Non-POU Domain-Containing Octomer-Binding (NONO) protein expression and stability promotes the tumorigenicity and activation of Akt/MAPK/ß-catenin pathways in human breast cancer cells.

Breast cancer is one of the most common cancers with a high mortality rate, underscoring the need to identify new therapeutic targets. Here we report that non-POU domain-containing octamer-binding (NONO) protein is overexpressed in breast cancer and validated the interaction of the WW domain of PIN1 with c-terminal threonine-proline (thr-pro) motifs of NONO. The interaction of NONO with PIN1 increases the stability of NONO by inhibiting its proteasomal degradation, and this identifies PIN1 as a positive regulator of NONO in promoting breast tumor development. Functionally, silencing of NONO inhibits the growth, survival, migration, invasion, epithelial to mesenchymal transition (EMT), and stemness of breast cancer cells in vitro. A human metastatic breast cancer cell xenograft was established in transparent zebrafish (Danio rerio) embryos to study the metastatic inability of NONO-silenced breast cancer cells in vivo. Mechanistically, NONO depletion promotes the expression of the PDL1 cell-surface protein in breast cancer cells. The identification of novel interactions of NONO with c-Jun and ß-catenin proteins and activation of the Akt/MAPK/ß-catenin signaling suggests that NONO is a novel regulator of Akt/MAPK/ß-catenin signaling pathways. Taken together, our results indicated an essential role of NONO in the tumorigenicity of breast cancer and could be a potential target for anti-cancerous drugs. Video Abstract.

Knockdown of CSNK2ß suppresses MDA-MB231 cell growth, induces apoptosis, inhibits migration and invasion.

Breast cancer is the most common cancer among women worldwide. Among different types of breast cancer known, treatment of triple-negative breast cancer is a major challenge because of its aggressiveness and poor prognosis; thus, identification of specific drivers is required for targeted therapies of breast cancer malignancy. Protein Casein Kinase (CSNK) is a serine/threonine kinase that exists as a tetrameric complex consisting of two catalytic (α and /or α') and two regulatory ß subunits. CSNK2ß can also function independently without catalytic subunits and exist as a distinct population in cells. This study aims to elucidate the role of Casein Kinase 2ß (CSNK2ß) gene in cell proliferation, cell cycle, migration and apoptosis of triple-negative breast cancer MDA-MB-231 cells. The silencing of CSNK2ß in MDA-MB-231 cells resulted in decreased cell viability and colony formation. Cell cycle analysis showed a significant arrest of cells in G2M phase. Hoechst and CM-H2DCFDA staining showed nuclear condensation and augmented intracellular reactive oxygen species (ROS) production. Furthermore, silencing of CSNK2ß in MDA-MB-231 cells modulated the apoptotic machinery- BAX, Bcl-xL, and caspase 3; autophagy machinery-Beclin-1 and LC3-1; and inhibited the vital markers (p-ERK, c-Myc, NF-κB, E2F1, PCNA, p38-α) associated with cell proliferation and DNA replication pathways. In addition, knockdown of CSNK2ß also affected the migration potential of MDA-MB-231, as observed in the wound healing and transwell migration assays. Altogether, the study suggests that CSNK2ß silencing may offer future therapeutic target in triple-negative breast cancer.

SUPT5H Post-Transcriptional Silencing Modulates PIN1 Expression, Inhibits Tumorigenicity, and Induces Apoptosis of Human Breast Cancer Cells.

BACKGROUND/AIMS: Breast cancer (BrCa) is one of the most common cancers and a highly heterogenous disease, both at the pathological and molecular levels. A common element for the progression of cancer is the presence of aberrant transcription. Targeting the misregulation of transcription may serve as a tool for cancer therapeutics. SUPT5H (Suppressor of Ty 5 homolog) is a highly conserved RNA polymerase II-associated transcription elongation and processivity factor. However, few studies have examined the relationship between SUPT5H and cancer. METHODS: Yeast two-hybrid and colocalization by immunofluorescence were performed to investigate protein-protein interaction. Colony formation assay, CTG assay, and crystal violet assays were performed for cell viability, clonogenicity, and cell proliferation study. Data mining was performed for expression analysis of SUPT5H in breast cancers. Flow cytometry was performed for the assessment of cell cycle and apoptosis. The Transwell chambers were employed for the migration and invasion assays. Quantitative real-time polymerase chain reaction (qRT PCR) and Western blotting were performed to measure the mRNA and protein levels of SUPT5H and other markers related to viability, migration, cell cycle, and apoptosis. Silent mutations were generated for rescue experiments. The biological function of SUPT5H was investigated through siRNA depletion of SUPT5H mRNA in vitro. RESULTS: We showed that SUPT5H is upregulated in breast cancer tissue as compared with the adjacent normal tissue in breast cancer patients. In human breast cancer cells, the levels of SUPT5H and PIN1 are positively correlated with each other. Our biochemical analysis showed that PIN1 interacts with SUPT5H through WW domain, that was required to promote SUPT5H protein stability. Depletion of SUPT5H by siRNA technology reduced the tumorigenic and metastatic properties, promoted s-phase cell cycle arrest and apoptosis of MDA-MB-231 cells. Moreover, depletion of SUPT5H abrogated MAPK molecules thereby regulates the oncogenic behavior of breast cancer cells. CONCLUSION: Our findings demonstrated an essential role of SUPT5H in BrCa tumorigenicity by regulating the expression levels of genes that control proliferation, migration, cell cycle, and apoptosis of breast cancer MDA-MB-231 cells.

Inhibition of prostate cancer cell line (PC-3) by anhydrodihydroartemisinin (ADHA) through caspase-dependent pathway.

Cancer is a generic term for a large group of diseases characterized by the growth of abnormal cells, which is the second leading cause of death globally. To treat cancer, currently, a number of anticancer drugs belonging to various classes chemically are available. The discovery of artemisinin and its derivatives such as artesunate, arteether, and artemether became a milestone in the cure for malaria. Here, we report the anti-cancer property of anhydrodihydroartemisinin (ADHA) - a semisynthetic derivative of artemisinin against prostate cancer cell line PC-3. ADHA was found to be inhibiting growth of PC-3 cells. ADHA was also found to be inhibiting migration of PC-3 cells. At molecular level, ADHA was found to be inhibiting the expression of c-Jun, p-c-Jun, p-Akt and NF-κB and activated caspase 3 and 7. The results show that ADHA like few other artemisinin derivatives hold potential to be used as an anti-cancer agent against prostate cancer cells.

Isaria tenuipes Peck, an entomopathogenic fungus from Darjeeling Himalaya: Evaluation of in-vitro antiproliferative and antioxidant potential of its mycelium extract.

BACKGROUND: Isaria tenuipes is one of the potent species in the members of the genus Isaria, which is well reported to possess multiple bioactive substances of therapeutic importance. Therefore, an in vitro experimental study was carried to evaluate the bioactivities of the crude methanolic extract from the mycelium of this fungus. METHODS: The fungus was authenticated through morphological characters and the species discrepancy was resolved using the nuclear rDNA ITS sequence. The methanolic extract was fingerprinted by FTIR. The antioxidant components in terms of total phenols and flavonoids were determined as gallic acid and quercetin equivalents respectively. Antioxidant activities of the methanolic extract was assessed using 1, 1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2/-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) radical cation (ABTS0+), Fe2+chelating activity, and hydroxyl radical scavenging assays. Cytotoxicity of the extract was determined by [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (MTT) assay on three cancer cell lines: HeLa, HepG2, and PC3. Apoptosis was further studied by propidium iodide (PI) and Annexin-V/PI staining flow cytometric analysis. Anti-proliferation capacity was studied by colony-forming assay. RESULTS: In the present study total phenol content of the dried methanol extract was 148.09 ± 3.51µg gallic acid equivalent/mg and flavonoid was 9.02±0.95 µg quercetin/mg. The antioxidant activities of methanol-water extract (8:2 v/v) from cultured mycelia of I. tenuipes investigated and evaluated with 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay revealed IC50 value of 5.04mg/ml with an inhibition rate of 74.77% at 10mg/ml and with an iron-chelating assay the chelating ability was recorded to be 86.76% where the IC50 value was 4.43 mg/ml. In comparison among the antioxidant assays, 2,2/-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) radical cation (ABTS0+) and hydroxyl assay exhibited radical scavenging rate of 44.42% and 49.82% respectively at a concentration of 10 mg/ml. The IC50 value of the extract in MTT assay was 43.45µg/ml with HeLa cells, 119.33µg/ml with PC3 cells, and 125.55µg/ml with HepG2 cells. CONCLUSION: In this study, it can be concluded that the crude methanolic extract exhibited potent antioxidant and antiproliferative activities suggesting natural antioxidative and antiproliferative agents.

Knockdown of PTOV1 and PIN1 exhibit common phenotypic anti-cancer effects in MDA-MB-231 cells.

BACKGROUND: Earlier, we have identified PTOV1 as a novel interactome of PIN1 in PC-3 cells. This study aims to explore the functional similarity and the common role of both genes in breast cancer cell proliferation. METHODS: CTG, crystal violet assay, clonogenic assay, wound healing assay, cell cycle analysis, Hoechst staining and ROS measurement were performed to assess cell viability, colony forming potential, cell cycle arrest, nuclear condensation and ROS production after knocking down of PTOV1 and PIN1 by siRNAs in MDA-MB-231 and MCF-7 cells. CO-IP, qPCR and western blot were performedto study interaction, transcriptional and translational regulation of both genes. RESULTS: Knockdown of PTOV1 and PIN1 inhibited the cell proliferation, colony formation, migration, cell cycle, and induced nuclear condensation as well as ROS production. Interaction of PTOV1 and PIN1 was validated by Co-IP in MDA-MB-231 cells. Genes involved in cell proliferation, migration, cell cycle, and apoptosis were regulated by PIN1 and PTOV1. PTOV1 knockdown inhibited Bcl-2, Bcl-xL and inducedBAX, LC3 and Beclin-1expression. Overexpression of PIN1 increased the expression of PTOV1. Knockdown of both genes inhibited the expression of cyclin D1, c-Myc, and ß-catenin. CONCLUSIONS: PTOV1 and PIN1 interact and exert oncogenic role in MDA-MB-231 cells by sharing the similar expression profile at transcriptional and translational level which can be a promising hub for therapeutic target.

CRISPR/Cas9 System: A Bacterial Tailor for Genomic Engineering.

Microbes use diverse defence strategies that allow them to withstand exposure to a variety of genome invaders such as bacteriophages and plasmids. One such defence strategy is the use of RNA guided endonuclease called CRISPR-associated (Cas) 9 protein. The Cas9 protein, derived from type II CRISPR/Cas system, has been adapted as a versatile tool for genome targeting and engineering due to its simplicity and high efficiency over the earlier tools such as ZFNs and TALENs. With recent advancements, CRISPR/Cas9 technology has emerged as a revolutionary tool for modulating the genome in living cells and inspires innovative translational applications in different fields. In this paper we review the developments and its potential uses in the CRISPR/Cas9 technology as well as recent advancements in genome engineering using CRISPR/Cas9.