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

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

p300/CBP acetyl transferases interact with and acetylate the nucleotide excision repair factor XPG.

Nucleotide excision repair (NER) is an important DNA repair mechanism through which cells remove bulky DNA lesions. Following DNA damage, the histone acetyltransferase (HAT) p300 (also referred to as lysine acetyltransferase or KAT) is known to associate with proliferating cell nuclear antigen (PCNA), a master regulator of DNA replication and repair processes. This interaction, which results in HAT inhibition, may be dissociated by the cell cycle inhibitor p21(CDKN1A), thereby restoring p300 activity; however, the role of this protein interplay is still unclear. Here, we report that silencing p300 or its homolog CREB-binding protein (CBP) by RNA interference (RNAi) significantly reduces DNA repair synthesis in human fibroblasts. In addition, we determined whether p300 and CBP may associate with and acetylate specific NER factors such as XPG, the 3'-endonuclease that is involved in the incision/excision step and is known to interact with PCNA. Our results show that p300 and CBP interact with XPG, which has been found to be acetylated in vivo. XPG is acetylated by p300 in vitro, and this reaction is inhibited by PCNA. Knocking down both p300/CBP by RNAi or by chemical inhibition with curcumin greatly reduced XPG acetylation, and a concomitant accumulation of the protein at DNA damage sites was observed. The ability of p21 to bind PCNA was found to regulate the interaction between p300 and XPG, and an abnormal accumulation of XPG at DNA damage sites was also found in p21(-/-) fibroblasts. These results indicate an additional function of p300/CBP in NER through the acetylation of XPG protein in a PCNA-p21 dependent manner

Mutations in CREBBP and EP300 genes affect DNA repair of oxidative damage in Rubinstein-Taybi syndrome cells

Rubinstein-Taybi syndrome (RSTS) is an autosomal-dominant disorder characterized by intellectual disability, skeletal abnormalities, growth deficiency, and an increased risk of tumors. RSTS is predominantly caused by mutations in CREBBP or EP300 genes encoding for CBP and p300 proteins, two lysine acetyl-transferases (KAT) playing a key role in transcription, cell proliferation and DNA repair. However, the efficiency of these processes in RSTS cells is still largely unknown. Here we have investigated whether pathways involved in the maintenance of genome stability are affected in lymphoblastoid cell lines (LCLs) obtained from RSTS patients with mutations in CREBBP or in EP300 genes. We report that RSTS LCLs with mutations affecting CBP or p300 protein levels or KAT activity, are more sensitive to oxidative DNA damage and exhibit defective base excision repair (BER). We have found reduced OGG1 DNA glycosylase activity in RSTS compared to control cell extracts, and concomitant lower OGG1 acetylation levels, thereby impairing the initiation of the BER process. In addition, we report reduced acetylation of other BER factors, such as DNA polymerase \u3b2 and PCNA, together with acetylation of histone H3. We also show that complementation of CBP or p300 partially reversed RSTS cell sensitivity to DNA damage. These results disclose a mechanism of defective DNA repair as a source of genome instability in RSTS cells

Comparison of StemPrintER, a novel biology-based genomic predictor of distant recurrence in breast cancer, with Oncotype DX in the TransATAC cohort.

Abstract 1020 from 2020 ASCO Annual Meeting