The effect of oridonin and LNT on the growth of MRC-5 and A549 cells
Cell viabilities were determined using MTT assay. The viabilities of MRC-5 and A549 cells were not affected by 0-20 µg/ml oridonin. 0-300 µg/ml LNT did not affect the viability of MRC-5 cells, but 50-400 µg/ml LNT significantly inhibited the viability of A549 cells. The oridonin, LNT, or the combination of both did not affect MRC-5 cell viability. The oridonin had no effect on A549 cell viability but LNT significantly suppressed A549 cell viability, and the used of oridonin increased the suppression effect of LNT on A549 cells. (Fig. 1)
20 µg/ml oridonin was used in the subsequent study because it was the highest concentration that had no significant effect on cell viability. In addition, 300 µg/ml LNT was selected for the subsequent study as a high concentration group (LNT-H) because it had the strongest potency in the viability of cancer cells while had no effect on the viability of normal cells. As a comparison, we also used a low concentration group (LNT-L) which was 100 µg/ml LNT.
The effect of oridonin and LNT on mRNA and protein expression of apoptosis-associated proteins in A549
LNT at both concentrations tested increased the mRNA and protein expressions of caspase-3, caspase-8, and caspase-9 in A549 cells with a larger increase in LNT-H. Oridonin further increased the mRNA and protein expression of caspase-3, caspase-8, and caspase-9 in LNT treated A549 cells. (Fig. 2 AB) Besides, LNT at both concentrations tested increased the mRNA and protein expression of Bax in A549 cells with more increase in LNT-H (LNT high concentration group). Oridonin further increased the mRNA and protein expression of Bax in LNT treated A549 cells. On the other hand, LNT at both concentrations tested decreased the mRNA and protein expression of Bcl-2 and Bcl-xL in A549 cells with more decrease in LNT-H. Oridonin further decreased the mRNA and protein expression of Bcl-2 and Bcl-xL in LNT treated A549 cells. (Fig. 2 CD) These results revealed that apoptosis might mediate the effects.
The effect of oridonin and LNT on mRNA and protein expression of the p53/p21 pathway proteins in A549
We suggested the viability effect was associated with p53/p21 signaling, thus we also tested them. LNT at both concentrations tested increased the mRNA and protein expression of p53 and p21 in A549 cells. Oridonin further increased the mRNA and protein expression of p53 and p21 in LNT treated A549 cells (Fig. 2 EF). These results suggested that the p53/p21 pathway was involved.
The effect of oridonin and LNT on mRNA and protein expression of NF-κB and IκB-α in A549
Additionally, LNT at both concentrations tested increased the mRNA and protein expression of NF-κB in A549 cells with a more dramatic increase in LNT-H. Oridonin further increased the mRNA and protein expression of NF-κB in LNT treated A549 cells. On the other hand, LNT at both concentrations tested decreased the mRNA and protein expression of IκB-α in A549 cells with more decrease in LNT-H. Oridonin further decreased the mRNA and protein expression of IκB-α in LNT treated A549 cells (Fig. 2 GH). These results suggested that the NF-κB and IκB-α signaling was involved.
The effects of oridonin and LNT on lung tumor metastasis in mice
We established lung cancer metastasis mice model and treatment them with LNT-L, oridonin, oridonin + LNT-L, LNT-H, and oridonin + LNT-H. Mice without cancer or any treatment and mice with lung cancer but any treatment were used for comparison. As showed in Table 1, after 10 days, the oridonin alone had no effect on short time lung cancer metastasis. LNT treatment decreased the metastasis with a higher inhibitory rate in LNT-H group than in LNT-L group. Oridonin improved the suppression of LNT against lung cancer metastasis in both groups. These results suggested that oridonin help LNT decrease lung tumor metastasis in mice.
The effects of oridonin and LNT on the overall survival of mouse with lung tumor cells
We also conducted a survival assay to test the effect of oridonin and LNT. Results showed that oridonin alone had almost no effect on the survival of the animals. Both LNT-L and LNT-H improved the survival and LNT-H had a better outcome than LNT-L. Oridonin increased the improvement effect of LNT on the survival rate (Fig. 3). Results indicated that the combined use of both oridonin and LNT optimized survival.
The effects of oridonin and LNT on the mRNA and protein expression of caspase-3, caspase-8, and caspase-9 in mice lung tissues
After the lung cancer cell injection, the mRNA and protein expression of caspase-3, caspase-8, and caspase-9 significantly decreased in lung tissue samples. The LNT at both concentrations tested increased the mRNA and protein expression of caspase-3, caspase-8, and caspase-9 in lung tissue samples with a more significant increase in LNT-H. Oridonin further increased the mRNA and protein expression of caspase-3, caspase-8, and caspase-9 in the lung of LNT treated animals (Fig. 4 AB). These results were similar to those in A549, which confirmed the effect of oridonin and LNT on lung cancer.
The effects of oridonin and LNT on the mRNA and protein expression of Bax, Bcl-2, and Bcl-xL in lung tissues
In addition, after cancer induction, the mRNA and protein expressions of Bax significantly increased while Bcl-2 and Bcl-xL significantly decreased in lung tissue samples. LNT at both concentrations tested increased the mRNA and protein expressions of Bax while LNT-H increased more than LNT-L. Oridonin further increased the mRNA and protein expression of the expression of Bax in the lung. On the other hand, LNT at both concentrations tested decreased the mRNA and protein expressions of Bcl-2 and Bcl-xL with more decrease in the LNT-H group. Oridonin further decreased the mRNA and protein expressions of Bcl-2 and Bcl-xL in LNT treated groups (Fig. 4 CD). These results were similar to those in A549, which confirmed the effect of oridonin and LNT on lung cancer.
The effects of oridonin and LNT on the mRNA and protein expression of p53 and p21 in mice lung tissues
Besides, the cancer induction significantly decreased the mRNA and protein expressions of p53 and p21 in lung tissue samples. The LNT at both concentrations tested increased the mRNA and protein expressions of p53 and p21 in lung tissue samples with a more increased rate in LNT-H. Oridonin further increased the mRNA and protein expressions of p53 and p21 in the lung (Fig. 4 EF). These results were similar to those in A549, which confirmed the effect of oridonin and LNT on lung cancer.
The effects of oridonin and LNT on the mRNA and protein expression of NF-κB and IκB-α in lung tissues
Additionally, the mRNA and protein expression of NF-κB significantly increased while IκB-α significantly decreased in lung tissue samples. LNT at both concentrations tested decreased the mRNA and protein expression of NF-κB with more decrease in LNT-H. Oridonin further decreased the mRNA and protein expression of NF-κB in the lung of LNT treated animals. On the other hand, LNT at both concentrations tested increased the mRNA and protein expression of IκB-α with a larger increase in LNT-H. Oridonin further increased the mRNA and protein expression of IκB-α in LNT treated lung tissues (Fig. 4 GH). These results were similar to those in A549, which confirmed the effect of oridonin and LNT on lung cancer.