Alpha-Tomatine Induces Apoptosis and Inhibits Nuclear Factor-Kappa B Activation on Human Prostatic Adenocarcinoma PC-3 Cells

BACKGROUND: Alpha-tomatine (α-tomatine) is the major saponin in tomato (Lycopersicon esculentum). This study investigates the chemopreventive potential of α-tomatine on androgen-independent human prostatic adenocarcinoma PC-3 cells. METHODOLOGY/PRINCIPAL FINDINGS: Treatment of highly aggressive human prostate cancer PC-3 cells with α-tomatine resulted in a concentration-dependent inhibition of cell growth with a half-maximal efficient concentration (EC(50)) value of 1.67±0.3 µM. It is also less cytotoxic to normal human liver WRL-68 cells and normal human prostate RWPE-1 cells. Assessment of real-time growth kinetics by cell impedance-based Real-Time Cell Analyzer (RTCA) showed that α-tomatine exhibited its cytotoxic effects against PC-3 cells as early as an hour after treatment. The inhibitory effect of α-tomatine on PC-3 cancer cell growth was mainly due to induction of apoptosis as evidenced by positive Annexin V staining and decreased in mitochondrial membrane potential but increased in nuclear condensation, polarization of F-actin, cell membrane permeability and cytochrome c expressions. Results also showed that α-tomatine induced activation of caspase-3, -8 and -9, suggesting that both intrinsic and extrinsic apoptosis pathways are involved. Furthermore, nuclear factor-kappa B (NF-κB) nuclear translocation was inhibited, which in turn resulted in significant decreased in NF-κB/p50 and NF-κB/p65 in the nuclear fraction of the treated cells compared to the control untreated cells. These results provide further insights into the molecular mechanism of the anti-proliferative actions of α-tomatine. CONCLUSION/SIGNIFICANCE: α-tomatine induces apoptosis and inhibits NF-κB activation on prostate cancer cells. These results suggest that α-tomatine may be beneficial for protection against prostate cancer development and progression

Increased Expression of PcG Protein YY1 Negatively Regulates B Cell Development while Allowing Accumulation of Myeloid Cells and LT-HSC Cells

Ying Yang 1 (YY1) is a multifunctional Polycomb Group (PcG) transcription factor that binds to multiple enhancer binding sites in the immunoglobulin (Ig) loci and plays vital roles in early B cell development. PcG proteins have important functions in hematopoietic stem cell renewal and YY1 is the only mammalian PcG protein with DNA binding specificity. Conditional knock-out of YY1 in the mouse B cell lineage results in arrest at the pro-B cell stage, and dosage effects have been observed at various YY1 expression levels. To investigate the impact of elevated YY1 expression on hematopoetic development, we utilized a mouse in vivo bone marrow reconstitution system. We found that mouse bone marrow cells expressing elevated levels of YY1 exhibited a selective disadvantage as they progressed from hematopoietic stem/progenitor cells to pro-B, pre-B, immature B and re-circulating B cell stages, but no disadvantage of YY1 over-expression was observed in myeloid lineage cells. Furthermore, mouse bone marrow cells expressing elevated levels of YY1 displayed enrichment for cells with surface markers characteristic of long-term hematopoietic stem cells (HSC). YY1 expression induced apoptosis in mouse B cell lines in vitro, and resulted in down-regulated expression of anti-apoptotic genes Bcl-xl and NFκB2, while no impact was observed in a mouse myeloid line. B cell apoptosis and LT-HSC enrichment induced by YY1 suggest that novel strategies to induce YY1 expression could have beneficial effects in the treatment of B lineage malignancies while preserving normal HSCs

3D bioactive composite scaffolds for bone tissue engineering

Bone is the second most commonly transplanted tissue worldwide, with over four million operations using bone grafts or bone substitute materials annually to treat bone defects. However, significant limitations affect current treatment options and clinical demand for bone grafts continues to rise due to conditions such as trauma, cancer, infection and arthritis. Developing bioactive three-dimensional (3D) scaffolds to support bone regeneration has therefore become a key area of focus within bone tissue engineering (BTE). A variety of materials and manufacturing methods including 3D printing have been used to create novel alternatives to traditional bone grafts. However, individual groups of materials including polymers, ceramics and hydrogels have been unable to fully replicate the properties of bone when used alone. Favourable material properties can be combined and bioactivity improved when groups of materials are used together in composite 3D scaffolds. This review will therefore consider the ideal properties of bioactive composite 3D scaffolds and examine recent use of polymers, hydrogels, metals, ceramics and bio-glasses in BTE. Scaffold fabrication methodology, mechanical performance, biocompatibility, bioactivity, and potential clinical translations will be discussed

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

In Vitro and In Vivo pro-apoptotic and chemosensitizing effects of alpha-tomatine on human prostate adenocarcinoma / Lee Sui Ting

Alpha (α)-tomatine is a major saponin found in tomatoes (Lycopersicon esculentum). The present study investigates the molecular mechanisms by which α-tomatine exerts its anti-cancer effect on human prostatic adenocarcinoma cells. Treatment of human androgen-dependent LNCaP and androgen-independent PC-3 prostate cancer cells with α-tomatine resulted in a concentration-dependent inhibition of cell growth with a halfmaximal efficient concentration (EC50) value of 2.65 ± 0.1 μM and 1.67 ± 0.3 μM, respectively. PC-3 cells appear to be more sensitive to α-tomatine-induced growth inhibition compared to LNCaP cells. Importantly, α-tomatine treatment is also less cytotoxic to non-tumorigenic human liver WRL-68 and human prostatic epithelial RWPE-1 cells. Due to the higher sensitivity of PC-3 cells to α-tomatine and significant morbidity of metastatic androgen-independent prostate cancer, it is of interest to study in greater detail the mechanisms of action of α-tomatine in PC-3 cells. Results from the present study showed that the inhibitory effect of α-tomatine on PC-3 cell growth was mainly due to the induction of apoptosis via the inhibition of nuclear factor-kappa B (NF-κB) pathway. Alpha-tomatine suppresses both basal constitutive and tumor necrosis factor-alpha (TNF-α)-induced NF-κB activation. The suppression of NF-κB activation by α-tomatine occurs through the inhibition of Akt, leading to the inhibition of IκBα kinase (IKK) activity and subsequently suppression of NF-κB nuclear translocation in PC-3 cells. The inhibition of NF-κB signaling pathway by α-tomatine was accompanied by significant reduction in the expression of NF-κB-dependent antiapoptotic proteins. The anti-tumor study of α-tomatine against PC-3 cells was extended to subcutaneous xenograft and orthotopic mouse models. Intraperitoneal administration of α-tomatine significantly attenuates the growth of PC-3 cell tumors grown at both sites without significant body weight loss. In agreement to the in vitro data, analysis of tumor materials showed an increase in tumor cell apoptosis and a decrease in the basal nuclear localization of NF-κB p50 and p65. The present study further investigated the efficacy of α-tomatine in combination with low-dose of paclitaxel in PC-3 cells. Treatment with sub-toxic dose of α-tomatine in combination with low-dose paclitaxel resulted in a decrease in cell viability with concomitant increase in apoptosis in PC-3 cells but not in non-tumorigenic human prostatic epithelial RWPE-1 cells. Results from these in vitro experiments indicated that the induction of apoptosis by the combined treatment was accompanied by the inhibition of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pro-survival signaling, which is an upstream mediator of NF-κB and known to confer chemoresistance in prostate cancer. The combined treatment also completely suppressed subcutaneous tumor growth in mouse xenograft without apparent body weight loss. Analysis of tumor materials showed an increase in tumor cell apoptosis with a reduction in the protein expression of activated PI3K/Akt. In summary, results from the present study provide comprehensive evidence that α- tomatine is an effective naturally-derived anti-tumor agent against androgenindependent prostate cancer and when used in combination, it can enhance the efficacy of taxane-based agent. The clinical applications of α-tomatine in prostate cancer treatment should be further explored

Alpha-tomatine synergises with paclitaxel to enhance apoptosis of androgen-independent human prostate cancer PC-3 cells in vitro and in vivo

Alpha (α)-tomatine, a major saponin found in tomato has been shown to inhibit the growth of androgen-independent prostate cancer PC-3 cells. The effects of α-tomatine in combination with the chemotherapeutic agent paclitaxel against PC-3 cells were investigated in the present study. Combined treatment with a sub-toxic dose of α-tomatine and paclitaxel significantly decreased cell viability with concomitant increase in the percentage of apoptotic PC-3 cells. The combined treatment, however, had no cytotoxic effect on the non-neoplastic prostate RWPE-1 cells. Apoptosis of PC-3 cells was accompanied by the inhibition of PI3K/Akt pro-survival signaling, an increase in the expression of the pro-apoptotic protein BAD but a decrease in the expressions of anti-apoptotic proteins, Bcl-2 and Bcl-xL. Results from a mouse xenograft model showed the combined treatment completely suppressed subcutaneous tumor growth without significant side effects. Consistent with its in vitro anti-cancer effects, tumor materials from mice showed increased apoptosis of tumor cells with reduced protein expression of activated PI3K/Akt. These results suggest that the synergistic anti-cancer effects of paclitaxel and α-tomatine may be beneficial for refractory prostate cancer treatment

Alpha-Tomatine Attenuation of In Vivo Growth of Subcutaneous and Orthotopic Xenograft Tumors of Human Prostate Carcinoma PC-3 Cells Is Accompanied by Inactivation of Nuclear Factor-Kappa B Signaling

Background: Nuclear factor-kappa B (NF-κB) plays a role in prostate cancer and agents that suppress its activation may inhibit development or progression of this malignancy. Alpha (α)-tomatine is the major saponin present in tomato (Lycopersicon esculentum) and we have previously reported that it suppresses tumor necrosis factor-alpha (TNF-α)-induced nuclear translocation of nuclear factor-kappa B (NF-κB) in androgen-independent prostate cancer PC-3 cells and also potently induces apoptosis of these cells. However, the precise mechanism by which α-tomatine suppresses NF-κB nuclear translocation is yet to be elucidated and the anti-tumor activity of this agent in vivo has not been examined. Methodology/ Principal Findings: In the present study we show that suppression of NF-κB activation by α-tomatine occurs through inhibition of I kappa B alpha (IκBα) kinase activity, leading to sequential suppression of IκBα phosphorylation, IκBα degradation, NF-κB/p65 phosphorylation, and NF-κB p50/p65 nuclear translocation. Consistent with its ability to induce apoptosis, α-tomatine reduced TNF-α induced activation of the pro-survival mediator Akt and its inhibition of NF-κB activation was accompanied by significant reduction in the expression of NF-κB-dependent anti-apoptotic (c-IAP1, c-IAP2, Bcl-2, Bcl-xL, XIAP and survivin) proteins. We also evaluated the antitumor activity of α-tomatine against PC-3 cell tumors grown subcutaneously and orthotopically in mice. Our data indicate that intraperitoneal administration of α-tomatine significantly attenuates the growth of PC-3 cell tumors grown at both sites. Analysis of tumor material indicates that the tumor suppressing effects of α-tomatine were accompanied by increased apoptosis and lower proliferation of tumor cells as well as reduced nuclear translocation of the p50 and p65 components of NF-κB. Conclusion/ Significance: Our study provides first evidence for in vivo antitumor efficacy of α-tomatine against the human androgen-independent prostate cancer. The potential usefulness of α-tomatine in prostate cancer prevention and therapy requires further investigation