Relebactam br Mar C reduces tumor growth
3.2. Mar-C reduces tumor growth potential and prolongs the survival of tumor bearing mice
To determine whether Mar-C had effects on tumor growth due to the induction of cancer cell senescence in vivo, human A549-luc xenografts (A549 Relebactam expressing luciferase) were developed in mice. As shown in Fig. S1B, all tumor-bearing mice exhibited ~108 photons·s−1·cm−2·sr−1 in total photon intensity before treatment. Tumor-bearing mice in ve-hicle control exhibited 4.17 × 1010 photons·s−1·cm−2·sr−1, however, the fluorescence signal dropped to 1.71 × 1010 photons·s−1·cm−2·sr−1 after Mar-C treatment, similar to that of DOX exposure (Fig. 2A), im-plying that Mar-C reduced the tumor growth with impaired cell pro-liferation activity. The average tumor size of vehicle control group was (0.9 ± 0.3) cm3 (Fig. 2B), whereas it notably decreased in Mar-C-group (0.6 ± 0.2) cm3 or DOX-treated group (0.5 ± 0.3) cm3
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(B) Cell cycle protein cyclinD1 was inhibited by Mar-C (2 μM) in 5 days. (C) Western blot analysis showed significantly enhanced expression of p53 and p21 after Mar-C (2 μM) treatment in 5 days. (D) Phosphorylation of Rb protein was decreased by Mar-C (2 μM) in 5 days. (E) The efficacy of PFT-α on inhibiting the function of p53. (F) SA-β-gal staining were performed after treatment with Mar-C, PFT-α or both for 5 days. PFT-α did not affect the cellular senescence induced by Mar-C (2 μM). (G) The A549 cells were transfected with siRNA-ctrl or siRNA-p21 for 24 h following the exposure to 2 μM of Mar-C for 5 days. Fluorescent images of EdU incorporation assays after knock-down of p21 were observed. (H) Western blot analysis of p21 knock-down efficiency. (I) Western blot analysis showed that decreased phos-phorylation of Rb protein by Mar-C (2 μM) was rescued after p21 knock-down. Data information: In A and B, data represent mean ± SEM (n = 3); *P < .05 or **P < .01.
(Fig. 2B). Remarkably, administration of Mar-C had no impairment on body weight. But, the sharp drop of body weight was clearly observed in DOX-treated group although it exerted better antitumor efficacy than Mar-C (Fig. 2C). Mar-C caused limited toxicity in mice as indicated by the decreased releases of enzyme aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT) and alanine aminotransferase (ALT) in serum, compared to the group treated with DOX (Fig. 2D). More im-portantly, Mar-C greatly increased the animal survival time compared with control and DOX (Fig. 2E). Positive SA-β-Gal staining were also observed in tumor sections obtained from the Mar-C-group, but not from the vehicle group (Fig. 2F). And Mar-C noticeably inhibited cell proliferation, as indicated by positively stained Ki-67 cells in tumor sections (Fig. S1C). Thus, Mar-C functioned as a potential antitumor agent with high efficacy and low toxicity.
3.3. Mar-C triggers cell cycle arrest in a p21-dependent manner
As senescent cells are characteristic with cell cycle arrest, flow cy-tometry analysis indicated that Mar-C increased the proportion of G0/ G1 phase cells as compared with control in Fig. 3A. Also, reduced cy-
clinD1 was observed upon treatment with Mar-C (Fig. 3B). Analysis of the expressions of p53-p21WAF1/CIP1 (p21) and p16INK4A (p16)-Rb, which are critical in cell cycle arrest and senescence induction [17–19], revealed that Mar-C significantly increased the protein level of p53 and p21 in A549 cells (Fig. 3C), and decreased phospho-Rb following ex-posure to Mar-C (Fig. 3D). We noted that total Rb was reduced in re-sponse to Mar-C treatment (Fig. 3D). The expression of p27 was induced after 1-day exposure to Mar-C, but less potent than the p21, and dropped down to basal level after 5-day exposure (Fig. 3D). PFT-α, an inhibitor of p53 activity, was employed to confirm the role of p53-p21 due to the significant changes in response to Mar-C. As shown in Fig. 3E and F, PFT-α was unable to suppress p21 expression, and had limited effect on senescence of A549 cells in the presence of Mar-C, indicating that induction of p21, at least in part, was p53-independent. The effect of Mar-C on induction of senescence in a panel of cells including H1299 and H446 lacking wild type p53 (Fig. S1A), supported that p53 may not be critical in Mar-C-induced senescence. To identify the role of p21 in Mar-C-induced cellular senescence, we knocked-down p21 with siRNA in A549 cells. EdU incorporation assays showed that silencing of p21 could partly impair the impact of Mar-C on A549 cells (Fig. 3G and H). Besides, p21 is also involved in senes-cence through the regulation of Rb. Inhibition of phospho-Rb and total Rb by Mar-C was recovered after p21 depletion (Fig. 3I). These results indicated that p21, but not p53, was required in driving Mar-C-induced senescence.