3.1 Tannic acid inhibited cell proliferation in bladder cancer cells
Cells were treated with TA at different concentrations (0 μM, 25 μM, 50 μM, 100 μM, 200 μM) for 24 hours. Compared to untreated cells, TA treated cells dose dependently decreased UMUC3 cell growth (Figure 1a). Treatment with TA showed high toxicity in UMUC3 cells, and the growth inhibition was evident from lower concentration. In the normal urinary cells -SV-HUC1, TA treatment did not show any significant changes in cell proliferation (Figure 1b).
When UMUC3 cells were treated with TA and observed after incubation for 14 days, it was found that, compared to the control cells, the treated cells had significantly reduced number and size of colonies, and this variation was dose dependent. These results indicated that TA can inhibit colony formation in UMUC3 cells.
3.3 Tannic acid induced apoptosis in UMUC3 cells
After treatment of UMUC3 cells with different concentrations of TA for 24 hours, the cells were collected and analyzed to check whether TA induced apoptosis in cancer cells. As shown in Figure 2, treatment with TA induced apoptosis in a dose-dependent manner in UMUC3 cells. This data confirmed that TA inhibits proliferation of cancer cells by promoting apoptosis. To further validate the effects of TA, TUNEL assay was performed and analyzed for TUNEL-positive cells while treated with TA. As expected, the number of TUNEL-positive cells were found to be higher when the cells were treated with TA (Figure 3). Therefore, cancer cells exhibited higher rates of apoptosis when treated with TA.
3.4 Tannic acid promoted apoptosis through the intrinsic mitochondrial pathway
As TA elevated the occurrence of apoptosis in UMUC3 cells, we checked whether apoptosis was regulated by the intrinsic or extrinsic pathway. We first analyzed the caspase-3 pro- and cleaved- form in UMUC3 cells after treating the cells with TA for 24 hours. We found that the expression of the cleaved-form of caspase-3 was remarkably higher in cells treated with higher concentrations of TA, while the pre-form was reduced in a dose-dependent manner (Figure 4).
We also checked the protein expression of the Bcl-2 family (anti-apoptotic) proteins such as Bcl-2, Bcl-xL and Mcl-1, which are important for regulating cell survival. These protein expressions were significantly downregulated after treatment with TA, which further proved that the cells were affected due to TA treatment.
3.5 Tannic acid affected the stemness of UMUC3 cells
TA promoted apoptosis in UMUC3 cells and also reduced the colony-formation ability of the cancer cells. Therefore, next, we investigated whether TA treatment has any effects on the expression of stem cell markers. As expected, we found that TA treatment lowered the expression of stem cell markers such as SOX2, OCT4, and NANOG protein expression in a dose-dependent manner. Thus, TA was found to be effective to block the proliferation of cancer stem cells (Figure 5).
3.6 Tannic acid promoted apoptosis in UMUC3 cells through PI3K/AKT pathway
As the PI3K/AKT pathway plays a very important role in proliferation activity, we then analyzed whether AKT protein level is altered after TA treatment. As shown in Figure 6, phosphorylation level of AKT was lowered in a dose depend manner Additionally, we found that total AKT did not show any significant changes. These results showed that TA can inhibit the proliferation of UMUC3 cells via AKT pathway.