It is well known that the resistant to current therapies is one of the poor prognostic outcomes of human bladder cancer and it is the leading cause of cancer-related death [39
]. Currently, the successful treatment with chemotherapeutic agents is based on these agents to trigger cell death in tumor cells [41
]. Until now, investigators have been focusing on the novel inducers of apoptosis to provide a new therapeutic approach for cancer patients and some of the compounds for treatment of cancer are from natural products [44
Results showed that cucurbitacin E induced G2
/M phase arrest () in T24 cells. The protein levels of phospho-STAT3, CDK1, and cyclin B were decreased, and the levels of p53 and p21 were increased in cucurbitacin E-treated T24 cells in a time-dependent manner (Figures and ). Furthermore, curcumin E also inhibited the CDK1 activity in T24 cells (). It is reported that activated STAT3 is formed through downregulating p53 level and affects the p53 promoter in in vitro
and in vivo
]. Moreover, CDKs regulate the cell-cycle progression in mammalian cells [48
]. In the G2
/M phase progression which is regulated with CDK1 and CDK2 kinases that are activated primarily in association with cyclins A and B [50
]; it is reported that distributing cell-cycle progression by alterations in cell cycle-related protein expression plays important roles in the proliferation of cancer cells [50
]. Therefore, we suggest that alterations in STAT3/p21/p53 signaling and G2
/M phase-associated protein levels occurred in cucurbitacin E-treated T24 cells. This is also in agreement with other reports addressing that inhibitory effect of STAT3 level on cucurbitacin E and triterpene-derived natural products treated human tumor cells and umbilical vascular endothelial cells in vitro
Our results also showed that cucurbitacin E induced apoptotic death of T24 cells and this effect is a dose- and time-dependent response (). Apoptosis is a programmed cell death which is a multiple regulated process which can be divided into the caspases and mitochondrial signaling pathways [52
]. Cucurbitacin E treatment in T24 cells promoted the activations of caspase-8, -9, and -3 in a dose-dependent manner (). Otherwise, cells were pretreated with a general caspase inhibitor (Z-VAD-FMK) and exposed to cucurbitacin E, leading to increase the percentage of viable cells when compared to the cucurbitacin E-treated only cells (). The data indicated that the activation of caspase cascade is involved in cucurbitacin E-induced cell death
Result from Figures and indicated that cucurbitacin E decreased the level of ΔΨm
in a time-dependent response. Bcl-2 family proteins (antiapoptotic and proapoptotic proteins) have been reported to regulate cytochrome c
release from mitochondria [53
]. Our results also showed that cucurbitacin E promoted the level of cytochrome c
(released from mitochondria) (). Cucurbitacin increased the levels of truncated BID which is related to the dysfunction of mitochondria (). also shows an increase in the level of AIF which is released from mitochondria. Based on these observations, we may suggest that cucurbitacin-induced apoptosis of T24 cell is carried out through the Fas/CD95 and mitochondrial signaling pathways.
In conclusion, cucurbitacin E decreased the percentage of viable T24 cells through the cell cycle arrest and induction of apoptosis in human bladder T24 cancer cells in vitro. Cucurbitacin E-induced G2/M phase arrest was associated with the inhibitions of phosphorylation STAT3, promotion of p53 and p21, and reduction of CDK1 and cyclin B. Cucurbitacin E-induced apoptosis was accompanied with upregulation of Fas/CD95, decrease the level of ΔΨm and then led to cytochrome c release from mitochondria and promoted the activations caspase-8, caspase-9, and caspase-3, leading to apoptosis of T24 cells. The proposed signaling pathways can be seen in .
The possible signaling pathways for cucurbitacin E-induced G2/M phase arrest through STAT3/p53/p21 signaling and apoptosis via Fas/CD95 and mitochondria-dependent pathways in human bladder cancer T24 cells.