The effect of soy isoflavone crude extract and daidzein on the proliferation of normal and lung cancer cells
The effects of daidzein on the proliferation of HELF and H1299 cells were evaluated by CCK8 assay, which measures the impedance of the attached cells to determine cell viability.
We found a significant proliferation promoted for 10.00% (95%CI: 2.66, 17.336, P = 0.037) when the concentration increase 10 times from 10 to 1000 µg/L in HELF cells, an average increase of 64.5%±0.71 in 10− 1 and 1 µg/L isoflavone crude extract in H1299 cells (Fig. 1a). Cellular activity increases by an average of 5.06% (95%CI: 1.95, 8.18, P = 0.009) per hour in HELF cells treated with 100 µg/L isoflavone crude extract from 0 to 24 hours, and no significant time-dependent observed in H1299 cells, however, significantly 21% decreased appeared in H1299 cells at 24 h (Fig. 1b).
The effect of daidzein on the proliferation of HELF and H1299 cells were observed. For every 10 times, the concentration of daidzein was increased, cellular viability was increased by an average of 29.47% (95%CI: 22.06, 36.87, P < 0.001) in HELF cells at the different concentration (from 0 to 1000 µM) of daidzein for 6 hours respectively, and 2.3% (95%CI: -3.33, -1.29, P = 0.01) decrease in H1299 cells (Fig. 1c). And we observed a significantly altered in proliferation, cellular viability increases by an average of 1.83% (95%CI: 1.11, 2.55, P = 0.002) in HELF cells treated with 10 µM daidzein from 0 to 24 h, and a significant time-dependent decrease in H1299 cells from 0 to 24 h was not observed, however, 62% and 49% were significantly decreased at 12 and 24 hours respectively (Fig. 1d).
The effect of Isoflavone crude extract and Daidzein induced on apoptosis in HELF and H1299 cells
Apoptotic cells were counted as ‘‘late’’ or ‘‘early’’ apoptotic cells, which are shown respectively in the upper right (UR) and lower right (LR) quadrants of the histograms. The relative apoptosis rate decreased to 76.32%±7.86 (P < 0.05) in HELF cells in the experimental group treated with isoflavone crude extract controlled by non-isoflavone crude extract, while non-significantly changed in H1299 cells in the experiment group which was treated with isoflavone crude extract controlled by non-treatment (Fig. 2a). The relative rate of apoptosis was decreased to 86.13%±4.94 (P < 0.05) in HELF cells induced by 10 µM daidzein versus the control group without daidzein. Unfortunately, no significant effect of decreased or increased inducing apoptotic in H1299 cells induced by daidzein (Fig. 2b). Experiments were repeated three times and the resultant data are summarized as the mean of total apoptotic cells (early + late) ± SD.
Daidzein induced apoptosis-related protein expression in HELF and H1299 cells
Gene expression microarray detected significantly altered expression profiles between the two groups which were treated with 10 µM daidzein and non-treatment in H1299 cells for 6 hours. Microarray results suggest the P53 signaling pathway plays an important role in the apoptosis of H1299 cells treated with 10 µM daidzein compared with the control group. There are 5 genes (Bax, FANCC, TP53, PIGS, CASP9) up-regulated and 3 genes (IGF1, FAS, CASP8) down-regulated that were involved in the P53 signaling pathway, and all of these genes were related to apoptosis.
Differences in gene expression between H1299 cells treatment with 10 µM daidzein and H1299 cells without treatment are encoded in the heat map from low to high. Transcripts that show similar expression patterns are clustered together, as indicated by the colored groups to the right of the heat map (Fig. 3a).
These proteins regulate a wide variety of cellular functions. String 10.0 (http://string-db.org) was used to examine associations among the groups of proteins that were negatively or positively correlated with TP53 (listed in Fig. 3b).
RT-qPCR was performed for the expression of apoptosis-related genes of HELF and H1299 cells with daidzein treatment. As shown in Fig. 4a, the mRNA expression of TP53 and Caspase9 were significantly increased, compared to non-treatment, the mRNA expression level of the Caspase9 was significantly increased to 12.35 times in the HELF, and that was 28.08 times in the H1299 compared with the control group. At the same time, the expression level of the TP53 was significantly increased to 1.63 times in the HELF, and 2.31 times in the H1299 compared with the group of blank control(Fig. 4a).
As shown in Fig. 4b, Western blot analysis revealed that the levels of caspase9 were increased after the treatment of HELF and H1299 cells for 6 h with 10 µM daidzein. The relative expression of caspase9 was significantly decreased to 76% in HELF cells while non-significantly changed in H1299 cells. Similar effects on the levels of TP53 was increased 30% in HELF cells but no increase in H1299 cells (Fig. 4b).
All of these results indicated that daidzein may promote apoptosis of lung cancer cells through the P53 apoptosis pathway. Moreover, the mRNA expression of pro-apoptosis factor TP53 was significantly increased. In conclusion, daidzein induced apoptosis involved in the regulation of multiple apoptosis-related genes in HELF and H1299 cells.
Verified the absence of p53, then detected the apoptosis in HELF and H1299 cells
To determine whether the TP53 expression level in the H1299 and HELF cells was associated with their sensitivity to daidzein induced inhibition of proliferation and induction of apoptosis, H1299, and HELF cells were treated with pifithrin-α (10 µM) and daidzein, respectively. As shown in Fig. 5a, the relative mRNA expression level of the TP53 was significantly increased to 7.20 times in the HELF, and the expression in the H1299 was 3.53 times which were treated with 10 µM pifithrin-α and 10 µM daidzein while 10 µM pifithrin-α were controlled. As shown in Fig. 5b, western blot analysis revealed that the relative expression of P53 was significantly increased by 78% in H1299 cells, but the positive result was not observed in HELF cells.
To investigate whether daidzein induced apoptosis was affected by p53 expression in the HELF and H1299 cells
Both HELF and H1299 cells were treated with 10 µM daidzein and 10 µM pifithrin-α. It was found that daidzein treatment of HELF and H1299 cells resulted in non-significantly promote or inhibit in HELF cells than H1299 cells in the numbers of fluorescence-positive cells (Fig. 6).
With PFTα added, the proliferation of HELF and H1299 cells were induced by daidzein
To verify our hypothesis that the effects on the proliferation of lung cancer cells induced by 10 µM daidzein may be produced by P53 pathway genes, the 10 µM pifithrin-α was added into HELF and H1299 cells. Pictures were taken when the HELF and H1299 cells were incubated at 37℃ with 5% CO2 for 6 h. HELF and H1299 cells without treatment in the DMEM medium only served as controls (Fig. 7a, d). The proliferation of HELF and H1299 cells with PFTα were more obvious than the control group (Fig. 7b, e). Added PFTα and daidzein, the proliferation of HELF and H1299 cells were more obvious than the control group but not more obvious than PFTα (Fig. 7c, f). The results of cell count displayed that the proliferation was significantly promoted when PFTα was added, 10% and 11% were promoted in HELF and H1299 cells in the group of PFTα; 12% was significant promoted in the groups of PFTα and DD in HELF cells (Fig. 7g).