In Vitro Anti-cancer Effect of Crataegus oxyacantha Berry Extract on Hormone Receptor Positive and Triple Negative Breast Cancers via Regulation of Canonical Wnt Signaling Pathway.

Breast cancer treatment strategy depends mainly on the receptor status. Our aim was to identify a herbal preparation, effective against breast cancer, irrespective of hormone sensitivity, and to understand its molecular mechanism. The rich antioxidant composition of hawthorn (Crataegus oxyacantha) makes it a promising anti-cancer drug candidate. Polyphenol-rich methanolic extract of C. oxyacantha berry (M.Co) was found to be cytotoxic on hormone receptor positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines, at a dose (75 µg/ml) safe on normal cells. It could effectively inhibit tumor cell proliferation and arrest cell cycle at G1/S transition in both cell lines. Molecular targets were selected from different levels of canonical Wnt signaling pathway (such as autocrine and antagonistic ligands, receptor, effector, cytoplasmic components, downstream targets, and pathway antagonist), since they are frequently found dysregulated in all breast cancers and their aberrant activation is associated with cancer stem cell expansion. M.Co could significantly downregulate the expression of Wnt pathway agonists and upregulate that of Wnt antagonists at transcriptional and translational levels, in both cell lines. To conclude, C. oxyacantha berry extract is effective against breast cancer irrespective of its hormone dependency, and cancer growth inhibition at stem cell level can be expected.

A Novel Approach of Transducing Recombinant Baculovirus into Primary Lymphoid Cells of Penaeus monodon for Developing Continuous Cell Line.

Cell line development from shrimp is not a novel venture as researchers across the globe have been trying to have crustacean cell lines over 30 years. The reason for not attaining a crustacean or precisely a shrimp cell line is believed to be the replicative senescence and the inability to maintain telomere length in vitro. Moreover, spontaneous in vitro transformations do not happen in shrimp cells. Oncogenic induction in primary cell culture is one of the ways to attain in vitro transformation by way of disrupting the mechanisms which involve cellular senescence. In this context, a recombinant baculovirus with shrimp viral promoter IHHNV-P2 was used for the transduction aimed at immortalization. An oncogene, H-ras, was successfully amplified and cloned in to the baculoviral vector, downstream to shrimp viral promoter IHHNV-P2 and upstream to GFP. Recombinant baculovirus with H-ras was generated and used for transduction into shrimp lymphoid cells during early dividing stage. Accordingly, fibroblast-like primary cell culture got developed, and H-ras and GFP expression could be confirmed. The study suggests that the simple method of incubating recombinant baculovirus with minced tissue enables in vitro transduction during early dividing stage of the cells, and the transduction efficiency gets enhanced by adding 5 mM sodium butyrate to the culture medium.

Immortalization of shrimp lymphoid cells by hybridizing with the continuous cell line Sf9 leading to the development of 'PmLyO-Sf9 '.

Shrimp progressively gets more attention among marine invertebrates from researchers all over the world due to it being a healthy food as well as having economic importance. There were a lot of attempts to develop a continuous cell line from shrimp but none successful. In this context a novel hybrid cell line named 'PmLyO-Sf9' could be developed by fusing shrimp lymphoid organ cells with Sf9 cells after to metabolic blocking of Sf9 cells using puromycin and actinomycin D and effecting the fusion by way of PEG application. The cells are maintained and multiplied in a mixture of SCCM and TNM-FH having osmolality 550 mOsm kg-1 and pH 6.8. Transmission electron microscopy of the hybrid cells revealed the presence of two nuclei during the initial stages and a single nucleus subsequently. The cell line is with shrimp and Sf9 genomic components and shrimp specific protein and is susceptible to WSSV. Shrimp elongation factor, Sf9 beta-actin, shrimp STAT and peroxinectin could be expresses through RT-PCR in the cell line. This is the first successful report of a hybrid cell line with shrimp genomic components and envisaged to be recognized a model system for multitudes of biomedical research in vitro. The cell line is in the National Cell Line Repository of ICAR - National Bureaue of Fish Genetic Resources, Lucknow, India.

'PmLyO-Sf9 - WSSV complex' could be a platform for elucidating the mechanism of viral entry, cellular apoptosis and replication impediments.

White spot syndrome virus (WSSV) is the most devastating pathogen found in shrimp aquaculture. The lack of certified continuous/established cell lines from penaeid shrimp restricts in vitro studies on the viruses to bring out effective prophylactic and therapeutic measures. In this context, a novel hybrid cell line named, PmLyO-Sf9, consisting of shrimp and Sf9 genomes has been established and employed to study WSSV susceptibility and multiplication. The hybrid cells were exposed to the shrimp virus WSSV and cytopathic effects (CPE) such as (a) enlargement of cells, (b) cessation cell division, (c) granulation of cytoplasm, (d) rounding off of cells, shortening and disappearance of tail-like structures and (e) detachment from the flask. Expression of immediate early genes such as ie 1, dnapol, rr1, tk-tmk, and pk 1could be confirmed indicating that viral DNA replication in the PmLyO-Sf9 took place followed by the expression of late genes such as VP-28, VP-26, VP-15 and VP-19. Electron micrograph of WSSV infected cells demonstrated marginated dense zones in the nucleus with clumped chromatin, and the mid zone with virus-like particles. However, neither discrete virus particles nor the culture supernatant having infectivity could be observed suggesting that virions were not getting formed in the cells. This is the first report of the susceptibility of PmLyO-Sf9 to WSSV, and the 'PmLyO-Sf9 - WSSV Complex' formed, defined as the infected status of PmLyO-Sf9 with WSSV, could be of use for unraveling at molecular level the mechanism of viral entry, replication impediments and cellular apoptosis.