Epigallocatechin-3-gallate suppresses cell proliferation and promotes apoptosis and autophagy in oral cancer SSC-4 cells

Alexandra Iulia Irimie,1 Cornelia Braicu,2 Oana Zanoaga,2 Valentina Pileczki,2,3 Claudia Gherman,2,4 Ioana Berindan-Neagoe,2,4–6 Radu Septimiu Campian7 1Department of Prosthodontics and Dental Materials, Faculty of Dental Medicine, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania; 2Research Center for Functional Genomics, Biomedicine and Translational Medicine, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania; 3Department of Analytical Chemistry, Faculty of Pharmacy, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania; 4Department of Functional Genomics and Experimental Pathology, The Oncology Institute “Prof Dr. Ion Chiricuta”, Cluj-Napoca, Romania; 5Department of Immunology, Faculty of Medicine, University of Medicine and Pharmacy, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania; 6Department of Experimental Therapeutics MD Anderson Cancer Center Houston, TX, USA; 7Department of Oral Rehabilitation, Faculty of Dental Medicine, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania Abstract: Epigallocatechin-3-gallate (EGCG) is the major bioactive component of green tea. Our experimental data indicated that EGCG treatment suppresses cell proliferation of SSC-4 human oral squamous cell carcinoma (OSCC), the effect being dose- and time-dependent. In parallel was observed the activation of apoptosis and autophagy, in response to EGCG exposure in SSC-4 cells. Treatment with EGCG activates the expression of the BAD, BAK, FAS, IGF1R, WNT11, and ZEB1 genes and inhibits CASP8, MYC, and TP53. All of these results suggest that EGCG has an excellent potential to become a therapeutic compound for patients with OSCC, by inducing tumor cell death via apoptosis and autophagy. Keywords: oral squamous carcinoma, time dependent cell proliferation, gene expressio

Double gene siRNA knockdown of mutant p53 and TNF induces apoptosis in triple-negative breast cancer cells

Valentina Pileczki,1,2 Laura Pop,1 Cornelia Braicu,1 Livia Budisan,1 Gabriela Bolba Morar,3 Paloma del C Monroig-Bosque,4 Robert V Sandulescu,2 Ioana Berindan-Neagoe1,5,6 1The Research Center for Functional Genomics, Biomedicine and Translational Medicine, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania; 2Department of Analytical Chemistry, Faculty of Pharmacy, “Iuliu Hatieganu” University of Medicine and Pharmacy, 3Department of Senology, the Oncology Institute “Prof Dr Ion Chiricuta”, Cluj-Napoca, Romania; 4University of Puerto Rico School of Medicine, San Juan, Puerto Rico; 5MedFuture Research Center for Advanced Medicine, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania; 6Department of Functional Genomics and Experimental Pathology, the Oncology Institute “Prof Dr Ion Chiricuta”, Cluj-Napoca, Romania Abstract: Apoptosis is the major downregulated pathway in cancer. Simultaneous inhibition using specific small interfering RNA (siRNA) of two key player genes, p53 and TNF, is an interesting and feasible strategy when it comes to investigating various molecular pathways and biological processes in triple-negative breast cancer (TNBC), which is one of the most aggressive and therapeutically unresponsive forms of breast cancers. Our present research focuses on evaluating the impact of double p53-siRNA and TNF-siRNA knockdown at a cellular level, and also evaluating cell proliferation, apoptosis, induction of autophagy, and gene expression by using reverse transcription polymerase chain reaction array approaches. Simultaneous inhibition of p53 and TNF in Hs578T TNBC human cell line revealed a panel of up- and downregulated genes involved in apoptosis. Furthermore, the effects of double gene knockdown were validated in a second TNBC cell line, MDA-MB-231, by using reverse transcription polymerase chain reaction TaqMan assay. All our findings help in understanding the functional mechanisms of extrinsic apoptosis, cell signaling pathways, and the mechanisms involved in tumor cell survival, growth, and death in TNBC. Keywords: apoptosis, double gene silencing, mut-p53, TNF, TNB

Normalization of gene expression measurement of tissue samples obtained by transurethral resection of bladder tumors

Laura A Pop,1,* Valentina Pileczki,1,2,* Roxana M Cojocneanu-Petric,1 Bogdan Petrut,3,4 Cornelia Braicu,1 Ancuta M Jurj,1 Rares Buiga,5 Patriciu Achimas-Cadariu,6,7 Ioana Berindan-Neagoe1,8 1The Research Center for Functional Genomics, Biomedicine and Translational Medicine, Iuliu Haţieganu University of Medicine and Pharmacy, Cluj-Napoca, Cluj, Romania; 2Department of Analytical Chemistry, Iuliu Haţieganu University of Medicine and Pharmacy, Cluj-Napoca, Cluj, Romania; 3Department of Surgery II – Urology, The Oncology Institute “Prof Dr Ion Chiricuţă”, Cluj-Napoca, Cluj, Romania; 4Department of Urology, Iuliu Haţieganu University of Medicine and Pharmacy, Cluj-Napoca, Cluj, Romania; 5Department of Pathology, The Oncology Institute “Prof. Dr Ion Chiricuţă”, Cluj-Napoca, Cluj, Romania; 6Department of Surgery, The Oncology Institute “Prof Dr Ion Chiricuţă”, Cluj-Napoca, Cluj, Romania; 7Department of Surgical Oncology and Gynecological Oncology, Iuliu Haţieganu University of Medicine and Pharmacy, 8Department of Functional Genomics and Experimental Pathology, The Oncology Institute “Prof Dr Ion Chiricuţă”, Cluj-Napoca, Cluj, Romania *These authors contributed equally to this work Background: Sample processing is a crucial step for all types of genomic studies. A major challenge for researchers is to understand and predict how RNA quality affects the identification of transcriptional differences (by introducing either false-positive or false-negative errors). Nanotechnologies help improve the quality and quantity control for gene expression studies. Patients and methods: The study was performed on 14 tumor and matched normal pairs of tissue from patients with bladder urothelial carcinomas. We assessed the RNA quantity by using the NanoDrop spectrophotometer and the quality by nano-microfluidic capillary electrophoresis technology provided by Agilent 2100 Bioanalyzer. We evaluated the amplification status of three housekeeping genes and one small nuclear RNA gene using the ViiA 7 platform, with specific primers. Results: Every step of the sample handling protocol, which begins with sample harvest and ends with the data analysis, is of utmost importance due to the fact that it is time consuming, labor intensive, and highly expensive. High temperature of the surgical procedure does not affect the small nucleic acid sequences in comparison with the mRNA. Conclusion: Gene expression is clearly affected by the RNA quality, but less affected in the case of small nuclear RNAs. We proved that the high-temperature, highly invasive transurethral resection of bladder tumor procedure damages the tissue and affects the integrity of the RNA from biological specimens. Keywords: bladder cancer, transurethral resection, RNA quality, real-time PC

Small molecules against B-RAF (BRAF) Val600Glu (V600E) single mutation

Florin Zaharie,1,* Roxana Cojocneanu-Petric,1,* Mihai Muresan,1 Ioana Frinc,2 Delia Dima,2 Bobe Petrushev,3 Alina Tanase,4 Cristian Berce,1 Mariana Chitic,2 Ioana Berindan-Neagoe,1 Valentina Pileczki,1 Alexandru Irimie,5 Ciprian Tomuleasa2 1Iuliu Hatieganu University of Medicine and Pharmacy, 2Department of Hematology, Ion Chiricuta Oncology Institute, 3Department of Pathology, Emergency University Hospital, Cluj Napoca, 4Department of Stem Cell Transplantation, Fundeni Clinical Institute, Bucharest, 5Department of Surgery, Ion Chiricuta Oncology Institute, Cluj Napoca, Romania *These authors contributed equally to this communicationWe have read with great interest the paper by Tang and Chen1 published in the most recent issue of the International Journal of Nanomedicine, in which the authors describe the protocol by which scientists constructed the ideal BRAF (V600E)-modeled structure through homology modeling and introduced the method of structure-based docking or virtual screening from a large compound database. They concluded that BRAF (V600E) has a quite prominent structural or conformational variation when compared to the wild-type BRAF protein by matrix of root mean square fluctuation and principal component analysis. On the basis of structure-based virtual screening, ligand-based quantitative structure activity relationship models, and molecular dynamics simulation, we recommend aknadicine and 16beta-hydroxy-19s-vindolinine N-oxide as potent compounds for developing novel inhibitors in the future. Read the original article&nbsp