Recently, emerging evidence suggests that PCSCs may play a pivotal role in the development and progression of prostate cancer [15, 16]. The Notch signaling pathway is vital to tumorigenicity of the CSCs, and thus receiving increased attention as a target to eliminate CSCs [9, 10]. Therefore, targeting PCSCs through inhibition of Notch pathway could become a novel strategy for better treatment of patients diagnosed with prostate cancer. PCSCs has been isolated from PC-3 cell line and demonstrate resistance to chemotherapy in our previous study [6]. In the present study, we found that the expression of Notch-1 was increased in PC-3 PCSCs compared to parental cells. Furthermore, inhibition of Notch pathway using GSI enhanced the anti-tumor effect of DOX in PC-3 PCSCs. Thus, targeting PCSCs and enhancing DOX activity would provide a major benefit to patients with advanced prostate cancer.
The Notch signaling pathway controls cell-fate decisions during development, including differentiation, proliferation, stem cell maintenance, and self-renewal of various cell types [17]. Moreover, increasing evidence have shown that Notch pathway critically regulates the self-renewal and survival of CSCs in breast cancer, embryonal brain tumors, and gliomas [18]. In terms of prostate cancer, Oktem and colleagues identified CD133high/CD44high DU145 prostate CSCs and found that Jagged1, Delta‑like 3 and Notch-1 were respectively upregulated genes in the Notch signaling pathway appearing to be due to malignancy and tumor progression [19]. Furthermore, Liu and colleagues revealed that the protein level of activated form of Notch-1 was significantly higher in PCSCs isolated from LNCaP and PC-3 cell lines, and inhibition of Notch-1 with shRNA could improve chemosensitivity in PCSCs.[20] We have proved that PCSCs are resistant to DOX in our previous study [6]. In the present study, we also found the expression of Notch-1 mRNA was significantly increased in PC-3 PCSCs compared to parental cells, and inhibition of Notch pathway by GSI can enhance the chemosensitivity of PCSCs to DOX. These findings suggest that Notch pathway may play a key role in the tumorigenicity and chemoresistant of PCSCs. Therefore, targeting Notch signaling pathway as a therapeutic strategy for treating cancer has attracted increasing interest.
γ-Secretase is a key mediator of Notch signaling [21]. The activation of Notch pathway mainly depends on the γ-secretase enzyme activity that helps in the proteolytic cleavage of the receptors resulting in the release of the active intracellular fragment [10]. Thus, GSI is a promising target for Notch inhibition. PF-03084014 is a small molecule reversible, non-competitive and selective GSI, which has been shown to inhibit Notch pathway through blocking cleaved-notch receptor formation in various types of cancer [14, 22–24]. Recently, a few studies have found that inhibition of Notch signaling using PF-03084014 sensitized DOX-resistant prostate cancer cells to DOX [12, 25]. However, it is not completely clear that pharmacological targeting of the Notch pathway by PF-03084014 could impact DOX chemoresistance in prostate CSCs. In this study, we revealed that PF-03084014 enhanced the chemosensitivity of PC-3 PCSCs to DOX, promoted apoptosis and cell cycle arrest induced by DOX, and inhibited the self-renewal potentiality of PC-3 PCSCs in vitro. In addition, combination therapy of GSI with DOX reduced the PC-3 PCSCs-derived tumor growth in vivo, which was associated with the decreased Notch-1 expression in tumor tissues.
DOX is an antimitotic chemotherapy drug that interferes with cell division, and widely used for treatment of multiple types of cancer. However, recent studies have suggested CSCs as a main player for chemoresistance against a variety of drugs including DOX [26]. In our study, PF-03084014 enhanced the chemosensitivity and promoted apoptosis of PC-3 PCSCs to DOX due to the suppression of PCSCs by the inhibition of Notch pathway. In prostate cancer cells, DOX caused phosphorylation and hence inactivation of CDC2 kinase resulting in G2/M arrest [27]; whereas, targeting the Notch pathway induced cell cycle arrest at S phase [28]. The combination of DOX and GSI induced S and G2/M arrest in PC-3 PCSCs, suggesting that combination of the two agents may complement each other to result in enhanced arrest.
CSCs have self-renewal capacity and are considered linked to elevated Notch pathway signal [18]. The sphere formation assay is a classic method for the analysis of their self-renewal ability [3]. PC-3 PCSCs were resistant to chemotherapy, and DOX treatment didn’t inhibit the sphere formation of PC-3 PCSCs. However, the combination of DOX with GSI decreased the sphere formation and resulted in smaller sphere size in PC-3 PCSCs compared with monotherapy. The sphere formation results suggested a synergistic effect of DOX and GSI in inhibiting self-renew of PC-3 PCSCs.
The well know toxicities of GSI is associated with gastrointestinal tract leading to severe adverse diarrhea [10]. The intermittent dosing of PF-03084014 reduced gastrointestinal toxicity [14]. Thereby, we used the intermittent dosage regimen of PF-03084014 in the animal study, and there were no significant decreases in the mean body weights of mice treated with DOX, GSI, or combination therapy compared with control group, which indicates no severe toxicity due to monotherapy or combination therapy. Thus, DOX combined with GSI was not only effective but safe and well tolerated.
However, it should be recognized that tissue-specific stem cell therapy for prostate cancer is still in its infant stage and much more studies and optimizations are required to execute its full power in clinical treatment of prostate cancer patients [15]. Therefore, the specific mechanism of combination therapy of DOX with GSI on PCSCs needs further investigation, and this combination therapy should be evaluated in clinical trials for the therapy of advanced prostate cancer.