As previously noted, prostate cancer is the second most frequent cancer in men (1). Routine therapies for prostate cancer include chemotherapy, radiotherapy, and androgen deprivation therapy. However, the development of castration resistance, cancer recurrence, metastatic disease, and therapeutic regimen adverse effects are the main problems and have limited the treatment efficacy (5, 20). Hence, it’s vital to find new potential therapy that has no toxic effects on healthy cells. In the current study, prostate cancer cell lines, PC3 and LNCaP were treated with AP (10 µM) and Hesperidin (50 and 100 µM) alone and in combination groups. To the best of our knowledge, this is the first study that is aimed to assess the synergism effects of AP and Hesperidin on cancer cells. Our results showed that the combinations of 50 µM Hesperidin + 10 µM AP and 100 µM Hesperidin + 10 µM AP induce a significantly higher rate of apoptosis in both PC3 and LNCaP cell lines. On contrary, Hesperidin did not affect the normal cell line (HFF-1). The IC50 of the normal cell line (HFF-1) after treatment with Hesperidin was almost 2 times and 20 times higher than LnCaP and PC3 cell lines respectively. These results showed the safety of Hesperidin on the normal cell line, while it induces apoptosis in prostate cancer cells.
The mechanism underlying the synergism effect of AP and Hesperidin can be due to several molecular pathways. ROS is one of the potential pathways, especially for AP. ROS formation during metabolism has been shown in different physiological functions (21). In fact, the balance between ROS production and its scavenging with antioxidants has been properly maintained in healthy cells (22). However, cancer cells have dysregulated ROS hemostasis, leading to a higher ROS generation (22). A higher level of ROS has an antiapoptotic effect as a result of redox-sensitive transcription activation which includes nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) (23). NF-κB is located in the cytosol of healthy cells which is bonded to IκBα as inactive forms. Nevertheless, IκBα phosphorylation forms active NF-κB in cancer cells which can inhibit apoptosis and result in uncontrol cell growth (24). In fact, NF-κB can inhibit apoptosis by elevating anti-apoptotic genes including Bcl-2 and survivin (25). Previous studies showed the overexpression of Bcl-2 in prostate cancer, B-cell lymphomas, colorectal cancer, and breast cancer (26). Therefore, reducing the ROS level in prostate cancer cells can lead to reduced antiapoptotic effects as a result of lower Bcl-2 and survivin gene expression. The results of the current study showed that the combination of 10 µM AP + 50 µM Hesperidin significantly reduced the ROS level in the PC3 cell line. Also, Bcl-2 and Survivin gene expression significantly decreased with the combination of 50 µM Hesperidin + 10 µM AP, and 100 µM Hesperidin + 10 µM AP in the PC3 cell line. Almost similar results were found in the LNCaP cell line. This mechanism can be one of the potential anti-cancer effects of Hesperidin and AP on prostatic cancer cells. On contrary, in a study done by Ning et al on prostate cancer cells., it was shown that hesperidin can decrease cell growth and viability in a dose-dependent manner as a result of ROS elevation and MMP reduction (17). These results can be explained by the double sword feature of ROS, in which both lowering and elevating the ROS level in cancer cells can induce apoptosis and were identified to be potent therapeutic approaches in the cancer management (27).
On the other hand, P53 and P21 pathways seem to be another potential anti-cancer mechanism, especially for Hesperidin on prostatic cancer cells. Various cell functions are regulated with P53 tumor suppressor factor including cell growth, invasion, and migration (28). In addition, P21 plays an important role in the cell-growth arrest, senescence, and suppression of the cell invasion (29). Since cancer cells have dysregulated cell cycles, cell cycle arrest can be a potential target for anti-cancer treatments. The results of our study demonstrated that P53 and P21 gene expression significantly increased after treatment with 50 and 100 µM Hesperidin with or without 10 µM AP in the PC3 cell line. Also, 50 and 100 µM Hesperidin significantly increased the gene expression of P53 and P21 in the LnCaP cell line. Previous studies have shown the role of Hesperidin in cell cycle arrest in various cancer cells in different checkpoints including the G1, G2/M, or S phases (30, 31). Its cell cycle arrest role can be due to the modulation of cell cycle regulatory proteins such as cyclins, cyclin-dependant kinases (CDK), and CDK inhibitors (32). In addition, it was reported that Hesperidin can increase P21 in various cancer cells including leukemia cell lines, colon cancer, breast cancer, and lung cancer (33). On the other hand, it was shown that AP can cause cell cycle arrest in G2/M and significantly decrease cyclin B1, as well as, increase P21 in other types of cancer cell lines (34). Besides, it was stated that AP can cause cancer cell death by regulating different pathways such as cell cycle-related genes (c-Myc, cyclin D1, cyclin B1, p21), P53, PI3K / Akt/ NF-kB, and apoptosis target genes (Bcl-2 and Bax) (35). However, our results did not show significant changes in P53 and P21 in prostate cancer cells after treatment with 10 µM AP alone.
Furthermore, SP and its primary receptor, NK1R can be another pathway for the anti-cancer effect. As previously noted, SP’s binding to NK1R can induce cancer cell progression, metastasis, and angiogenesis (10). Further studies showed that prostate cancer cells overexpress NK1R (9). Based on the role of SP/NK1R system in initiating and progression of cancer cells, it seems to be a potential target for anti-cancer treatments. Toward this end, Ebrahimi et al. stated SP/NK1R can induce proliferation and migration in prostate cancer cells by affecting apoptosis-related genes, cell cycle-related proteins, and increasing MMP-2 and MMP-9 expression (11). They showed Aprepitant can reverse these effects in both in vitro and in vivo experiments on prostate cancer cells (11).
Although our results for the first time, showed the synergic anti-cancer effect of Hesperidin and AP on prostate cancer cells in both PC3 and LNCaP cell lines, this effect can be well explained by P53, P21, Bcl-2, Survivin, and ROS pathways only in the PC3 cell line. However, our results for these possible pathways of the anti-cancer effect of Hesperidin and AP were not straightforward in the LnCaP cell line. One possible explanation for these results can be the natural difference between the PC3 and LNCaP cell lines. For instance, a prior study showed Hesperidin can inhibit the testosterone-induced cell proliferation of the LNCaP cell line, while it had no effect on hormone-independent prostate cancer cells, PC3 (16). Therefore, the hormonal effects of these treatments can be an explanation for this difference and another potential pathway for their anti-cancer effects. In addition, the following limitations should be considered in this study. First, SP/NK1R pathway in prostate cancer cells was not fully investigated in the current study. Second, the signaling pathways of NF-κB were not evaluated. Finally, this is in vitro investigation and further in vivo studies are needed on this subject.