous implantation, grafts in vehicle-treated group grew to average (1.78 ± 0.24) g, whereas tumor weight was markedly diminished to (0.65 ± 0.07) g in the group treated with Mar-C. We noted that Mar-C plus curdlan sulfate (CS), an im-munomodulator, showed more antitumor effect than that of vehicle-group (Fig. 6A and B), but without significant difference than that of Mar-C alone. Additionally, the staining of Ki-67 in Fig. 6C indicated that Mar-C and Mar-C plus CS had an obvious inhibitory effect on tumor cells proliferation. Although DOX exerted better anti-tumor efficacy than Mar-C, DOX showed higher toxicity as the sharp drop of body weight shown in DOX-group, administration of Mar-C or Mar-C plus CS had no impairment on body weight in tumor-bearing mice (Fig. 6D). DOX had an obvious damage on the spleen of mice compared with vehicle (Fig. 6E), while Mar-C or Mar-C plus CS could both elevate the splenic indexes without impairment on spleen (Fig. 6E). Besides, the liver function reflected by related serum enzymes, including aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), and alanine aminotransferase (ALT), in Mar-C-treated group also displayed lower toxicity than DOX-group (Fig. 6F).These data provided evidence that Mar-C had low immunosuppressive toxicity and obvious anti-tumor effect both in vitro and in vivo, and might ameliorate some side effects of 
    4. Discussion
    In the present study, we explored a new anti-cancer mechanism of Mar-C, suppressing cancer cell growth by inducing senescence, and studied the effect of Mar-C on SASP components. The results showed that in vitro Mar-C selectively induces cancer cell senescence at low concentrations, and in vivo Mar-C not only showed anti-cancer effect with lower toxicity, but also prolonged the survival time of the tumor-bearing mice as well. It means that Mar-C can play its anti-cancer role via inducing cancer cell senescence without significant effect on SASP.
    Cellular senescence is triggered by diverse stressors and executed by multiple molecular pathways [35]. The p53-p21 and p16-Rb signaling pathways function as the central points. Which pathway was crucial in Mar-C induced senescence of lung cancer cells? Our results showed that Mar-C significantly increased the p53 and p21 in A549 cells (p53 wild type), instead of p16 (A549 null) [36,37]. However, the inhibition of p53 activity could not change the senescence induced by Mar-C, and the cells with mutated p53 (H1299, H446) also exhibited senescent phe-notype. To some extent, p21, but not p53, is responsible for Mar-C-induced senescence in this study. There were examples of senescence that appeared to be independent of p53 and Rb pathways [38–40]. For example, knockdown of SETD8 had been reported to trigger cellular senescence with irrespective of p53 status in cells [40]. Moreover, cells carrying or lacking p21 both can undergo senescence [41,42]. In our study, p21 appeared to be important in Mar-C-mediated senescence. Elucidating the mechanism of enhanced p21 in different kinds of cells treated with Mar-C still needs further investigation.