br Antineoplastic activity br Live
3.3. Antineoplastic activity
Live/dead staining and CCK were conducted to qualitatively and quantitatively evaluate the eﬀects of APS on the growth of MCF-7 cells, respectively (Fig. 4a and b). The results of live/dead staining indicated that APS groups failed to inhibit cell growth as an overwhelming pro-portion of living NCT501 were positively stained by calcein-AM with strong green fluorescence, and sporadic dead cells exhibited red fluorescence with PI staining compared with those of untreated cells. Meanwhile, a great many living cells also exhibit slight blue fluorescence in nuclei stained by Hoechst. Instead, a small number of living cells were iden-tified in positive control group accompanied by numerous dead cells. In addition, the results of CCK also suggested that no cytotoxicity on MCF-7 cells was observed after the treatment with diﬀerent doses of APS for 3 days, particularly on day 1 with the higher cell viabilities in the ex-perimental group than that in the negative control group. Although better viabilities were identified in the negative control group on day 1 and day 3, there were no significant diﬀerences in comparison with
those in the experimental group. Conversely, 5-FU presented significant toxicity eﬀects on MCF-7 cells. The cell viability was obviously de-creased after exposure to 5-FU in a time-dependent manner compared with those in experimental and negative control group (P < 0.001) (Fig. 4b).
To investigate the possible anti-tumor activity from the increased production of NO and TNF-α in CM mediated by APS on macrophages, MCF-7 cells were also subjected to CM for 24, 48, and 72 h, respec-tively. After exposure to CM for 2 days, the number of living cells gradually decreased and that of dead cells increased in a dose-depen-dent manner. However, the cytotoxicity eﬀect of CM was weaker than that in 5-FU group (Fig. 4c). The results of CCK indicated that CM ex-erted growth inhibitory eﬀects on MCF-7 cells in a time- and dose-de-pendent manner, with the most remarkable toxicity eﬀect (41%) at the concentration of 1000 μg/mL on day 3. Significant inhibitory eﬀects of CM were identified at the concentration of 1000 μg/mL as compared to those at the concentration of 50–200 μg/mL at 24, 48, and 72 h (P < 0.001). However, the toxicity eﬀect of CM at the highest con-centration was markedly inferior compared than that in 5-FU group at 24 h (P < 0.01). Furthermore, the diﬀerence between two groups be-came even more pronounced over time (P < 0.001, Fig. 4d).
The 6-well plate-based colony formation assays were carried out to uncover the eﬀect of CM on the long-term growth of MCF-7 cells. Upon 14-d incubation with CM, the numbers of Giemsa-stained colonies were remarkably decreased with the increase of APS concentration in CM. Notably, no colony was noticed in the positive control group since the 5-FU dose on each cell was enhanced with the lower cell-seeding den-sity (Fig. 4e). Compared to untreated cells, CM significantly inhibited the colony formation at the concentration of 200–1000 μg/mL of APS
Fig. 4. Inhibitory eﬀect of CM on proliferative activity of MCF-7 cells. (a,c) Live/dead staining to evaluate the eﬀect of APS and CM on cell viability after incubation for 72 h, respectively. Cells were stained with calcein-AM, PI and Hoechst 33342 after exposure to APS and APS mediated CM (100–1000 μg/mL), and 50 μg/mL 5-FU (scale bar = 100 μm). (b, d) The viability and inhibitory of MCF-7 cell cultured with diﬀerent dosages of APS and APS-mediated CM were evaluated by CCK-8 assay after incubation for 24, 48, and 72 h, respectively, (n = 4). (e) Colony formation of MCF-7 cells in the presence of APS mediated CM with diﬀerent concentration and 5-FU. (f) Colony number of MCF-7 cells after incubation with CM and 5-FU. *** P < 0.001 compared between CM groups and negative control group; ## P < 0.01, ### P < 0.001 compared between 5-FU and APS mediated CM at the concentration of 1000 μg/mL.
(P < 0.001). Nevertheless, the colony number in the presence of 5-FU was markedly lower than that of highest concentration of APS in CM (Fig. 4f, P < 0.001). These findings indicated that APS was capable to active macrophages to inhibit MCF-7 cells growth, even if the cyto-toxicity was inferior in comparison with 5-FU.
To examine the mechanism responsible for CM-mediated cell pro-liferation inhibition, cell cycle phase distribution and proliferation index were analyzed. CM treatment induced cell cycle arrest in G1 phase in a dose-dependent manner (Fig. 5a and b). Compared to the negative control group, significant elevation was identified in the CM group mediated by APS at the concentration of 500 μg/mL (P < 0.05) and 1000 μg/mL (P < 0.01). The formed sub-G1 peak appeared in the cell cycle histogram as a result of apoptosis with the characteristic of karyopyknosis and DNA cleavage in apoptotic cells. As is presented in Fig. 5a, an apoptotic peak with the sub-G1 fraction of 3.19% appeared after exposure to CM with APS (1000 μg/mL), while the fraction was 9.01% after the treatment with 5-FU. Meanwhile, APS mediated CM down-regulated proliferation index of MCF-7 cells in particular at the concentration of 500 μg/mL (35.94%, P < 0.05) and 1000 μg/mL (24.52%, P < 0.01) compared to those of untreated cells (44.02%, Fig. 5c), which further appeared to inhibit cell proliferation. In