MATERIALS AND METHODS: Thirty five female Sprague Dawley rats at age 21-day old were divided into 4 groups; Group 1 (control, n=10), Group 2 (PF4, n=5), Group 3 (rapamycin, n=10) and Group 4 (rapamycin+PF4, n=10). MNU was administered intraperitionally, dosed at 70 mg/kg body weight. The rats were treated when the tumors reached the size of 14.5 ± 0.5 mm and subsequently sacrificed after 5 days. Rapamycin and PF4 were administered as focal intralesional injections at the dose of 20 μg/lesion. The tumor tissue was then subjected to histopathological examinations for morphological appraisal and immunohistochemical assessment of the pro-apoptotic marker, Bax and anti-apoptotic markers, Bcl-2 and survivin.
RESULTS: The histopathological pattern of the untreated control cohort showed that the severity of the malignancy augments with mammary tumor growth. Tumors developing in untreated groups were more aggressive whilst those in treated groups demonstrated a transformation to a less aggressive subtype. Combined treatment resulted in a significant reduction of tumor size without phenotypic changes. Bax, the pro-apoptotic marker, was significantly expressed at higher levels in the rapamycin-treated and rapamycin+PF4-treated groups compared to controls (p<0.05). Consequently, survivin was also significantly downregulated in the rapamycin-treated and rapamycin+PF4-treated group and this was significantly different when compared to controls (p).
CONCLUSIONS: In our rat model, it could be clearly shown that rapamycin specifically affects Bax and survivin signaling pathways in activation of apoptosis. We conclude that rapamycin plays a critical role in the induction of apoptosis in MNU-induced mammary carcinoma.
Methods: In this study, the dystrophin gene from M. rosenbergii (MrDys) was first characterised followed by the characterization of dystrophin gene from a closely related shrimp species, Penaeus monodon (PmDys). Dystrophin sequences from different phyla were then used for evolutionary comparison through BLAST analysis, conserved domain analysis and phylogenetic analysis. The changes in mRNA expression levels of dystrophin and the alteration of intracellular calcium concentrations in WSSV infected muscle cells were then studied.
Results: A 1246 base pair long dystrophin sequence was identified in the giant freshwater prawn, Macrobrachium rosenbergii (MrDys) followed by 1082 base pair long dystrophin sequence in P. monodon (PmDys). Four conserved domains were identified from the thirteen dystrophin sequences compared which were classified into 5 different phyla. From the phylogenetic analysis, aside from PmDys, the characterised MrDys was shown to be most similar to the invertebrate phylum of Nematoda. In addition, an initial down-regulation of dystrophin gene expression followed by eventual up-regulation, together with an increase in intracellular calcium concentration [Ca2+]
i
were shown upon WSSV experimental infection.
Discussion: Both the functionality of the dystrophin protein and the intracellular calcium concentration were affected by WSSV infection which resulted in progressive muscle degeneration. An increased understanding of the role of dystrophin-calcium in MrDys and the interactions between these two components is necessary to prevent or reduce occurrences of muscle degeneration caused by WSSV infection, thereby reducing economic losses in the prawn farming industry from such disease.