Suppression of NFAT5-mediated Inflammation and Chronic Arthritis by Novel ??B-binding Inhibitors

Nuclear factor of activated T cells 5 (NFAT5) has been implicated in the pathogenesis of various human diseases, including cancer and arthritis. However, therapeutic agents inhibiting NFAT5 activity are currently unavailable. To discover NFAT5 inhibitors, a library of >. 40,000 chemicals was screened for the suppression of nitric oxide, a direct target regulated by NFAT5 activity, through high-throughput screening. We validated the anti-NFAT5 activity of 198 primary hit compounds using an NFAT5-dependent reporter assay and identified the novel NFAT5 suppressor KRN2, 13-(2-fluoro)-benzylberberine, and its derivative KRN5. KRN2 inhibited NFAT5 upregulation in macrophages stimulated with lipopolysaccharide and repressed the formation of NF-??B p65-DNA complexes in the NFAT5 promoter region. Interestingly, KRN2 selectively suppressed the expression of pro-inflammatory genes, including Nos2 and Il6, without hampering high-salt-induced NFAT5 and its target gene expressions. Moreover, KRN2 and KRN5, the latter of which exhibits high oral bioavailability and metabolic stability, ameliorated experimentally induced arthritis in mice without serious adverse effects, decreasing pro-inflammatory cytokine production. Particularly, orally administered KRN5 was stronger in suppressing arthritis than methotrexate, a commonly used anti-rheumatic drug, displaying better potency and safety than its original compound, berberine. Therefore, KRN2 and KRN5 can be potential therapeutic agents in the treatment of chronic arthritis.ope

Validating Antimetastatic Effects of Natural Products in an Engineered Microfluidic Platform Mimicking Tumor Microenvironment

Development of new, antimetastatic drugs from natural products has been substantially constrained by the lack of a reliable in vitro screening system. Such a system should ideally mimic the native, three-dimensional (3D) tumor microenvironment involving different cell types and allow quantitative analysis of cell behavior critical for metastasis. These requirements are largely unmet in the current model systems, leading to poor predictability of the in vitro collected data for in vivo trials, as well as prevailing inconsistency among different in vitro tests. In the present study, we report application of a 3D, microfluidic device for validation of the antimetastatic effects of 12 natural compounds. This system supports co-culture of endothelial and cancer cells in their native 3D morphology as in the tumor microenvironment and provides real-time monitoring of the cells treated with each compound. We found that three compounds, namely sanguinarine, nitidine, and resveratrol, exhibited significant antimetastatic or antiangiogenic effects. Each compound was further examined for its respective activity with separate conventional biological assays, and the outcomes were in agreement with the findings collected from the microfluidic system. In summary, we recommend use of this biomimetic model system as a new engineering tool for high-throughput evaluation of more diverse natural compounds with varying anticancer potentials

Berberine, an Epiphany Against Cancer

Alkaloids are used in traditional medicine for the treatment of many diseases. These compounds are synthesized in plants as secondary metabolites and have multiple effects on cellular metabolism. Among plant derivatives with biological properties, the isoquinoline quaternary alkaloid berberine possesses a broad range of therapeutic uses against several diseases. In recent years, berberine has been reported to inhibit cell proliferation and to be cytotoxic towards cancer cells. Based on this evidence, many derivatives have been synthesized to improve berberine efficiency and selectivity; the results so far obtained on human cancer cell lines support the idea that they could be promising agents for cancer treatment. The main properties of berberine and derivatives will be illustrated

Increased levels of p21CDKN1A do not inhibit the recruitment of NER factors at DNA damage sites.

P21CDK1NA is a cyclin-dependent kinase inhibitor playing multiple roles also in the DNA damage response. Therapeutic trials have been developed to contrast tumor cell proliferation, by exploiting the p21 ability to arrest the cell cycle; in particular, proteasome inhibitors increase p21 protein levels, impairing tumor cell growth. However, this approach is may be potentially dangerous because high p21 levels inhibit the apoptotic response and allow DNA repair, rendering tumor cells resistant to chemotherapy. We have investigated whether the accumulation of p21 levels, induced by the inhibitor of proteasome MG132, may affect nucleotide excision repair (NER) and apoptosis. The results have shown that MG132 induced persistent increased levels of XPC, PCNA and p21 proteins at local DNA damage sites, together with accumulation of XPG, DNA polymerase δ and CAF-1, suggesting that the presence of p21 protein did not block the recruitment of NER factors interacting with PCNA. Immunoprecipitation experiments have shown that DNA pol δ interacts with an ubiquitinated form of p21. These results indicate that p21 regulates steps of NER before degradation

Increased levels of p21CDKN1A do not inhibit the recruitment of NER factors at DNA damage sites.

P21CDK1NA is a cyclin-dependent kinase inhibitor playing multiple roles also in the DNA damage response. Therapeutic trials have been developed to contrast tumor cell proliferation, by exploiting the p21 ability to arrest the cell cycle; in particular, proteasome inhibitors increase p21 protein levels, impairing tumor cell growth. However, this approach is may be potentially dangerous because high p21 levels inhibit the apoptotic response and allow DNA repair, rendering tumor cells resistant to chemotherapy. We have investigated whether the accumulation of p21 levels, induced by the inhibitor of proteasome MG132, may affect nucleotide excision repair (NER) and apoptosis. The results have shown that MG132 induced persistent increased levels of XPC, PCNA and p21 proteins at local DNA damage sites, together with accumulation of XPG, DNA polymerase δ and CAF-1, suggesting that the presence of p21 protein did not block the recruitment of NER factors interacting with PCNA. Immunoprecipitation experiments have shown that DNA pol δ interacts with an ubiquitinated form of p21. These results indicate that p21 regulates steps of NER before degradation

Involvement of the cell cycle inhibitor p21CDKN1A in DNA repair

A variety of chemical and physical agents may induce the formation of different lesions in the DNA molecule. These types of DNA damage may be genotoxic to the cells, and must be removed in order to avoid genomic instability, and to prevent cancer formation. To this end, virtually every organism has developed highly conserved genome surveillance and signaling mechanisms, collectively known as the DNA damage response. This pathway consists of DNA damage signaling cascade (cell cycle checkpoints), and of DNA repair processes able to recognize and remove a great number of DNA lesions. Recent findings have shown that cell cycle checkpoints and DNA repair systems are strictly connected each other. However, the role and the molecular mechanisms underlying these connections are not yet completely understood. Among cell cycle regulatory proteins that are activated following DNA damage, the cyclin-dependent kinase inhibitor p21CDKN1A plays fundamental roles in the DNA damage response by inducing cell cycle arrest, direct inhibition of DNA synthesis, as well as by regulating transcription and apoptosis. During the last years, several lines of evidence have also indicated that p21 may be directly involved in DNA repair. Participation of p21 in DNA repair pathways, like nucleotide excision repair (NER), and base excision repair (BER), is thought to occur thanks to its interaction with Proliferating Cell Nuclear Antigen (PCNA), a crucial protein involved both in DNA replication and repair. In addition, a direct involvement of p21 in DNA trans-lesion synthesis, has been postulated to keep within low levels the mutagenesis intrinsic in this process. In this review, all relevant findings supporting the participation of p21 protein in NER and BER will be presented. In particular, the ability of p21 to interact with PCNA seems to be required for regulating interaction of DNA repair factors with PCNA. Examples of this role will be discussed together with other aspects of the DNA damage response in which p21 is also involved. A special attention will be given to the dynamics of p21 recruitment to sites of DNA damage. In fact, a common feature of checkpoint and DNA repair factors is their accumulation at nuclear sites where DNA damage has occurred. The involvement of p21 in various DNA repair pathways supports its important function of protein barrier against genome instability