Inhibiting WNT and NOTCH in renal cancer stem cells and the implications for human patients.

Current treatments for clear cell renal cell cancer (ccRCC) are insufficient because two-thirds of patients with metastases progress within two years. Here we report the identification and characterization of a cancer stem cell (CSC) population in ccRCC. CSCs are quantitatively correlated with tumor aggressiveness and metastasis. Transcriptional profiling and single cell sequencing reveal that these CSCs exhibit an activation of WNT and NOTCH signaling. A significant obstacle to the development of rational treatments has been the discrepancy between model systems and the in vivo situation of patients. To address this, we use CSCs to establish non-adherent sphere cultures, 3D tumor organoids, and xenografts. Treatment with WNT and NOTCH inhibitors blocks the proliferation and self-renewal of CSCs in sphere cultures and organoids, and impairs tumor growth in patient-derived xenografts in mice. These findings suggest that our approach is a promising route towards the development of personalized treatments for individual patients.

VEGF - Supplemented extracellular matrix is sufficient to induce endothelial differentiation of human iPSC.

Extracellular matrix (ECM) provides a scaffold for cells and tissues, but also supports organogenesis and tissue remodeling. The required instructive properties of the ECM to interact with cells depend on matrix architecture, structural proteins and functional matrix components such as growth factors, providing spatial, chemical and functional cues. Decellularized ECM (dECM) has been proposed as an instructive material that promotes tissue regeneration. We investigated the instructive ECM elements preserved in dECM and necessary to promote endothelial differentiation of human induced pluripotent stem cells (hiPSC). We show that detergent-decellularized human kidney ECM remains structurally intact and carries a number of heparin-binding growth factors, including FGF2, VEGF, BMP2, HGF, EGF, PDGF-BB and TGFß, albeit at reduced levels compared to native tissues. Clearance of these heparin-binding factors, or heparan-sulfate proteoclycans from ECM resulted in massively reduced differentiation of hiPSC, suggesting that remaining structural dECM proteins such as laminin, collagen or fibronectin alone are not instructive. In contrast, replenishing dECM with VEGF replaced medium-supplemented VEGF and resulted in more efficient differentiation of hiPSC into endothelial cells, and even in the absence of other culture-supplemented differentiation factors dECM alone was superior to geltrex. In conclusion, conditioning of dECM with specific growth factors acting as functional cues may allow to generate functional niches by selective promotion of cell attachment, survival and differentiation.

Punicalagin, a polyphenol from pomegranate fruit, induces growth inhibition and apoptosis in human PC-3 and LNCaP cells.

Prostate cancer (PCa) is an international health problem and search for its effective treatment is in progress. Punicalagin (PN), polyphenol from pomegranate fruit, is known to exhibit potent anticancer activity in lung, breast and cervical cells. However, there is paucity of information on its effect in PCa. This study evaluated anti-proliferative effects of PN and its effects on extrinsic pathway of apoptosis in PCa cells, and angiogenesis in chicken chorioallantoic membrane (CAM). Antioxidant activities of PN were determined by 2,2-diphenyl-1-picryhydrazyl (DPPH) radical scavenging and inhibition of lipid peroxidation (LPO) methods. PCa (PC-3 and LNCaP) and normal prostate (BPH-1) cells were cultured and treated with PN (10, 50 and 100 µM). Cytotoxicity and viability effects of PN were determined by lactate dehydrogenase (LDH) and XTT assays, respectively. Antiangiogenic effects were measured using CAM assay, while apoptosis was assessed by DNA fragmentation, enrichment factor by Cell Death Detection ELISA kit and expressions of caspases-3 and -8. Results showed that PN (10-200 µM) significantly scavenged DPPH and inhibited LPO in a concentration-dependent manner. Furthermore, PN (10-100 µM) concentration-dependently inhibited viability in PC-3 and LNCaP, while viability in BPH-1 was insignificantly affected. PN had low toxicity on cells in vitro at concentrations tested. Also, PN (100 µM) increased enrichment factor in PC-3 (2.34 ± 0.05) and LNCaP (2.31 ± 0.26) relative to control (1.00 ± 0.00). In addition, PN (50 µM) decreased the network of vessels in CAM, suggesting its anti-angiogenic effect. Moreso, PN increased the expressions of caspases-3 and -8 in PC-3. Overall, PN exerts anti-proliferative activity in PCa cells via induction of apoptosis and anti-angiogenic effect.

Antioxidant and antiproliferative potentials of methanol extract of Xylopia aethiopica (Dunal) A. Rich in PC-3 and LNCaP cells.

BACKGROUND: Our previous studies showed that fruit methanol extract from Xylopia aethiopica (MEXA) exhibited antiproliferative activity in human cervical cancer cells via the induction of apoptosis. The present study was designed to assess the antiproliferative, antiangiogenic and antioxidant effects of MEXA on prostate cancer (PCa) cells (PC-3 and LNCaP). METHODS: PC-3 and LNCaP cells were cultured and treated with MEXA (10, 50 and 100 µg/mL). The sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) and lactate dehydrogenase (LDH) assays were used to evaluate cell viability and cytotoxicity, respectively. DNA fragmentation was determined by cell death detection ELISA plus, and angiogenesis was assessed by chicken chorioallantoic membrane (CAM) assay. The antioxidant activities of MEXA were determined by DPPH and hydroxyl (OH) radicals' scavenging methods as well as through the inhibition of lipid peroxidation (LPO) in rats' liver homogenate. RESULTS: MEXA at 100, 250 and 500 µg/mL scavenged DPPH by 48%, 62%, 70% and OH radical by 39%, 58%, 67%, respectively. MEXA significantly (p<0.05) inhibited LPO in a concentration-dependent manner. In addition, MEXA had antiproliferative effects on PC-3 and LNCaP with IC50 of 62.1 and 73.6 µg/mL, respectively, at 96 h. The LDH assay showed that MEXA had low toxicity in vitro at its IC50 values. The extent of DNA fragmentation by MEXA showed higher values in PC-3 and LNCaP, suggesting the possible induction of apoptosis. In contrast, MEXA did not affect the network of vessels in CAM, thus lacking anti-angiogenic property. CONCLUSIONS: These findings suggest that MEXA induces antiproliferative activity in PCa cells through a mechanism that involves apoptosis. Therefore, MEXA may be a potential therapeutic agent for PCa.

Antioxidant, antiangiogenic and antiproliferative activities of root methanol extract of Calliandra portoricensis in human prostate cancer cells.

OBJECTIVE: Prostate cancer (PCa) is a major health concern. Calliandra portoricensis (CP) is traditionally known for its analgesic, anti-ulcerogenic and anticonvulsant properties. However, its antiproliferative properties for PCa still need to be investigated. METHODS: Antioxidant activities of CP were determined by 1,1-diphenyl-2-picryhydrazyl (DPPH) and hydroxyl (OH(-)) radicals-scavenging methods. PC-3 and LNCaP (androgen-refractory and androgen-dependent PCa-derived cell lines) were cultured and treated with CP (10, 50 and 100 µg/mL). Effects of CP on cells were determined by cytotoxicity assay (lactate dehydrogenase, LDH) and viability assay (sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate, XTT). DNA fragmentation was detected by cell death detection enzyme-linked immunosorbent assay plus kit. CP was tested as an inhibitor of angiogenesis using chicken chorioallantoic membrane (CAM) assay. RESULTS: CP showed significant scavenging of DPPH and OH(-) radicals. CP significantly (P<0.05) inhibited lipid peroxidation in a dose-dependent manner. Precisely, CP (10, 50 and 100 µg/mL) inhibited PC-3 and LNCaP growth by 7%, 74% and 92%, and 27%, 73%, and 85% respectively at 48 h. CP had low toxicity in vitro at its half inhibitory concentration dose. Detection of cell death induced by CP at 50 µg/mL showed higher enrichment factors in LNCaP (7.38±0.95) than PC-3 (3.48±0.55). Also, treatment with CP (50 µg/mL) significantly reduced network of vessels in CAM, suggesting its antiangiogenic potential. CONCLUSION: Calliandra portoricensis elicited antioxidant, antiangiogenic and antiproliferative effects in PCa cells.