Development and Pilot Screen of Novel High Content Assay for Down Regulators of Expression of Heterogenous Nuclear Ribonuclear Protein H2.

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late-stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al., Cell Physiol Biochem 2019;53:656-686). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H2 that can be used for melanoma therapy and research. METHODS: We established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. RESULTS: All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z' = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis. CONCLUSION: We developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.

Dengue virus infection induces upregulation of hn RNP-H and PDIA3 for its multiplication in the host cell.

The pathogenic mechanism of Dengue virus (DENV) infection is related to the host responses within target cells and therefore, we assessed intracellular changes in host cell proteins following DENV infection. This study provides evidence that heterogeneous nuclear ribonucleoprotein H (hnRNP-H) and protein disulfide isomerase A3 (PDIA3) helps in DENV multiplication by suppressing TNF-α production in human monocytic THP1 cells. Proteomic analysis of infected cells, identified upregulation of the host cell proteins PDIA3 and hnRNP-H in comparison to mock infected cells. The functional role of hnRNP-H and PDIA3 in DENV infection was identified by down regulating hnRNP-H and PDIA3 genes with their specific siRNA duplexes which lead to decreased intracellular viral load. It also resulted in increased TNF-α level which mediates antiviral effect. This is the first study, which reports the role of PDIA3 and hnRNP-H in TNF-α production in DENV infected cells. Collectively, these results suggest that increased level of hnRNP-H and PDIA3 expression in DENV infected THP1 cells assist in the viral replication by suppressing the TNF-α production.

Altered expression and localization of creatine kinase B, heterogeneous nuclear ribonucleoprotein F, and high mobility group box 1 protein in the nuclear matrix associated with colon cancer.

Identification of biomarkers could lead to the development of effective screening tests for colorectal cancer. A previous study from our laboratory showed specific alterations of nuclear structure in colon cancer. In an effort to characterize these biomarkers, protein spots were selected from separations made by two-dimensional gel electrophoresis, which were analyzed by mass spectrometry. The sequences obtained from the isolated spots revealed that they have close similarity to creatine kinase B (CKB) isoforms, heterogeneous nuclear ribonucleoprotein F (hnRNP F) and high mobility group box 1 protein (HMGB1) isoforms. To determine the expression of these proteins in colon cancer, expression was studied in 9 tumor and matched adjacent normal pairs, 5 donor colons, 16 polyps, 4 metastatic liver lesions and matched adjacent normal pairs, and 3 liver donors. CKB and hnRNP F were expressed in 78% and 89% of colon tumors, respectively. hnRNP F had a higher frequency of expression than CKB in premalignant polyps. With the establishment of differential expression of the proteins in colon cancer, their subcellular localization was analyzed. The subcellular fractions studied both showed high protein levels of hnRNP F in colon tumors compared with normal colon tissues. Surprisingly, subcellular levels of CKB were decreased in colon tumors, suggesting that the observed high CKB levels in nuclear matrix extracts are caused by the enhanced localization of CKB to the nuclear matrix during colon tumorigenesis. These results suggest an involvement of hnRNP F and CKB in colorectal cancer. Additionally, they suggest that hnRNP F is a potential marker for colorectal cancer progression.