Development and progression of PCa to the most aggressive state mCRPC makes prostate cancer cells more resistant to approved chemotherapies (Docetaxel and Cabazitaxel) (3). Many theories have evolved to elucidate mechanisms of such resistance. Abnormal activation of multiple signaling pathways has been described as one of the well-studied mechanisms for mCRPC progression despite continual hormonal therapy (17).
Several signaling pathways have been shown to be implicated in PCa disease progression. Among them, PI3K/AKT/mTOR and Hh/GLI pathways have been considered as two pathways that can participate either separately or through pathway crosstalk in mCRPC development (18). In this regard, the current work aims to assess the antitumor effects resulting from the co-targeting of PI3K/AKT/mTOR and Hh/ GLI pathways in PC3 cells using Dactolisib and GANT61 as PI3K/AKT/mTOR and Hh/ GLI pathways inhibitors, respectively and also to investigate whether their combination add therapeutic benefit over their single treatments.
Pertaining to GLI1 gene expression, it was found that Dactolisib, GANT61 and their combination down-regulated such expression level. As a target gene for GLI1 transcriptional factor (19), GLI1 gene expression is normally down-regulated by GANT61. However, down-regulation of such gene expression by Dactolisib and to a better extent by GANT61/Dactolisib combination may support the existence of a non-canonical activation of Hh/GLI pathway by PI3K/AKT/mTOR pathway as reported by previous studies (20–21). Moreover, low p-AKT (a downstream kinase to PI3K (22)) and pS6K1 (a downstream kinase to mTORC1 (23)) protein levels have been observed due to Dactolisib, GANT61 and their combination treatment when compared with their levels that are found in vehicle control cells. This may clarify the capability of GLI1 protein to activate PI3K/AKT/mTOR pathway through different mechanisms (18, 24).
Regarding the results of the effect of the treatment protocol on caspase-3 activity, both drugs were unable to initiate apoptotic cell death in PC3 cells when used individually while their combination induced apoptosis in PC3 cells. Our observation is concordant with the results of a previous study that has been conducted to elucidate the exact mechanism by which Dactolisib induced cell death in different PCa cell lines (25). However, the inability of GANT61 to initiate apoptosis in PC3 cells has been opposed by the results of a study conducted to assess the effect of inhibition of GLI1 on the proliferation of PC3 cells (26).
Concerning LC3 protein level, only Dactolisib but not GANT61 was able to initiate autophagy in PC3 cells. It was reported that Dactolisib can induce cell death in PC3 cells through initiation of autophagy as long as PC3 cells are PTEN-null in contrast to other PCa cell lines like DU145 which harbors wild-type PTEN (25). However, the statistically significant increase in LC3 level in the combination-treated group compared to that in Dactolisib-treated group may indicate that GANT61 augmented Dactolisib in initiating autophagy in PC3 cells.
Considering the effect of the treatment protocol on cell cycle progression, the results demonstrated that both drugs and their combination have proven their ability to arrest cell cycle in PC3 cells. Taking into consideration the studies (27, 28) that have reported cyclin D1 as one of the downstream genes for GLI1 transcriptional factor, inhibition of GLI1 by GANT61 could explain our finding regarding the suppression of Hh/ GLI pathway activity. Additionally, the cell cycle arrest that resulted from the dual inhibition of PI3K/mTOR by Dactolisib could be explained by the activation of glycogen synthase kinase 3 (29) which phosphorylates and promotes the degradation of cyclin D1 (30) and also by the inhibition of mTOR-related translational pathways resulting in the down-regulation of cyclin D1 protein expression (31).
Concerning the ability of PC3 cells for angiogenesis, treating cells with Dactolisib/GANT61 combination or with Dactolisib or GANT61 as single treatments has led to suppression of angiogenesis as documented by lower levels of VEGF1. Our results herein could definitely match the finding of another study that has reported VEGF1 as one of the downstream genes for GLI1 transcriptional factor (9). Therefore, inhibiting GLI1 activity by GANT61 has decreased the angiogenesis potentiality of PC3 cells. Moreover, suppression of angiogenesis capability of PC3 cells after treatment with Dactolisib may be attributed to the reduction of the activity of Hypoxia-inducible factor 1 (HIF1), another transcriptional factor that controls the expression of VEGF1 gene as described by a previous study that has clarified the role of PI3K and mTOR in the stabilization of HIF1α subunit (32).
To sum up, the present findings support the implication of PI3K/AKT/mTOR and Hh/GLI pathways and their crosstalk in PC3 cell cycle progression and in its ability for angiogenesis. Also, the current findings highlighted the possible additive/synergistic effect of Dactolisib and GANT61 in suppressing the viability of PC3 cells and in initiating programmed cell death through apoptosis and/or autophagy pathways. However, these findings still need further in-vitro and in-vivo studies to guarantee the applicability of implementing such drug combination in treating PCa.