Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial-to-mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E-cadherin and ZO-1 and increased expression of N-cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC-0449, and after knockdown by Shh-siRNA, and led to reversal of the EMT phenotype. The results with HTB-9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF-β1 treated HTB-9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF-β1 they did not exhibit tumorigenicity. The TGF-β1 treated HTB-9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF-β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki-67, Shh, Gli2, and N-cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF-β1-induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF-β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the ability to reverse the EMT phenotype of tumor cells and potentially inhibit bladder cancer progression and metastasis.

Activation of fibroblasts and their differentiation into myofibroblasts, excessive collagen production and fibrosis occurs in a number of bladder diseases. Similarly, conversion of epithelial cells into mesenchymal cells (EMT) has been shown to increase fibroblasts like cells. TGF-β1 can induce the EMT and the role of TGF-β1-induced EMT during bladder injury leading to fibrosis and possible organ failure is gaining increasing interest. Here we show that EMT and fibrosis in porcine bladder urothelial (UC) cells are Smad dependent. Fresh normal porcine bladder urothelial cells were grown in culture with or without TGF-β1 and EMT markers were assessed. TGF-β1 treatment induced changes in cellular morphology as depicted by a significant decrease in the expression of E-cadherin and corresponding increase in N-cadherin and α-SMA. We knocked down Smad2 and Smad3 by Smad specific siRNA. Downregulation of E-cadherin expression by TGF-β1 was Smad3-dependent, whereas N-cadherin and α-SMA were dependent on both Smad2 and Smad3. Connective tissue growth factor (CTGF/CCN2), matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) has been shown to play important roles in the pathogenesis of fibrosis. Induction of these genes by TGF-β1 was found to be time dependent. Upregulation of CTGF/CCN2 by TGF-β1 was Smad3 dependent; whereas MMP-2 was Smad2 dependent. Smad2 and Smad3 both participated in MMP-9 expression. TGF-β1 reprogrammed mesenchymal fibroblast like cells robustly expressed collagen I and III and these was inhibited by SB-431542, a TGF-β receptor inhibitor. Our results indicate that EMT of porcine bladder UC cells is TGF-β1 dependent and is mediated through Smad2 and Smad3. TGF-β1 may be an important factor in the development of bladder fibrosis via an EMT mechanism. This identifies a potential amenable therapeutic target.

Eighty-three species of finfish belonging to 13 Orders, 35 Families, and 69 genera including three exotic species, and 10 species of shellfish (crustaceans) under one order, 3 families and 3 genera were identified from the River Halda during September 2004 to December 2011. Thus a total of 93 species of Ichthyofauna (finfish and shellfish) were recorded from this river during the seven years (2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011) of investigation. Maximum number of finfish species were recorded under the family Cyprinidae (19 species) followed by the family Gobiidae (11 species), whereas maximum number of shellfish species were recorded under the family Palaemonidae (8 species) followed by Penaeidae (1 species) and Portunidae (1 species). Of the 83 finfish species, three belonged to the critically endangered, nine to the endangered and eight to the vulnerable category according IUCN (2000). Four species of finfish were new records from inland waters of Bangladesh. A systematic list of the ichthyofauna with local and English names, and habitat preference are provided.

These findings show that the prevalence of SEN-V-D/H and TTV is higher than healthy blood donors. Also, these results indicate that the prevalence of SEN-V and TTV infections in our region is similar with that in other countries.

Acute toxicity tests were carried out with aqueous and ethanolic extracts of three botanicals Lagerstroemia speciosa (L.) Pers, Dipterocarpus turbinatus Gaertn and Hevea brasiliensis (Willd. Ex Juss) on Heteropneustes fossilis (Bloch) under normal laboratory conditions. Extracts of L. speciosa were more potent than the extracts of D. turbinatus and H. brasiliensis. Histopathological changes in gill, liver and intestine of exposed fishes caused subtle cellular damages like disintegration of gill filaments and lamellae, degeneration of hepatocytes and blood vessels and necrosis of the intestinal epithelia. Different seed extracts showed histopathological variations in the different organs depending on the toxicity of the plant extracts. On the basis of histopathology, effectivity of the extract was in the order: Absolute ethyl alcohol > 50% ethyl alcohol > distilled water and the effectivity of the seed extracts on the studied organs was: L. speciosa > D. turbinatus > H. brasiliensis seed extracts. DOI: http://dx.doi.org/10.3329/jasbs.v38i2.15612 J. Asiat. Soc. Bangladesh, Sci. 38(2): 217-226, December 2012