br specifically br E ect of IL GFE on
3.2. Eﬀect of IL-GFE on MCF-7 cell growth kinetics
The growth kinetics of untreated (control) and treated MCF-7 with IL-GFE (4.75 μg/mL) and Taxol (0.99 μg/mL) was studied and com-pared. Fig. 4 shows the growth behaviour and the changes of MCF-7 (S)-Mephenytoin after treatment. The number of cells was initially inoculated at 2 × 105 cells/mL. The lag phase was similar for the untreated and the treated MCF-7 cells at first 16 h. Then the cells started proliferation and showed clear log phase after 24 h. However, the treated cells did not show a clear log phase and took 88 h to reach the late log phase. The taxol showed clear log phase but produced less total cell number. It can be observed that the IL-GFE and Taxol treated MCF-7 shows a great reduction in the number of cell proliferation compared to the control. The highest number of cells obtained at the stationary phase for the IL-GFE and Taxol-treated cells were only 6.4 × 105 and 9.1 × 105 cells/ mL, respectively, as compared to the control 26.3 × 105.
Based on the fourth equation that was obtained from the growth curve (Fig. 4), cell growth at exponential phase and death phase (Fig. 5) and the cell generation number (Table 1) were obtained for all treat-ments.
The number of cell generation was reduced from 3.71 generations in the untreated cells to 1.67 generations in IL-GFE treated cells. While Taxol reduced cell generation from 3.71 to 2.18 generations, this output Journal of Ethnopharmacology 236 (2019) 466–473
Fig. 5. MCF-7 Cells Growth at A: Exponential Phase and B: Death Phase, for untreated MCF-7 and the treated MCF-7 with IL-GFE and Taxol.
is significantly (p ≤ 0.05) deferent when the cell generations number were compared between diﬀerent treatments and the control.
The exemplary photographs of the untreated and the IL-GFE-treated MCF-7 at 24, 72 and 120 h were presented in Fig. 6. The longer the treatment time, the less density of MCF-7 observed. This probably due to the antiproliferative eﬀect of IL-GFE toward MCF-7 cells growth.
Cancer growth model in some types of cancers can give a good approximation of the volume of cancer cells using specific growth rate (Mehrara et al., 2007). Therefore, to evaluate the eﬀect of IL-GFE on the specific growth of MCF-7, the graph of the log of viable cells number vs. time was plotted, and the regression slope was taken as a specific growth rate (μ). The growth rate of IL-GFE treated cells and taxol were observed to reduce significantly 0.0035 h−1 and 0.012 h−1, respec-tively compared to 0.0077 h−1 in control. r> Table 1 Number of Generations (X), specific growth rate (μ) and doubling time (td) for Untreated MCF-7 Cells (control), MCF-7 Cells Treated with IL-GFE and Taxol.
Cell growth Maximum cells volume (N) (cells/mL) Number of generations (X) Specific growth rate μ (h−1) Doubling time td (h)
Initial cells number (N0) was constant at 2 × 105 cells/mL for all experiments. * Identical superscripts in the same column indicate significant diﬀerence (p ≤ 0.05).
Fig. 6. Representative photographs of A; Untreated MCF-7 cells, and B; MCF-7 cells treated with IL-GFE at a designated time point.
3.3. Cell cycle distribution and apoptosis
Cell cycle progression is well-correlated to the proliferation of the cell. The untreated and the treated MCF-7 cells with IL-GFE were ana-lyzed using RNase A/PI staining in combination with flow cytometry. The results show that IL-GFE treatment at IC50 for 24, 48 and 72 h in-creased the cells in the G0/G1 phase from 59.07% in control into 72.82% in GFE-treated cells for 72 h, while decreased in the S and G2M phase compared with the control. Moreover, cell number at the sub G0 phase of the treated MCF-7 cells increased in a time-dependent manner compared to the control, taking cells at the sub G0 phase as apoptotic cells (Agu et al., 2018); there was a remarkable increase in the number of apoptotic cells starting from 24 h of IL-GFE treatment (Fig. 7).