6-Azauridine Induces Autophagy-Mediated Cell Death via a p53- and AMPK-Dependent Pathway.

6-Azauridine (6-AZA), a pyrimidine nucleoside analogue, is known to exhibit both antitumor and antiviral activities. Although 6-AZA was discovered more than 60 years ago, the cellular effects of this compound are yet to be elucidated. Here, we report that 6-AZA regulates autophagy-mediated cell death in various human cancer cells, where 6-AZA treatment activates autophagic flux through the activation of lysosomal function. Furthermore, 6-AZA exhibited cytotoxicity in all cancer cells studied, although the mechanisms of action were diverse. In H460 cells, 6-AZA treatment induced apoptosis, and the extent of the latter could be reduced by treatment with chloroquine (CQ), a lysosomal inhibitor. However, 6-AZA treatment resulted in cell cycle arrest in H1299 cells, which could not be reversed by CQ. The cytotoxicity associated with 6-AZA treatment could be linearly correlated to the degree of autophagy-mediated cell death. In addition, we demonstrated that the cytotoxic effect of 6-AZA was dependent on AMPK and p53. These results collectively indicate that autophagy-mediated cell death triggered by 6-AZA contributes to its antitumor effect.

Combination of ionizing radiation and 2-thio-6-azauridine induces cell death in radioresistant triple negative breast cancer cells by downregulating CD151 expression.

BACKGROUND: Triple-negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer and is frequently resistant to therapy, ultimately resulting in treatment failure. Clinical trials have demonstrated the potential of sensitizing radiation therapy (RT)-resistant TNBC through the combination of chemotherapy and RT. This study sought to explore the potential of CD151 as a therapy response marker in the co-treatment strategy involving ionizing radiation (IR) and the repurposed antiviral drug 2-Thio-6-azauridine (TAU) for sensitizing RT-resistant TNBC (TNBC/RR). METHODS: The investigation encompassed a variety of assessments, including viability using MTT and LDH assays, cell proliferation through BrdU incorporation and clonogenic assays, cell cycle analysis via flow cytometry, cell migration using wound scratch and Boyden chamber invasion assays, DNA damage assessment through γH2AX analysis, apoptosis evaluation through acridine-orange and ethidium bromide double staining assays, as well as caspase 3 activity measurement using a colorimetric assay. CD151 expression was examined through ELISA, flow cytometry and RT-qPCR. RESULTS: The results showed a significant reduction in TNBC/RR cell viability following co-treatment. Moreover, the co-treatment reduced cell migration, induced apoptosis, downregulated CD151 expression, and increased caspase 3 activity in TNBC/RR cells. Additionally, CD151 was predicted to serve as a therapy response marker for co-treatment with TAU and IR. CONCLUSION: These findings suggest the potential of combination treatment with IR and TAU as a promising strategy to overcome RT resistance in TNBC. Furthermore, CD151 emerges as a valuable therapy response marker for chemoradiotherapy.

Proteomic profiling and ROC analysis identify CD151 and ELAVL1 as potential therapy response markers for the antiviral drug in resistant TNBC.

Triple-negative breast cancer is high heterogeneous, aggressive, and metastatic with poor prognosis. Despite of advances in targeted therapies, TNBC has been reported to cause high morbidity and mortality. A rare subpopulation within the tumor microenvironment organized into a hierarchy of cancer stem cells is responsible for therapy resistance and tumor recurrence. Repurposing of antiviral drugs for cancer treatment is gaining momentum due to reduced cost, labour, and research time, but limited due to lack of prognostic, and predictive markers. The present study investigates proteomic profiling and ROC analysis to identify CD151 and ELAVL1 as potential therapy response markers for the antiviral drug 2-thio-6-azauridine (TAU) in resistant TNBC. The stemness of MDA-MB 231 and MDA-MD 468 adherent cells was enriched by culturing them under non-adherent and non-differentiation conditions. Then, CD151+ subpopulation was isolated and characterized for the enrichment of stemness. This study found that CD151 has overexpressed in stemness enriched subpopulations, and also showed CD44 high and CD24 low expression along with stem cell-related transcription factors octamer-binding transcription factor 4 (OCT4) and Sex determining Y-box 2 (SOX2). This study also found that TAU induced significant cytotoxicity and genotoxicity in the CD151+TNBC subpopulation and inhibited their proliferation by inducing DNA damage, cell cycle arrest at the G2M phase, and apoptosis. Further, a proteomic profiling study showed that the expression of CD151 along with ELAVL1, an RNA-binding protein, was significantly reduced with TAU treatment. KM plotter showed correlation of CD151 and ELAVL1 gene expression with a poor prognosis of TNBC. ROC analysis predicted and validated CD151 and ELAVL1 as best therapy response marker for TAU in TNBC. These findings provide new insight into repurposing antiviral drug TAU for treatment of metastatic and drug resistant TNBC.

Comparison of three dimensional synergistic analyses of percentage versus logarithmic data in antiviral studies.

Cell culture antiviral experiments were conducted in order to understand the relationship between percentage data generated by plaque reduction (PR) and logarithmic data derived by virus yield reduction (VYR) assays, using three-dimensional MacSynergy II software. The relationship between percentage and logarithmic data has not been investigated previously. Interpretation of drug-drug interactions is based on a Volume of Synergy (VS) calculation, which can be positive (synergy), negative (antagonistic), or neutral (no or minimal interaction). Interactions of two known inhibitors of vaccinia virus replication, cidofovir and 6-azauridine, used in combination by PR assay yielded a VS value of 265, indicative of strong synergy. By VYR, the VS value was only 37, or weak synergy using the same criterion, even though profound log10 reductions in virus titer occurred at multiple drug combinations. These results confirm that the differences in VS values is dependent of the measurement scale, and not that the degree of synergy differed between the assays. We propose that for logarithmic data, the calculated VS values will be lower for significant synergy and antagonism and that volumes of >10 µM2log10 PFU/ml (or other units such as µM2log10 genomic equivalents/ml or µM2log10 copies/ml) and <-10 µM2log10 PFU/ml are likely to be indicative of strong synergy and strong antagonism, respectively. Data presented here show that the interaction of cidofovir and 6-azauridine was strongly synergistic in vitro.