Data information In A B C
Data information: In A, B, C, and D, data represent mean ± SEM (n = 6); *P < .05 or **P < .01.
quantified by the RayBio® Analysis Tool software.
Subcutaneous tumor xenograft models were built to evaluate the anti-tumor effect of Mar-C on A549 Fosfomycin calcium in vivo. Balb/c athymic (nu +/nu+) female mice, 6 weeks old, were selected.
For bioluminescence imaging, A549-luc cells (1 × 107 cells in 1 mL of PBS) were injected into the lower right flank of nude mice for sub-cutaneous tumors. Mice were randomized to three groups and used for bioluminescence observation, efficacy studies and survival analysis. The vehicle, Mar-C (10 mg/kg), and DOX-group (4 mg/kg) were in-traperitoneally (i.p.) injected and body weight was weighed every other day. The tumor volume was regularly measured using vernier calipers per two days. The experiment was terminated on the day 17 after in-itiation of treatment and the tumors were harvested and stored.
2.11.2. Lewis lung carcinoma cells homograft model
Tumors were generated by subcutaneous injection into the right anterior flanks of C57BL/6 mice (female, 6 weeks of age) with 1 × 107 cells mL−1 of Lewis lung carcinoma (LLC) cells. The tumor-embedded C57BL/6 mice were divided into 5 group (n = 8 per group) to receive vehicle, DOX (4 mg/kg), Mar-C (10 mg/kg), CS (10 mg/kg), or Mar-C plus CS. After 14 d treatment as described above as A549-derived xenografts, C57BL/6 mice were anesthetized and the tumors were harvested for further studies.
LLC cells homograft were constructed for the evaluation of Mar-C toxicity. After tumor implantation, the mice (female, n = 6) were treated with vehicle, DOX or Mar-C for 14 d. The mice were euthanized and the serum were collected for measurement of the liver enzyme levels.
All animal experiments were approved by the Institutional Guidelines of Animal Care and Use Committee at Shandong University.
For in vivo bioluminescence imaging, the mice were injected with luciferin (150 μg·g−1 per mouse, i.p.) about 8 min. The mice were fully anesthetized with the 4% isoflurane gas about 5 min before imaging. Luminescence images were obtained using an IVIS imaging system and analyzed with Living Image version 4.1 (Caliper Life Sciences, USA). All mice were immobilized by isoflurane gas (1.5%) during imaging.
2.12. Statistical analysis
Data are presented as mean ± SEM and analyzed with GraphPad Prism 7 software (San Diego, USA). Statistical significance (P values) was determined by using unpaired Student's t-tests (twotailed) with Welch's correction or ANOVA. P values < .05 were considered statis-tically significant.
3.1. Mar-C induces cellular senescence of lung cancer cells at a low concentration with less effect on normal cells
We initiated our screening analysis to determine the concentrations of Mar-C which are required to achieve activity on cancer cell lines by titration. Similar to our previous study [12,13], Mar-C exerted cytotoxic effect on lung cancer cell proliferation with an IC50 of ~8 μM (Fig. 1B and C). Interestingly, the cells also responded to Mar-C as presented by enlarged and flattened morphologic changes at a low concentration (2 μM), displaying senescence-like phenotype (Fig. 1D and Fig. S1A). Lung cancer cells were selected as a model to further determine the effect of Mar-C on cellular senescence. After 5-day treatment with Mar-C, the accumulation of senescent cells was evident in both A549 and H1299 cells, as indicated by an increase in the senescence-associated-β-galactosidase (SA-β-gal) staining, a hallmark of senescence (Fig. 1E). The effect of Mar-C on the induction of cellular senescence was also observed in lung cancer H446, H1688, H460 and drug resistant H460/ RT cells (Fig. S1A), supporting the ability of Mar-C in triggering cancer cellular senescence.
As premature senescence is characteristic by loss of cell proliferation potential, cell colony formation assays were performed to examine the ability of Mar-C on cell proliferation. The results in Fig. 1F showed a ~80% decline in colony formation compared with that of DMSO-treated control after treatment with Mar-C for 5 days. However, Mar-C had no detectable effect on normal human fibroblasts (NHF) cell via-bility as shown in Fig. 1G. Also, NHF cell proliferation was not altered upon treatment with Mar-C, as indicated by EdU incorporation ex-amined by flow cytometry and immunofluorescence assays (Fig. 1H and I). Importantly, NHF cells maintained normal morphologic phenotype and presented no SA-β-gal staining after Mar-C treatment (Fig. 1J), while the senescent cells were evident changed with a senescence in-ducer doxorubicin (DOX), which served as a positive control. Further-more, Mar-C treatment had limited inhibitory effect on both fetal lung fibroblast IMR90 cells and human bronchial epithelial HBE cell lines (Fig. 1K), supporting the observations that Mar-C had limited cyto-toxicity on normal cells. Thus, the low concentration of Mar-C could selectively facilitate the premature senescence of cancer cells with little toxicity against normal cell proliferation.