Crocus sativusL. extracts (saffron) are rich in carotenoids. Preclinical studies have shown that dietary intake of carotenoids has antitumor effects suggesting their potential preventive and/or therapeutic roles. We have recently reported that saffron (SE) and crocin (CR) exhibit anticancer activity by promoting cell cycle arrest in prostate cancer (PCa) cells. It has also been demonstrated that crocetin esters are produced after SE gastrointestinal digestion by CR hydrolysis. The aim of the present report was to investigate if SE, crocetin (CCT), and CR affectedin vivotumor growth of two aggressive PCa cell lines (PC3 and 22rv1) which were xenografted in male nude mice treated by oral gavage with SE, CR, and CCT. We demonstrated that the antitumor effects of CCT were higher when compared to CR and SE and treatments reverted the epithelial-mesenchymal transdifferentiation (EMT) as attested by the significant reduction of N-cadherin and beta-catenin expression and the increased expression of E-cadherin. Additionally, SE, CR, and CCT inhibited PCa cell invasion and migration through the downmodulation of metalloproteinase and urokinase expression/activity suggesting that these agents may affect metastatic processes. Our findings suggest that CR and CCT may be dietary phytochemicals with potential antitumor effects in biologically aggressive PCa cells.

In this study, we investigated the antiproliferative effects of saffron extract (SE) and its major constituent crocin on 5 different malignant and 2 nonmalignant prostate cancer cell lines. Using high performance liquid chromatography (HPLC), the purity and content of crocin were determined. All cells were incubated with different concentrations of SE or crocin for 48 h. Cell cycle and apoptosis were also evaluated. In a time- and concentration-dependent manner, both SE and crocin reduced cell proliferation in all malignant cell lines with IC₅₀ values ranging between 0.4 and 4 mg/ml for SE and between 0.26 and 0.95 mM/ml for crocin. Nonmalignant cells were not affected. Flow cytometry profiles revealed that most cells were arrested at G₀/G₁ phase with a significant presence of apoptotic cells. Western blot analysis revealed that the expression of Bcl-2 was strikingly downregulated, whereas Bax was upregulated. Analysis of caspase activity indicated a caspase-dependent pathway with involvement of caspase-9 activation, suggesting an intrinsic pathway. Based on these findings, it can be concluded that both SE and crocin can inhibit cell proliferation, arrest cell cycle progression, inducing apoptosis in prostate cancer. Consequently, these agents could potentially be used as a chemopreventive as well as a chemotherapeutic agent for prostate cancer management.

Over recent years, many authors discussed the effects of different natural compounds on glioblastoma (GBM). Due to its capacity to impair survival and progression of different cancer types, saffron extract (SE), named crocetin (CCT), is particularly noteworthy. In this work, we elucidated the antitumor properties of crocetin in glioma in vivo and in vitro models for the first time. The in vitro results showed that the four tumor cell lines observed in this study (U251, U87, U138, and U373), which were treated with increasing doses of crocetin, showed antiproliferative and pro-differentiative effects as demonstrated by a significant reduction in the number of viable cells, deep changes in cell morphology, and the modulation of mesenchymal and neuronal markers. Indeed, crocetin decreased the expression of Cluster of Differentiation CD44, CD90, CXCR4, and OCT3/4 mesenchymal markers, but increased the expression of βIII-Tubulin and neurofilaments (NFH) neuronal linage-related markers. Epigenetic mechanisms may modulate these changes, since Histone Deacetylase, HDAC1 and HDAC3 were downmodulated in U251 and U87 cells, whereas HDAC1 expression was downmodulated in U138 and U373 cells. Western blotting analyses of Fatty Acid Synthase, FASN, and CD44 resulted in effective inhibition of these markers after CCT treatment, which was associated with important activation of the apoptosis program and reduced glioma cell movement and wound repair. The in vivo studies aligned with the results obtained in vitro. Indeed, crocetin was demonstrated to inhibit the growth of U251 and U87 cells that were subcutaneously injected into animal models. In particular, the Tumor To Progression or TTP values and Kaplan–Meier curves indicated that crocetin had more major effects than radiotherapy alone, but similar effects to temozolomide (TMZ). An intra-brain cell inoculation of a small number of luciferase-transfected U251 cells provided a model that was able to recapitulate recurrence after surgical tumor removal. The results obtained from the orthotopic intra-brain model indicated that CCT treatment increased the disease-free survival (DFS) and overall survival (OS) rates, inducing a delay in appearance of a detectable bioluminescent lesion. CCT showed greater efficacy than Radio Therapy (RT) but comparable efficacy to temozolomide in xenograft models. Therefore, we aimed to continue the study of crocetin’s effects in glioma disease, focusing our attention on the radiosensitizing properties of the natural compound and highlighting the ways in which this was realized.

Glioblastoma recurrence after treatment with the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab is characterized by a highly infiltrative and malignant behavior that renders surgical excision and chemotherapy ineffective. It has been demonstrated that anti-VEGF/VEGFR therapies control the invasive phenotype and that relapse occurs through the increased activity of CXCR4. We therefore hypothesized that combining bevacizumab or sunitinib with the novel CXCR4 antagonist, PRX177561, would have superior antitumor activity. The effects of bevacizumab, sunitinib, and PRX177561 were tested alone or in combination in subcutaneous xenografts of U87MG, U251, and T98G cells as well as on intracranial xenografts of luciferase tagged U87MG cells injected in CD1-nu/nu mice. Animals were randomized to receive vehicle, bevacizumab (4 mg/kg iv every 4 days), sunitinib (40 mg/kg po qd), or PRX177561 (50 mg/kg po qd). The in vivo experiments demonstrated that bevacizumab and sunitinib increase the in vivo expression of CXCR4, SDF-1α, and TGFβ1. In addition, we demonstrate that the co-administration of the novel brain-penetrating CXCR4 antagonist, PRX177561, with bevacizumab or sunitinib inhibited tumor growth and reduced the inflammation. The combination of PRX177561 with bevacizumab resulted in a synergistic reduction of tumor growth with an increase of disease-free survival (DSF) and overall survival (OS), whereas the combination of PRX177561 with sunitinib showed a mild additive effect. The CXC4 antagonist PRX177561 may be a valid therapeutic complement to anti-angiogenic therapy, particularly when used in combination with VEGF/VEGFR inhibitors. Therefore, this compound deserves to be considered for future clinical evaluation.

Exportin 1 (XPO1), also called chromosome region maintenance 1 (CRM1), is the sole exportin mediating transport of many multiple tumor suppressor proteins out of the nucleus. To verify the hypothesis that XPO1 inhibition affects prostate cancer (PCa) metastatic potential, orally available, potent and selective, SINE compounds, Selinexor (KPT- 330) and KPT-251, were tested in preclinical models known to generate bone lesions and systemic tumor spread. In vitro, Selinexor reduced both secretion of proteases and ability to migrate and invade of PCa cells. SINEs impaired secretion of pro-angiogenic and pro-osteolytic cytokines and reduced osteoclastogenesis in RAW264.7 cells. In the intra-prostatic growth model, Selinexor reduced DU145 tumor growth by 41% and 61% at the doses of 4 mg/Kg qd/5 days and 10 mg/Kg q2dx3 weeks, respectively, as well as the incidence of macroscopic visceral metastases. In a systemic metastasis model, following intracardiac injection of PCb2 cells, 80% (8/10) of controls, 10% (1/10) Selinexor- and 20% (2/10) KPT-251-treated animals developed radiographic evidence of lytic bone lesions. Similarly, after intra-tibial injection, the lytic areas were higher in controls than in Selinexor and KPT-251 groups. Analogously, the serum levels of osteoclast markers (mTRAP and type I collagen fragment, CTX), were significantly higher in controls than in Selinexor- and KPT-251-treated animals. Importantly, overall survival and disease-free survival were significantly higher in Selinexor- and KPT-251-treated animals when compared to controls. Selective blockade of XPO1-dependent nuclear export represents a completely novel approach for the treatment of advanced and metastatic PCa.

DNA methylation might be the earliest somatic genome changes in prostate cancer that also play an important role in the process of tumor invasion, growth and metastasis. In recent years, several inhibitors of DNA methyltransferases (DNMTis) have been developed and evaluated in pre-clinical models and in clinical trials. While these compounds are effective in the treatment of hematological conditions, clinical trials in solid tumors and in prostate cancer have shown limited or no efficacy. This may be attributed to inappropriate dose regimens leading to toxicity-related adverse events. As with other anti-target compounds, one of the obstacles encountered with DNMTis in prostate cancer could be the inability to select patients for the clinical studies as well as the inability to monitor the efficacy of the drug if not the conclusion of the study. Primary cultures derived from human prostatic tissues harvested from patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa) as well as neoplastic and non-neoplastic prostate cell lines were tested for DNMT expression/activity and to monitor azacitidine molecular efficacy. We observed that in primary cultures the levels of DNMT activity as well as the protein levels of DNMT1, DNMT3a and DNMT3b were higher in cultures derived from PCa compared to BPH tissue samples and significantly higher in cultures derived from PCa with Gleason scores ≥7 compared to those observed in cultures derived from Gleason scores <7. In addition, DNMT activity as well as DNMT1, DNMT3a and DNMT3b levels were higher in PCa cell lines compared to their non-neoplastic counterparts. Although DNMT activity was higher in high tumorigenic/aggressive PCa cell lines compared to low tumorigenic/aggressive cell lines, only the levels of DNMT3a and DNMT3b were significantly higher in the first group of cells, suggesting that DNMT1 activity is related to the transition to non-neoplastic versus neoplastic phenotype whereas the de novo methylation enzymes were mainly related to progression. Nevertheless, the comparison in the more aggressive PC3 cell derivatives (PC3-LN4 cells) also possessed higher levels of DNMT1 compared to PC3 and PC3M from which these cells were derived. Collectively, our results confirm previous data on the increased methylation in more aggressive tumors supporting the use of DNMTis in advanced prostate cancer. In addition, since glutathione S-transferase-π (GSTP1) was re-expressed or its protein levels were increased after treatment with non-toxic azacitidine doses and since GSTP1 can easily be measured in patient sera, the monitoring of this protein may aide in the evaluation of therapy in future clinical trials.