Exposure of environmental trace elements in prostate cancer patients: A multiple metal analysis.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths among men. To elucidate the connection between trace elements (arsenic: As, cadmium: Cd, lead: Pb, chromium: Cr, and nickel: Ni) and the risk of PCa, we analyzed trace element levels in the serum, urine, and tissues of PCa patients, while also examining their smoking status. We correlated these levels with their smoking habits. Notably, levels of Cd (P ≤ 0.05) and As (P ≤ 0.01) were significantly higher in the tumor tissue than in adjacent tissues. No significant differences were observed in the levels of Pb, Cr and Ni. Additionally, urinary Cd levels in 70% and arsenic levels in 2.3% of the PCa cohort were markedly higher than the CDC-reported cutoff (Cd ≤ 0.185 µg/L & As ≤100 µg/L). None displayed elevated levels of urinary Pb, Cr, and Ni. Conversely, in serum samples, the concentration of arsenic exceeded the CDC-determined limit (As ≤1.0 µg/L) in 31.69% of PCa patients. However, only 7.04% of patients had higher serum Cd levels than the CDC standard values (Cd ≤ 0.315 µg/L), while all PCa patients exceeded the Cr CDC limit (Cr ≤ 0.16 µg/L) and the Ni CDC limit (Ni ≤ 0.2 µg/L). On the contrary, no significant differences were observed in serum Pb (Pb ≤ 35.0 µg/L). Our findings establish a positive link between Cd and arsenic tissue concentrations and the risk of PCa. Subsequent studies are essential to determine whether elevated trace element levels pose a risk for the development of prostate carcinogenesis. Interestingly, among the PCa cohort comprising smokers, notably higher Cd levels were observed only in tumor tissues (P ≤ 0.01) and urine (P ≤ 0.05) compared to other elements or in other specimens.

The role of autophagy in metal-induced urogenital carcinogenesis.

Environmental and/or occupational exposure to metals such as Arsenic (As), Cadmium (Cd), and Chromium (Cr) have been shown to induce carcinogenesis in various organs, including the urogenital system. However, the mechanisms responsible for metal-induced carcinogenesis remain elusive. We and others have shown that metals are potent inducers of autophagy, which has been suggested to be an adaptive stress response to allow metal-exposed cells to survive in hostile environments. Albeit few, recent experimental studies have shown that As and Cd promote tumorigenesis via autophagy and that inhibition of autophagic signaling suppressed metal-induced carcinogenesis. In light of the newly emerging role of autophagic involvement in metal-induced carcinogenesis, the present review focuses explicitly on the mechanistic role of autophagy and potential signaling pathways involved in As-, Cd-, and Cr-induced urogenital carcinogenesis.

Nitric oxide restores peripheral blood mononuclear cell adhesion against hypoxia via NO-cGMP signalling.

Hypoxia is the most detrimental threat to humans residing at high altitudes, affecting multifaceted cellular responses that are crucial for normal homeostasis. Inhalation of nitric oxide has been successfully implemented to combat the hypoxia effect in the high altitude patients. We hypothesize that nitric oxide (NO) restores the peripheral blood mononuclear cell-matrix deadhesion during hypoxia. In the present study, we investigate the cellular action of exogenous NO in the hypoxia-mediated diminution of cell-matrix adhesion of PBMNC and NO bioavailability in vitro. The result showed that NO level and cell-matrix adhesion of PBMNC were significantly reduced in hypoxia as compared with normoxia, as assessed by the DAF-FM and cell adhesion assay, respectively. In contrast, cellular oxidative damage response was indeed upregulated in hypoxic PBMNC. Further, gene expression analysis revealed that mRNA transcripts of cell adhesion molecules (Integrin α5 and ß1) and eNOS expressions were significantly downregulated. The mechanistic study revealed that administration of NO and 8-Br-cGMP and overexpression of eNOS-GFP restored the basal NO level and recovers cell-matrix adhesion in PBMNC via cGMP-dependent protein kinase I (PKG I) signalling. In conclusion, NO-cGMP/PKG signalling may constitute a novel target to recover high altitude-afflicted cellular deadhesion. SIGNIFICANCE OF THIS STUDY: Cellular adhesion is a complex multistep process. The ability of cells to adhere to extracellular matrix is an essential physiological process for normal homeostasis and function. Hypoxia exposure in the PBMNC culture has been proposed to induce oxidative damage and cellular deadhesion and is generally believed to be the key factor in the reduction of NO bioavailability. In the present study, we demonstrated that NO donor or overexpression of eNOS-GFP has a protective effect against hypoxia-induced cellular deadhesion and greatly improves the redox balance by inhibiting the oxidative stress. Furthermore, this protective effect of NO is mediated by the NO-cGMP/PKG signal pathway, which may provide a potential strategy against hypoxia.

Physical activity and liver diseases.

UNLABELLED: Regular physical activity beneficially impacts the risk of onset and progression of several chronic diseases. However, research regarding the effects of exercising on chronic liver diseases is relatively recent. Most researchers focused on nonalcoholic fatty liver disease (NAFLD), in which increasing clinical and experimental data indicate that skeletal muscle crosstalking to the adipose tissue and the liver regulates intrahepatic fat storage. In this setting, physical activity is considered to be required in combination with calories restriction to allow an effective decrease of intrahepatic lipid component, and despite that evidence is not conclusive, some studies suggest that vigorous activity might be more beneficial than moderate activity to improve NAFLD/nonalcoholic steatohepatitis. Evidence regarding the effects of exercise on the risk of hepatocellular carcinoma is scarce; some epidemiological studies indicate a lower risk in patients regularly and vigorously exercising. In compensated cirrhosis, exercise acutely increases portal pressure, but in the longer term it has been proved safe and probably beneficial. Decreased aerobic capacity (VO2 ) correlates with mortality in patients with decompensated cirrhosis, who are almost invariably sarcopenic. In these patients, VO2 is improved by physical activity, which might also reduce the risk of hepatic encephalopathy through an increase in skeletal muscle mass. In solid organ transplantation recipients, exercise is able to improve lean mass, muscle strength, and, as a consequence, aerobic capacity. Few data exist in liver transplant recipients, in whom exercise should be an object of future studies given its high potential of providing long-term beneficial effects. CONCLUSIONS: Despite that evidence is far from complete, physical activity should be seen as an important part of the management of patients with liver disease in order to improve their clinical outcome.

Transcriptomic Analysis of Thalidomide Challenged Chick Embryo Suggests Possible Link between Impaired Vasculogenesis and Defective Organogenesis.

Since the conception of thalidomide as a teratogen, approximately 30 hypotheses have been put forward to explain the developmental toxicity of the molecule. However, no systems biology approach has been taken to understand the phenomena yet. The proposed work was aimed to explore the mechanism of thalidomide toxicity in developing chick embryo in the context of transcriptomics by using genome wide RNA sequencing data. In this study, we challenged the developing embryo at the stage of blood island formations (HH8), which is the most vulnerable stage for thalidomide-induced deformities. We observed that thalidomide affected the early vasculogenesis through interfering with the blood island formation extending the effect to organogenesis. The transcriptome analyses of the embryos collected on sixth day of incubation showed that liver, eye, and blood tissue associated genes were down regulated due to thalidomide treatment. The conserved gene coexpression module also indicated that the genes involved in lens development were heavily affected. Further, the Gene Ontology analysis explored that the pathways of eye development, retinol metabolism, and cartilage development were dampened, consistent with the observed deformities of various organs. The study concludes that thalidomide exerts its toxic teratogenic effects through interfering with early extra-embryonic vasculogenesis and ultimately gives an erroneous transcriptomic pattern to organogenesis.

sFRP4 signalling of apoptosis and angiostasis uses nitric oxide-cGMP-permeability axis of endothelium.

Nitric oxide (NO) plays a critical role in endothelial functions such as cellular migration, vascular permeability and angiogenesis. Angiogenesis, the formation of new blood vessels from "pre-existing" ones is a carefully regulated process and essential during reproduction, development and wound healing. Previously our lab group reported that Secreted Frizzled-Related Protein 4 (sFRP4) could inhibit angiogenesis in both in vitro and in vivo conditions. sFRP4 belongs to a family of secreted glycoproteins that function as antagonists of the canonical Wnt signalling pathway. Although the pro-apoptotic role of sFRP4 is well discussed in literature, little is known in regards to its anti-angiogenic property. The objective of this study was to elucidate sFRP4 implications in NO biology of the endothelium. Results demonstrate that sFRP4 causes endothelial dysfunction by suppressing NO-cGMP signaling and elevating corresponding ROS levels. The imbalance between NO and ROS levels results in apoptosis and subsequent leakiness of endothelium as confirmed in vivo (Texas red/Annxin - CAM assay) and in vitro (Monolayer permeability assay) conditions. Furthermore utilizing peptides synthesized from the CRD domain of sFRP4, our results showed that while these peptides were able to cause endothelial dysfunctions, they did not cause apoptosis of the endothelial cells. Thereby confirming that sFRP4 can mediate its anti-angiogenic effect independent of its pro-apoptotic property. In conclusion, the current study reports that sFRP4-mediated anti-angiogenesis occurs as a result of impaired NO-cGMP signaling which in turn allow for elevation of redox levels and promotion of apoptosis of endothelial cells.

Hepatocellular carcinoma and lifestyles.

The majority of hepatocellular carcinoma occurs over pre-existing chronic liver diseases that share cirrhosis as an endpoint. In the last decade, a strong association between lifestyle and hepatocellular carcinoma has become evident. Abundance of energy-rich food and sedentary lifestyles have caused metabolic conditions such as obesity and diabetes mellitus to become global epidemics. Obesity and diabetes mellitus are both tightly linked to non-alcoholic fatty liver disease and also increase hepatocellular carcinoma risk independent of cirrhosis. Emerging data suggest that physical activity not only counteracts obesity, diabetes mellitus and non-alcoholic fatty liver disease, but also reduces cancer risk. Physical activity exerts significant anticancer effects in the absence of metabolic disorders. Here, we present a systematic review on lifestyles and hepatocellular carcinoma.

Tipping off endothelial tubes: nitric oxide drives tip cells.

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a complex process that warrants cell migration, proliferation, tip cell formation, ring formation, and finally tube formation. Angiogenesis is initiated by a single leader endothelial cell called "tip cell," followed by vessel elongation by "stalk cells." Tip cells are characterized by their long filopodial extensions and expression of vascular endothelial growth factor receptor-2 and endocan. Although nitric oxide (NO) is an important modulator of angiogenesis, its role in angiogenic sprouting and specifically in tip cell formation is poorly understood. The present study tested the role of endothelial nitric oxide synthase (eNOS)/NO/cyclic GMP (cGMP) signaling in tip cell formation. In primary endothelial cell culture, about 40% of the tip cells showed characteristic sub-cellular localization of eNOS toward the anterior progressive end of the tip cells, and eNOS became phosphorylated at serine 1177. Loss of eNOS suppressed tip cell formation. Live cell NO imaging demonstrated approximately 35% more NO in tip cells compared with stalk cells. Tip cells showed increased level of cGMP relative to stalk cells. Further, the dissection of NO downstream signaling using pharmacological inhibitors and inducers indicates that NO uses the sGC/cGMP pathway in tip cells to lead angiogenesis. Taken together, the present study confirms that eNOS/NO/cGMP signaling defines the direction of tip cell migration and thereby initiates new blood vessel formation.

Regular exercise decreases liver tumors development in hepatocyte-specific PTEN-deficient mice independently of steatosis.

BACKGROUND & AIMS: Unhealthy lifestyles predispose people to non-alcoholic steatohepatitis (NASH), which may further result in the development of hepatocellular carcinoma (HCC). Although NASH patients benefit from physical activity, it is unknown whether regular exercise reduces the risk of developing HCC. Therefore, we studied the effect of regular exercise on the development of HCC in male hepatocyte-specific PTEN-deficient mice (AlbCrePten(flox/flox)), which develop steatohepatitis and HCC spontaneously. METHODS: Mice were fed a standardized 10% fat diet and were randomly divided into exercise or sedentary groups. The exercise group ran on a motorized treadmill for 60 min/day, 5 days/week during 32 weeks. RESULTS: After 32 weeks of regular exercise, 71% of exercised mice developed nodules larger than 15 mm(3)vs. 100% of mice in the sedentary group. The mean number of tumors per liver was reduced by exercise, as well as the total tumoral volume per liver. Exercise did not affect steatosis and had no effect on the non-alcoholic fatty liver disease (NAFLD) Activity Score (NAS). Exercise decreased tumor cell proliferation. Mechanistically, exercise stimulated the phosphorylation of AMPK and its substrate raptor, which decreased the kinase activity of mTOR. CONCLUSIONS: These data show a beneficial effect of regular exercise on the development of HCC in an experimental model of NASH and offer a rationale for encouraging predisposed patients to increase their physical activity for the prevention of HCC.

Cellular and molecular effects of the mTOR inhibitor everolimus.

mTOR (mechanistic target of rapamycin) functions as the central regulator for cell proliferation, growth and survival. Up-regulation of proteins regulating mTOR, as well as its downstream targets, has been reported in various cancers. This has promoted the development of anti-cancer therapies targeting mTOR, namely fungal macrolide rapamycin, a naturally occurring mTOR inhibitor, and its analogues (rapalogues). One such rapalogue, everolimus, has been approved in the clinical treatment of renal and breast cancers. Although results have demonstrated that these mTOR inhibitors are effective in attenuating cell growth of cancer cells under in vitro and in vivo conditions, subsequent sporadic response to rapalogues therapy in clinical trials has promoted researchers to look further into the complex understanding of the dynamics of mTOR regulation in the tumour environment. Limitations of these rapalogues include the sensitivity of tumour subsets to mTOR inhibition. Additionally, it is well known that rapamycin and its rapalogues mediate their effects by inhibiting mTORC (mTOR complex) 1, with limited or no effect on mTORC2 activity. The present review summarizes the pre-clinical, clinical and recent discoveries, with emphasis on the cellular and molecular effects of everolimus in cancer therapy.

Role of angiogenesis in bone repair.

Bone vasculature plays a vital role in bone development, remodeling and homeostasis. New blood vessel formation is crucial during both primary bone development as well as fracture repair in adults. Both bone repair and bone remodeling involve the activation and complex interaction between angiogenic and osteogenic pathways. Interestingly studies have demonstrated that angiogenesis precedes the onset of osteogenesis. Indeed reduced or inadequate blood flow has been linked to impaired fracture healing and old age related low bone mass disorders such as osteoporosis. Similarly the slow penetration of host blood vessels in large engineered bone tissue grafts has been cited as one of the major hurdle still impeding current bone construction engineering strategies. This article reviews the current knowledge elaborating the importance of vascularization during bone healing and remodeling, and the current therapeutic strategies being adapted to promote and improve angiogenesis.

Everolimus is a potent inhibitor of activated hepatic stellate cell functions in vitro and in vivo, while demonstrating anti-angiogenic activities.

Progression of liver fibrosis to HCC (hepatocellular carcinoma) is a very complex process which involves several pathological phenomena, including hepatic stellate cell activation, inflammation, fibrosis and angiogenesis. Therefore inhibiting multiple pathological processes using a single drug can be an effective choice to curb the progression of HCC. In the present study, we used the mTOR inhibitor everolimus to observe its effect on the in vitro activation of hepatic stellate cells and angiogenesis. The results of the present study demonstrated that everolimus treatment blocked the functions of the immortalized human activated hepatic stellate cell line LX-2 without affecting the viability and migration of primary human stellate cells. We also observed that treatment with everolimus (20 nM) inhibited collagen production by activated stellate cells, as well as cell contraction. Everolimus treatment was also able to attenuate the activation of primary stellate cells to their activated form. Angiogenesis studies showed that everolimus blocked angiogenesis in a rat aortic ring assay and inhibited the tube formation and migration of liver sinusoidal endothelial cells. Finally, everolimus treatment reduced the load of tumoral myofibroblasts in a rat model of HCC. These data suggest that everolimus targets multiple mechanisms, making it a potent blocker of the progression of HCC from liver fibrosis.

A comparative study of NONOate based NO donors: spermine NONOate is the best suited NO donor for angiogenesis.

Nitric oxide (NO) is a known modulator of angiogenesis. The NONOate subfamily of NO donors has long been used in experimental and clinical studies to promote angiogenesis. However, no studies have been conducted yet to compare the angiogenesis potential of these NO donors in respect to their pattern of NO release. We hypothesize that having different pattern of NO release, each of the NO donors in NONOate subfamily can promote key stages of angiogenesis in differential manner. To verify our hypothesis, NO donors with half life ranging from seconds to several hours and having very different pattern of NO release were selected to evaluate their efficacy in modulating angiogenesis. Endothelial tube formation using EAhy926 cells was maximally increased by Spermine NONOate (SP) treatment. SP treatment maximally induced both ex vivo and in vivo angiogenesis using egg yolk and cotton plug angiogenesis models respectively. Experiment using chick embryo partial ischemia model revealed SP as the best suited NO donor to recover ischemia driven hampered angiogenesis. The present study elaborated that differential release pattern of NO by different NO donors can modulate angiogenesis differentially and also suggested that SP have a unique pattern of NO release that best fits for angiogenesis.

Corrigendum: A small molecule inhibitor of Notch1 modulates stemness and suppresses breast cancer cell growth.

[This corrects the article DOI: 10.3389/fphar.2023.1150774.].

Breast cancer drugs dampen vascular functions by interfering with nitric oxide signaling in endothelium.

Widely used chemotherapeutic breast cancer drugs such as Tamoxifen citrate (TC), Capecitabine (CP) and Epirubicin (EP) are known to cause various cardiovascular side-effects among long term cancer survivors. Vascular modulation warrants nitric oxide (NO) signal transduction, which targets the vascular endothelium. We hypothesize that TC, CP and EP interference with the nitric oxide downstream signaling specifically, could lead to cardiovascular dysfunctions. The results demonstrate that while all three drugs attenuate NO and cyclic guanosine mono-phosphate (cGMP) production in endothelial cells, they caused elevated levels of NO in the plasma and RBC. However, PBMC and platelets did not show any significant changes under treatment. This implies that the drug effects are specific to the endothelium. Altered eNOS and phosphorylated eNOS (Ser-1177) localization patterns in endothelial cells were observed following drug treatments. Similarly, the expression of phosphorylated eNOS (Ser-1177) protein was decreased under the treatment of drugs. Altered actin polymerization was also observed following drug treatment, while addition of SpNO and 8Br-cGMP reversed this effect. Incubation with the drugs decreased endothelial cell migration whereas addition of YC-1, SC and 8Br-cGMP recovered the effect. Additionally molecular docking studies showed that all three drugs exhibited a strong binding affinity with the catalytic domain of human sGC. In conclusion, results indicate that TC, CP and EP cause endothelial dysfunctions via the NO-sGC-cGMP pathway and these effects could be recovered using pharmaceutical agonists of NO signaling pathway. Further, the study proposes a combination therapy of chemotherapeutic drugs and cGMP analogs, which would confer protection against chemotherapy mediated vascular dysfunctions in cancer patients.

Nitric oxide protects endothelium from cadmium mediated leakiness.

Cadmium targets the vascular endothelium causing endothelial dysfunction and leakiness of endothelial barrier. Nitric oxide plays a major role in mediating endothelial functions including angiogenesis, migration and permeability. The present study investigates the nitric oxide effects on cadmium induced endothelial leakiness. Results of ex vivo and in vitro permeability assays showed that even a sub-lethal dose of cadmium chloride (1 µM) was sufficient to induce leakiness of endothelial cells. Cadmium drastically altered the actin polymerisation pattern and membrane tension of these cells compared to controls. Addition of nitric oxide donor Spermine NONOate (SP) significantly blunted cadmium-mediated effects and recover endothelial cells integrity. Cadmium-induced cytoskeletal rearrangements and membrane leakiness are associated with the low nitric oxide availability and high reactive oxygen species generation. In brief, we show the protective role of nitric oxide against cadmium-mediated endothelial leakiness.

A small molecule inhibitor of Notch1 modulates stemness and suppresses breast cancer cell growth.

Although breast cancer stem cells (BCSCs) are well characterized, molecularly targeting and eradicating this sub-population remains a challenge in the clinic. Recent studies have explored several signaling pathways that govern stem cell activation: We and others established that the Notch1 signaling plays a significant role in the proliferation, survival, and differentiation of BCSCs. Earlier, we reported that a newly developed small molecule, ASR490, binds to the negative regulatory region (NRR: The activation switch of the Notch receptor) of Notch1. In vitro results demonstrated that ASR490 significantly inhibited BCSCs (ALDH+ and CD44+/CD24-) and breast cancer (BC) growth at nM concentrations, and subsequently inhibited the colony- and mammosphere-forming abilities of BCSCs and BCs. ASR490 downregulated the expressions of Notch1 intracellular domain (NICD: The active form of Notch1) and its downstream effectors Hey1 and HES1. Inhibition of Notch1-NICD facilitated autophagy-mediated growth inhibition by triggering the fusion of autophagosome and autolysosome in BCSCs. ASR490 was found to be non-toxic to healthy cells as compared to existing Notch1 inhibitors. Moreover, oral administration of ASR490 abrogated BCSC and BC tumor growth in the in vivo xenograft models. Together our results indicate that ASR490 is a potential therapeutic agent that inhibits BC tumor growth by targeting and abolishing Notch1 signaling in BCSCs and BC cells.

Corrigendum: A small molecule inhibitor of Notch1 modulates stemness and suppresses breast cancer cell growth.

[This corrects the article DOI: 10.3389/fphar.2023.1150774.].

Antiandrogen-Equipped Histone Deacetylase Inhibitors Selectively Inhibit Androgen Receptor (AR) and AR-Splice Variant (AR-SV) in Castration-Resistant Prostate Cancer (CRPC).

BACKGROUND: Epigenetic modification influences androgen receptor (AR) activation, often resulting in prostate cancer (PCa) development and progression. Silencing histone-modifying enzymes (histone deacetylases-HDACs) either genetically or pharmacologically suppresses PCa proliferation in preclinical models of PCa; however, results from clinical studies were not encouraging. Similarly, PCa patients eventually become resistant to androgen ablation therapy (ADT). Our goal is to develop dual-acting small molecules comprising antiandrogen and HDAC-inhibiting moieties that may overcome the resistance of ADT and effectively suppress the growth of castration-resistant prostate cancer (CRPC). METHODS: Several rationally designed antiandrogen-equipped HDAC inhibitors (HDACi) were synthesized, and their efficacy on CRPC growth was examined both in vitro and in vivo. RESULTS: While screening our newly developed small molecules, we observed that SBI-46 significantly inhibited the proliferation of AR+ CRPC cells but not AR- CRPC and normal immortalized prostate epithelial cells (RWPE1) or normal kidney cells (HEK-293 and VERO). Molecular analysis confirmed that SBI-46 downregulated the expressions of both AR+ and AR-splice variants (AR-SVs) in CRPC cells. Further studies revealed the downregulation of AR downstream (PSA) events in CRPC cells. The oral administration of SBI-46 abrogated the growth of C4-2B and 22Rv1 CRPC xenograft tumors that express AR or both AR and AR-SV in xenotransplanted nude mice models. Further, immunohistochemical analysis confirmed that SBI-46 inhibits AR signaling in xenografted tumor tissues. CONCLUSION: These results demonstrate that SBI-46 is a potent agent that inhibits preclinical models of CRPC by downregulating the expressions of both AR and AR-SV. Furthermore, these results suggest that SBI-46 may be a potent compound for treating CRPC.

Urolithin A analog inhibits castration-resistant prostate cancer by targeting the androgen receptor and its variant, androgen receptor-variant 7.

We investigated the efficacy of a small molecule ASR-600, an analog of Urolithin A (Uro A), on blocking androgen receptor (AR) and its splice variant AR-variant 7 (AR-V7) signaling in castration-resistant prostate cancer (CRPC). ASR-600 effectively suppressed the growth of AR+ CRPC cells by inhibiting AR and AR-V7 expressions; no effect was seen in AR- CRPC and normal prostate epithelial cells. Biomolecular interaction assays revealed ASR-600 binds to the N-terminal domain of AR, which was further confirmed by immunoblot and subcellular localization studies. Molecular studies suggested that ASR-600 promotes the ubiquitination of AR and AR-V7 resulting in the inhibition of AR signaling. Microsomal and plasma stability studies suggest that ASR-600 is stable, and its oral administration inhibits tumor growth in CRPC xenografted castrated and non-castrated mice. In conclusion, our data suggest that ASR-600 enhances AR ubiquitination in both AR+ and AR-V7 CRPC cells and inhibits their growth in vitro and in vivo models.